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A LABORATORY 'MANUAL
OF
PHYSIOLOGICAL CHEMISTRY
BY
D. WRIGHT WILSON
Benjamin Rush Professor of Physiological Chemistry,
University oj Pennsylvania... '
Sixth Edition
BALTIMORE
THE WILLIAMS & WILKINS COMPANY
1947
COPYRIGHT,
THE WILLIAMS
1947
& WILKINS COMPANY
Made in United States of America
First Edition, September, 1928
Second Edition, September, 1932
Third Edition, January, 1937
Fourth Edition, January, 1941
Fifth Edition, January, 1914
Sixth Edition, January, 1947
COMPOSED AND PR[NTED AT THE
WAVERLY PRESS, INC.
1M
THE WILLIAMS & WILKINS COMPANY
BALTIMORE, MD., U.S.A.
PREFACE TO THIRD EDITION
This laboratory manual of physiological chemistry is arranged for
students familiar with elementary inorganic, theoretical and organic
chemistry. It is intended to be used as a teaching manual and not as a
comprehensive reference book. Experiments have been described which
furnish knowledge and experience in biochemical technique. Methods
have been chosen which have didactic value and which may be carried out
without too extensive use of special apparatus.
The experience and generous suggestions of my colleagues have been
drawn upon freely in making revisions for this edition. The chapter on the
cell nucleus has been left as it was written by Professor Walter Jon~s, as
little or no improvement has been made in the methods described.
As there is little variation in the type of material presented in various
courses of physiologIcal chemistry, we have found no difficulty in using
this manual in our medical, dental and veterinary courses.
PREFACE TO SIXTH EDITION
Some additional quantitative methods of blood analysis involving the
use of the photoelectric photometer have been introduced. Both the visual
and photoelectric techniques are described for other methods. I have
adopted the words "Duboscq comparator)' instead of "Dub9scq colorimeter" and "photoelectric photometer" instead of "photoelectric' ~'olorimeter" because they seem best although not in common usage.
'
.. ,
I am greatly indebted to my colleagues, Drs. Drabkin, Jones and GUrin,
who have been helpful in suggesting changes.
'.
3
CONTENTS
PART
I.
INTRODUCTION
Inorganic Constituents .. " ..... ' .. .. .. . . . . .. . .... . .... . .. . . . .... .. .. . . . . .
Standard Acid and Alkali. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Electrolytic Dissociation.. . . . . . . . . . . .. .. . . . . . .. . .. . . .. . . . . .. . . . . . . .. .. . ..
Colloids..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Alcohols, Aldehydes and Esters ................ " ............ : . . . . . . .. . ...
Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Fats.....................................................................
PART
II.
8
18
22
40
4£
50
60
78
BODY TISSUES AND FLUIDS
Saliva. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Gastric Juice..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . .. . . . . . . . . .. . ..
Pancreatic Juice .........................................................
Milk .....................................................................
Blood: General. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Blood: Quantitative Methods ................ ,.............................
88
96
102
108
116
130
B~ ..................................................................... ~
Muscle ........................................................ ' ......... '
The Cell Nucleus .................................. .'. . . . . . . . .. . . . . . . . . . ..
Bile ......... '" ................... '" ......................... " .........
Normal Urine: Qualitative ...............................................
Pathological Urine: Qualitative ...........................................
Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
Urine: Quantitative Methods .............................................
Dietary Deficiencies ......................................................
Table of Logarithms .....................................................
Abridged Table of Atomic Weights .......... : ...........................
192
202
210
214
228
236
238
268
270
272
INDEX ••.•...........•.•............................. : . . . . . . . . . . . . . : ........ ~
273
5
.READ CAREFULLY THE FOLLOWING NOTES BEFORE
BEGINNING LABORATORY WORK
Avoid the neighborhood of flames when using ether. Work with ether
can be most safely done on the floor or at an open window. Never evaporate ether on a water bath heated by a free flame: heat the water and extinguish the flame before placing a dish containing ether on the water bath.
Do not evaporate alcohol over a free flame. Use a water bath heated
with a small flame.
N ever pipette strong acids or alkalis. When a measuring cylinder is
not sufficiently accurate, a suitable burette will be made available.
Should strong acid or alkali be accidentally introduced into the mouth
or splashed in the eye, rinse the affected part immediately with copious
quantities of cold water. If at all serious, notify an instructor.
Heating of strong acids, passage of CO or H 2S, and any work involving
unpleasant odors or noxious gases are done in a hood which is being ventilated. Before using H 2S, heat the solution to boiling in a flask, then take
it to the hood and pass in the H 2S. After the precipitation has been qompleted, b0il out the excess of H 2S before removing the solution from the
hood. Use strips of filter paper moistened with lead acetate to test
for the presence of H 2S in the mouth of the flask.
Materials intended for the refrigerator or incubator are to be labelled
and given to the storekeeper who will carry out any instructions concerning
their placement or removal.
Desk reagents should be kept in order. Special reagents may be found
on the stock shelves. These should not be removed to the desks; the
amount of material required should be taken out but the portion unused
must not be returned to the stock bottle.
See that the tops of desks are free from apparatus and clean before
leaving. Clean the reagent bottles and shelf about once a week.
The concentrations of the reagents on each student's desk are as follows.
Concentrated adds: H 2S04 , 36 N; HCI, 12 N; HN0 3 , 16 N; the dilute acids
and bases are 2 N.
7
PART
I.
INTRODUCTION
INORGANIC CONSTI'FUENTS
The following experiments not only constitute a review of qualitative
tests but illustrate procedures used in qualitative and quantitative analysis
of biological materials.
PHOSPHORUS
Ammonium Phosphomolybdate. When acid solutions of phosphates are heated
with ammonium molybdate, the phosphoric acid is precipitated as yellow ammonium
phosphomolybdate. The precipitate is said to be (NH4)3P04(Mo03)12·2HN03·H20
but the composition varies considerably with the conditions attending its precipitation. The yellow compound is somewhat soluble in water but it is practically insolUble in water containing sufficient nitric acid and ammonium nitrate.
(1) To 50 ml. of a 0.03 M solution of disodium phosphate add 10 ml. of
2 N nitric acid, about 10 g. of ammonium nitrate (solid) and 120 ml. of
3% ammonium molybdate. Boil for one or two minutes. Allow the
yellow precipitate to settle, filter while still hot and wash with ammonium
nitrate solution. l Treat the precipitate as directed below.
Magnesium Ammonium Phosphate. MgNH 4P0 4 ·6H 20. By the addition of a
soluble magnesium salt to a soluble phosphate in the presence of ammonia, a gelatinous precipitate is formed. But when the precipitation is done under proper conditions, the substance forms heavy crystalline needles of magnesium.ammonium phosphate which settle rapidly leaving a perfectly clear liquid. The crystals form in
the presence of ammonium molybdate, serum proteins, etc.
Bring the yellow precipitate (obtained as described above) into solution
with boiling hot water and a little ammonia. The volume should not
exceed 40 ml. Heat the colorless solution to the boiling point and add
magnesia mixture 2 a drop at a time agitating after each addition. Magnesium ammonium phosphate is precipitated in crystalline needles. Write
the reaction. Examine microscopically. Wash the precipitate with
water containing a little ammonium hydroxide. Test solubility in nitric
acid: in acetic acid.
1 Ammonium nitrate 200 g., 16 N mtric acid 40 m!.
Make up to 4000 m!. with
water.
2 Magnesium sulfate 200 g., ammonium sulfate 280 g., 15 N ammonia 700 mI., water
1500 mI.
8
Organic Phosphorus. When organic compounds are to be tested for phosphorus,
the material is first oxidized and the phosphorus at the same time changed to phosphoric acid. This can be done in either of two ways.
a. In the dry way with oxidation mixture (equal parts of sodium carbonate and
potassium nitrate).
b. In the wet way by heating with a mixture of concentrated sulfuric acid- and
potassium sulfate.
(2) Place 0.2 g. of casein in a 500 ml. Kjeldahl digestion flask with 7 g.
of potassium sulfate} 10 ml. of concentrated sulfuric acid and 5 drops of
0.3 M copper sulfate. Warm carefully until frothing ceases and then boil
gently until all carbon is oxidized and the solution has a greenish color.
After cooling dilute with about 50 m!. of water, add 5 g. of ammonium
nitrate and precipitate the phosphate with ammonium molybdate at the
boiling point. Convert the yellow precipitate into crystalline magnesium
ammonium pho~phate.
CALCIUM
Calcium Oxalate. This is the most important compound of calcium for analytical
purposes. It is scarcely soluble in ammonia or in acetic acid but easily soluble in
mineral acids. When heated for a short time just to redness it is converted into calcium carbonate
CaC 20 4
=
CaCO a + CO
When heated at a white heat the carbonate is decomposed forming the oxide
CaCO a
=
CaO
+ CO
2
(3) Treat 10 m!. of 0.01 M calcium chloride at the boiling point with
2-3 ml. of 0.1 M ammonium oxalate a drop at a time. Examine under the
microscope with the high power.
(4) Precipitate 10 m!. of 0.1 M calcium chloride with ammonium
oxalate. Filter off the calcium oxalate and allow to dry. Remove the
dry powder to a test tube and heat for a minute to glowing. When cool
add a few drops of dilute hydrochloric acid.
MAGNESIUM
The important analytical reaction for magnesium is the second reaction
described under phosphorus.
SEPARATION OF CALCIUM, MAGNESIUM AND
PHOSPHORIC ACID
(5) Mix 10 ml. of each of the following solutions: 0.1 M magn~sium
sulfate, 0.1 M calcium chloride, 0.1 M disodium phosphate. Add acetic
10
acid cautiousiy until the precipitate dissolves, then add slowly a small
excess of ammonium oxalate.
Filter off the calcium oxalate and add ammonium hydroxide, drop by
drop, to the clear filtrate, until strongly alkaline. Examine the crystals.
Filter off the precipitate. Dissolve in a little nitric acid and test for phosphate (with ammonium molybdate).
(6) Prepare a mixture of magnesium sulfate, calcium chloride and
disodium phosphate as described above. Add acetic acid cautiously until
the precipitate dissolves, then add about 3 g. of solid sodium acetate,
100 m!. of :water and, finally, 30 m!. of 0.05 M ferric chloride. Note the
color of the precipitate and solution. Heat to boiling and filter.
Test portions of the filtrate for iron and phosphate. The test for iron
may be carried out as follows: To about 5 ml. of filtrate, add a few drops
of potassium ferrocyanide. A blue color or precipitate of ferric ferrocyanide
(Prussian blue) indicates the presence of iron.
Heat the remainder of the filtrate to boiling and add a slight excess of hot
0.1 M ammonium oxalate. Examine the crystals under the microscope.
Filter, add disodium phosphate and make the filtrate markedly alkaline by
adding ammonia drop by drop. Examine the crystalline precipitate microscopically.
Dissolve the brown precipitate in dilute nitric acid, add some solid ammonium nitrate, treat with ammonium molybdate and boil.
Explain each step of the experiment.
SULFUR
(7) Sulfate. Treat a very dilute (0.01 N) solution of sulfuric acid
with a hot dilute solution of barium chloride drop at a time and boil after
each addition. Continue the boiling for a while. The cloudy solution
becomes clear and the heavy precipitate settles sharply.
(8) Organic Sulfur. Mix about 0.5 g. of casein with 2 g. of dry oxidation mixture (p. 10) and heat in a test tube until the organic matter is completely burned off. Plunge the hot tube into 10 m!. of water contained in a
beaker so that the tube is shattered and its contents dissolved. Divide the
solution into two portions. Acidify one portion with hydrochloric acid
and test for sulfate. Test the other portion for phosphate (p. 8).
(9) Sulfide. Warm 20 ml. of 0.03 M lead acetate in a flask (under the
hood). Add sodium sulfide, drop at a time. From time to time allow the
black precipitate to settle, take off a drop of the clear liquid from the
surface with a stirring rod and place .on a filter paper. Place near it on the
paper a drop of lead acetate and watch the two drops run together. The
formation of a brown contact shows that an excess of sodium sulfide has been
added.
12
(10) Unoxidized Sulfur. Treat some lead acetate with sodium hydroxide in sufficient amount to redissolve the precipitate first formed.
To this sodium plumbite solution add a little egg albumin and boil.
Sodium hydroxide splits off sulfur from the egg albumin and the sodium
sulfide formed reacts with the lead to form black lead sulfide.
CHLORIDE
Silver chloride is formed as a curdy white precipitate when a soluble chloride is
treated with a soluble silver salt. Purine bases and uric acid also form precipitates
with silver nitrate but these precipitates are gelatinous and differ from silver chloride
by their insolubility in ammonia.
Acidify 5 m!. of 0.1 M sodium chloride with a few drops of nitric
acid. Add a slight excess of 0.1 M silver nitrate and warm. Note the
change in color of the precipitate on standing. Add an excess of 2 N
ammonium hydroxide.
(11)
NITROGEN
Nitrogen is a constituent of countless chemical compounds any of which would
serve for the detection of the element, but one naturally selects for this purpose compounds that are easily recognized and at the same time easily formed.
Three such substances are:
Ammonia
(characteristic odor)
NH3
Pyrrol
(see test below)
HC--CH
II
HC
Prussian blue
(characteristic color)
Fej[Fe( CN)eh
II
CH
"-/
NH
All of these substances contain nitrogen, so that nitrogen is proven in a compound
if it can be made to yield ammonia, Prussian blue or pyrrol, provided that no compound of nitrogen has been employed in the procedure.
These tests are used especially for the detection of nitrogen in organic
compounds.
(12) Ammonia Test. Heat a small amount (about 0.1 g.) of casein
with soda lime over a free flame in a dry test tube loosely closed with a
cork having a piece of wet red litmus paper in a slit in its under surface.
Remove the cork and note the odor.
(13) Pyrrol Test. Heat a small amount of casein over a free flame in a
dry test tube and hold in the fumes a smooth pine splinter that has been
dipped into concentrated hydrochloric acid.
(14) Lassaigne Test. Heat a small piece of metallic sodium to dull
14
redness in a dry test tube and add a very small amountl of casein. Heat
again to dull redness for half a minute.
The sodium combines with carbon and nitrogen to form s~dium cyanide,
and with sulfur to form sodium sulfide. When the material has become
well charred immerse the hot tube in 10 ml. of cold water. The tube will
break and the remaining sodium will react violently with the water.
(Caution!) The filtrate should be colorless. Heat the liquid to boiling
and filter off the carbon and broken glass. Test 2-3 ml. of the filtrate for
sulfide with a drop of sodium nitroprusside. Use the remainder of the
solution to test for cyanide as follows:
Add a drop of a mixture of ferric chloride and ferrous sulfate, heat at the
boiling point for about a minute, cool and acidify with acetic acid. (How
did the solution become alkaline?) A precipitate of Prussian blue indicates
the presence of nitrogen. Write the reactions.
INORGANIC CONSTITUENTS OF URINE AND BONE
(15) Urine and bone may be examined qualitatively for the various
inorganic constituents. For this purpose, bone should be extracted over
night with dilute hydrochloric acid. The filtered solution is boiled down
to a small volume with nitric acid to remove the hydrochloric acid.
1 The piece of sodium should be about as large as a small pea and the amount of
casein should be no more than will stand on 5-6 mm. of the point of a knife.
16
STANDARD ACID AND ALKALJI
(1) Prepare a standard oxalic acid solution as follows: Calculate the
amount of oxalic acid (H2C20 4 • 2H 20) required to make 100 m!. of 0.4 N
solution. Weigh accurately in a small beaker an amount not far (within
10%) from that calculated. Dissolve in about 50 ml. of water, transfer
quantitatively to a 100 ml. volumetric flask, make the solution up to the
mark, and mix. Calculate the normality of the solution.
(2) Make up 2 liters of a sodium hydroxide solution that is somewhat
stronger than tenth normal using the calculated amount (use measuring
cylinder. Never suck strong acid or alkali into a pipette) of 1: 1 sodium
hydroxide (about 46% by weight; sp. gr. 1.50) that has been decanted from
the sediment of sodium carbonate. Standardize it by comparison with
the standard oxalic acid solution as follows: Measure out 10 ml. of the
acid with a pipette, add a drop of phenolphthalein for an indicator and
titrate with the alkali 2 from a burette. Repeat until three titrations are
obtained that do not differ from one another more than 0.3 ml.
From the known strength of the alkali solution, the volume of the solution may be calculated that will contain the amount of sodium hydroxide
required for 2 liters of a tenth normal solution. Measure out this volume
and dilute it to 2 liters. The diluted solution is approximately 0.1 normal.
Restandardize accurately by titration against the standard oxalic acid
and calculate the normality of the alkali.
(3) Make up 2 liters of hydrochloric acid that is somewhat stronger
than tenth normal using the calculated amount of concentrated hydrochloric acid (about 32% by weight; sp. gr. 1.15). Standardize it by comparison with the standard alkali just as the alkali was itself standardized
by comparison with the oxalic acid. Use sodium alizarine sulfonate as
the indicator instead of phenolphthalein. Dilute the hydrochloric acid
so that it will be about tenth normal and restandardize by titrating 30
or 40 m!. of the acid (accurately measured from a burette) with the standard
alkali. Repeat the titration until three results varying not more than 0.3
ml. are obtained. Calculate the normality of the acid:
1 For the use of the chemical balance and the procedures of quantitative analysis
refer to some standard textbook of inorganic quantitative analysis.
2 When using phenolphthalein and many other indicators, the color change at the
neutral point is observed most readily when the titration is made by adding the
alkali to the acid and not the reverse.
18
The bottle should be shaken each time before withdrawing portions.
The standard alkali dissolves alkali from the glass and should be rest andardized against the acid every week or two.
In certain types of work such as urine analysis, it is often convenient
to use the "nitrogen equivalent" of the standard acid. This is the weight
of nitrogen contained in the ammonia that 1 m!. of the standard acid will
neutralize.
ELECTROLYTIC DISSOCIATION
It is assumed that the student will be familiar with the following topics
when he carries out the experiments in this chapter: Theory of electrolytic
dissociation, strength of acids and alkalis, degree of dissociation, ionization
constant, ionization of water, hydrolysis of salts, (H+) and pH. Brief
discussions of tl;le above topics will be found in textbooks of elementary
physical chemistry.
The following discussion is given as a brief review.
Water consists of a relatively large number of molecules and a relatively very small
number of ions. These molecules and ions are continually changing back and forth
into one another. As the reaction is reversible, the equilibrium reached may be
represented by the following equation
H 20 p H+
+ OH-
The number of hydrogen ions in water at anyone instant exactly equals the number of hydroxyl ions. The fluid is therefore said to be neutral. Solutions containing
more hydrogen ions than hydroxyl ions are said to be acid and, conversely, solutions
containing more hydroxyl ions than hydrogen ions are said to be alkaline.
The equilibrium reaction illustrated above obeys the law of mass action and may
be formulated as follows:
a constantl
or
(Hi") X (OH-) = a constant X (H 20)
As the mass of the ions is so very much less than the mass of the undissociated water,
the latter may be assumed to be constant. Then
(H+) X (OH-) = a constant
At room temperature, this constant is about 0.000,000,000,000,01 or 1.10- 14 • As the
hydrogen ions and hydroxyl ions are present in equal numbers each has a concentration of 0.000,000,1 Normal (1.10-7 N).
Since the product, (H+) X (OH-), is a constant, one constituent must be correspondingly increased in amount whenever tne other is decreased, some of each
always being present. For this reason the acidity or alkalinity of a solution may be
expressed merely by the hydrogen ion concentration. For example, in acid solu1 The brackets ( ) indicate concentration in terms of gram molecular weight per
liter. For example, read (H+), hydrogen ion concentration.
22
tions the hydrogen ion concentration is greater than 1.10-7 N and the hydroxyl ion
concentration correspondingly less; conversely, in alkaline solutions where the hydroxyl ion concentration is greater than 1.10-7 N, the hydrogen ion concentration is
less than 1.10-7 N.
Acids are ionized in water forming hydrogen ions and thus cause an excess of hydrogen ions over hydroxyl ions. Strong acids like hydrochloric acid are ionized
almost completely in dilute solution. Acetic acid on the contrary is ionized only
about one per cent in 0.1 N solution. The strength of an acid may be expressed by
the degree of ionization at a definite concentration or by the ionization constant
which may be calculated therefrom.
Alkalis are ionized to yield hydroxyl ions. The strength of alkalis may be expressed in the same way as the strength of acids.
Salts are ionized in water to yield two or more ions. Acid salts may yield 80me
hydrogen ions:
NaHS04 ~ Na+ + H+ + S04'
Basic salts may yield some hydroxyl ions:
Fe(OH)2C 2H a0 2 t::! Fe+++
+ OH- + OH- + C 2H 30:-
Neutral salts yield directly neither hydrogen nor hydroxyl ions:
NaCl p Na+
+ Cl-
The ions from certain salts, however, combine with one or the other of the ions
from water and thus remove hydrogen or hydroxyl ions, as the case may be, leaving
the solution alkaline or acid. Thus when FeCla is dissolved in water, some of the
Fe+++ ions which are liberated combine with hydroxyl ions of the water to form molecules of Fe(OH)J
FeCb ~ Fe+++ + 3CI-
+
3 H 20 P 3 OH-
+ 3 H+
it
Fe(OH)a
thus removing hydroxyl ions from the solution and leaving an excess of hydrogen
ions. The solution is therefore acid.
On the other hand, when sodium carbonate is dissolved in water a part of the CO a
ions combines with hydrogen ions of the water to form HCO a- ions thus leaving an
excess of hydroxyl ions in solution and making the solution alkaline.
+ C0
+
OH- + H+
Na 2COa p 2 Na+
H 20 p
3-
JI
HCOaThese reactions illustrate the hydrolysis of neutral salts.
One liter of water contains 10 oo~ 000 of a gram of ionized hydrogen, or as stated
above, water is a 10 oo~ 000 normal'soiution of hydrogen ions. This may be expressed
in various ways as follows:
(H+) =
1
10,000,000
N
=
0.000,000,1 N
24
=
1.10-7 N
For convenience in comparing solutions of various hydrogen ion concentrations, only
the negative exponent in the last expression is used and is designated by the term
pH. (H+) = 1.10-7 N is the same as pH = 7,
1
or pH = log-(H+)
Hydrogen ion concentration may be determined by various means, the simplest
being by the use of indicators. The indicators generally used are compounds whose
solutions change color with certain concentrations of hydrogen ions. Each indicator chan gel) color within a definite range of hydrogen ion concentrations, but
these concentrations differ enormously for different indicators.
In carryiJ;lg out the experiments outlined below, care must be taken not to contaminate any of the test solutions with traces of acids or alkalis from dirty containers or
from fumes of strong acids and alkalis. Even the distilled water should be tested
before using it.
CHOICE OF INDICATORS
(1) Prepare approximately 0.1 N acetic acid and 0.1 N ammonium
hydroxide by dilution of desk reagents. Measure the desk reagents with
a pipette and measure the diluting water with a cylinder.
Titrate 10 m!. of 0.1 N acetic acid with your standard 0.1 N sodium
hydroxide using phenolphthalein as the indicator. Note the number of
ml. required for the titration and whether or not the end point is sharp.
Repeat using methyl orange as the indicator. Titrate in the same way
your 0.1 N hydrochloric acid with the 0.1 N ammonium hydroxide using
each indicator. Which indicator is the more satisfactory for titrating
acetic acid? ammonia?
INDICATORS
(2) In order to study the effect of various hydrogen ion concentrations
on the ~ame indicator and the effect of the same hydrogen ion concentration
on various indicators, prepare 100 ml. of each of the following solutions by
diluting the desk reagents appropriately: 0.1 N hydrochloric acid, 0.1 N
acetic acid, 0.01 N ammonium hydroxide. Take 10 ml. of the 0.1 N hydrochloric acid and dilute to 100 m!. In a similar manner prepare 0.01
Nand 0.001 N acetic acid and 0.001 N ammonium hydroxide. A measuring cylinder is accurate enough for these measurements.
Calculate the hydrogen ion concentration and pH of each solution from
the following values of ex (degree of dissociation). HCI: 0.1 N, 1; 0.01
N, 1. CH3COOH: 0.1 N, 0.013; 0.01 N, 0.041; 0.001 N, 0.12. NH40H:
0.01 N, 0.041; 0.001 N, 0.12.
Prepare solutions having pH 6.0 and 8.0 as follows. pH 6.0.-Take
26
10 ml. of 0.2 M KH 2P0 4 and add the equivalent of 2.26 ml. of 0.1 N sodium
hydroxide (use your standard sodium hydroxide). pH 8.O-Take 10 m!.
of 0.2 M KH.2P04 and add the equivalent of 18.74 m!. of 0.1 N sodium
hydroxide. Dilute each solution to about 50 ml.
Arrange the above fluids in order of their hydrogen ion concentrations.
Place 5 ml. of each solution in a separate test tube and to each tube add
1 drop of methyl orange: Note the colors produced and record the results
in a table. Repeat the experiment using sodium alizarine sulfonate
(alizarine red), methyl red, phenolphthalein, litmus (paper), brom crE;sol
purple, thymol ·blue, phenol red, brom thymol blue, brom cresol green.
These indicators are frequently used in biological work.
Make a table stating the colors of the indicators at the different hydrogen
ion concentrations. Note the pH region in which the color change occure
with each indicator.
REACTIONS OF SOLUTIONS OF NEUTRAL SALTS
(3) Place 5 ml. of 0.2 M solutions of the following neutral salts in
separate test tubes. Zinc sulfate, sodium chloride, sodium acetate and
sodium carbonate. Add 1 drop of methyl red and record the color variations. Repeat the test with brom cresol purple, phenol red and thymol
blue. Refer to the previous experiment and determine the approximate
(H+) of each salt solution. Do all neutral salts yield neutral solutions?
Show by ionic equations the source of the excess H+ or OH-.
BUFFER SOLUTIONS
(4) Place 10 ml. of distilled water in a test tube and add a drop of
phenolphthalein. Titrate with 0.1 N sodium hydroxide until the solution
is barely alkaline (what is the approximate pH of the final solution?).
Repeat the experiment using one drop of methyl orange and titrating with
0.1 N hydrochloric acid (what is the approximate pH of the resulting
solution?) .
Repeat the two titrations with 10 ml. portions of the following solutions:
0.1 M sodium chloride, 0.1 M acetate (a mixture containing 0.05 M acetic
acid and 0.05 M sodium acetate), 0.1 M phosphate (a mixture containing
0.05 M mono and 0.05 M disodium phosphate). Explain the results.
HYDROGEN ION CONCENTRATION
The hydrogen ion concentration of a solution may be determined by adding to a
portion a suitable indicator and matching the resulting color with those obtained
with the same concentration of indicator in solutions of known H ion concentrations.
The standard solutions contain buffer mixtures so that the reactions are not easily
altered. A series of such solutions, covering the range of reactions most frequently
met with, may be prepared as described below.
23
Stock Solutions. 1 0.2 M potassium chloride, 0.2 M acid potassium
phthalate, 0.2 M hydrochloric acid, 0.2 M sodium hydroxide, 0.2 M
monopotassium phosphate, 0.2 M boric acid in 0.2 M potassium chloride.
(The sodium hydroxide should be practically free from carbonates and
should not contain calcium or barium.) All of the stock solutions will
keep unaltered in resistance glass except the sodium hydroxide which
should be kept in a paraffined bottle equipped with a syphon and a sodalime tube.
Mix the stock solutions in the quantities shown in the following tables
and dilute each mixture to 200 ml. (except the KCl-HCl mixtures. See
below.) The buffer mixtures may be kept for some time without deterioration in pyrex flasks. Refer to Clark's book for discussion of the refinements
necessary for greatest precision in determining pH by colorimetric or
other methods.
Composition of mixtures giving pH values at 20°C. at intervals of 0.2
KCI-HCI mixtures (Dilute to 100 m!.)
pH
1.2
1.4
1.6
1.8
2.0
2.2
12.45 m!.
26.30 m!.
35.03 m!.
40.57 m!.
44.05 m!.
46.24 ml.
0.2 M
0.2 M
0.2 M
0.2 M
0.2]\1[
0.2 M
KCI
KCI
KCI
KCI
KCI
KCI
Phthalate-HCI mixtures
To 50 m!. 0.2 M KHphthaiate add
1
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
HCI
HCI
HCI
HCI
Hel
HCI
PhthaIate-NaOH mixtures
To 50 m!. 0.2 M KHphthaIate add
pH
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
37.55 m!.
23.70 ml.
14.95 m!.
9.43 m!.
5.95 ml.
3.76 ml.
pH
46.70 m!.
39.60 ml.
33.00 ml.
26.50 m!.
20.40 m!.
14.80 m!.
9.95 m!.
6.00 m!.
2.65 m!.
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
HCI
HCI
HCI
HCI
Hel
HCI
HCI
HCI
Hel
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
0.40 m!. 0.2 M
3.65 m!. 0.2 M
7.35 m!. 0.2 M
12.00 m!. 0.2 M
17.50 m!. 0.2 M
23.65 m!. 0.2 M
29.75 m!. 0.2 M
35.25 m!. 0.2 M
39.70 ml. 0.2 M
43.10 ml. 0.2 M
45.00 m!. 0.2 M
47.00 m!. 0.2 M
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
Q
Clark, W. M., The Determination of Hydrogen Ions. 3rd ed., 1928, Baltimore.
30
KH 2P0 4-NaOH mixtures
To 50 ml. 0.2 M KH 2P0 4 add-
Boric acid, KCl-NaOH mixtures
To 50 ml. 0.2 1\1 HaBO"
0.2M KCl add
pH
pH
5.8
3.66 ml. 0.2 M NaOH
6.0
5.64 ml. 0.21\1 NaOH
6.2
8.55 ml. 0.21\1 NaOH
6.4
12.60 rnl. 0.21\1 NaOH
6.617.74ml.O.21\1NaOH
6.8
23.60 rnl. 0.21\1 NaOH
7.0
29.. 54 ml. 0.2 M NaOH
7.2
34.90 rnl. 0.21\1 NaOH
7.4
39.34 m!. 0.21\1 NaOH
7.6
42.74 rnl. 0.2 M NaOH
7.8
45.17 ml. 0.2 M NaOH
8.0
46.85 rnl. 0.21\1 NaOH
7.8
8.0
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
2.65
4.00
5.90
8.55
12.00
16.40
21.40
26.70
32.00
36.85
40.80
43.90
rol.
rnl.
ro!.
rol.
rnl.
rol.
rn!.
rol.
rn!.
m!.
m!.
rn!.
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
~aOH
NaOH
NaOH
NaOH
NaOH
NaOH
The indicators listed in the following table have been suggested by Clark and Lubs
as the rnost satisfactory for pH determinations in the range of pH of the buffer
solutions given. The concentrations mentioned in the table are suitable when 5
drops of the indicator solution are added to 10 rn!. of the solution to be tested.
Common Name
Thymol blue (see below)
Brom phenol blue
Brom cresol green
Methyl red
Brom cresol purple
Brom thymol blue
Phenol red
Cresol red
Thymol blue
Cresol phthalein
Concentration
por <onl
0.04
0.04
0.04
0.02
0.04
0.04
0.02
0.02
0.04
0.02
Coior change
Red-yellow
Yellow-blue
Yellow-blue
Red-yellow
Yellow-purple
Yellow-blue
Yellow-red
Yellow-red
Yellow-bl ue
Colorless-red
Range pH
1.2-2.8
3.0-4.6
3.8-5.4
4.4-{).0
5.2-{).8
6.0-7.6
6.8-8.4
7.2-8.8
8.0-9.6
8.2-9.8
0.05 N NaOH
required per
100 mg.
4.30
2.98
2.86
3.70
3.20
5.64
5.24
4.30
Concentrated aqueous solutions of the indicators may be prepared by grinding
and warming 100 mg. of the dry powder on a watch glass with a suitable quantity of
0.05 N sodium hydroxide to form the monosodium salt. When solution is complete
dilute to 25 m!. with water. The 0.4% solution thus obtained may be diluted to the
appropriate concentration. 100 mg. portions of the indicators require the quantities
of 0.05 N sodium hydroxide given in the above table. Methyl red solution is prepared by dissolving 40 mg. in 100 m!. alcohol and diluting to 200 m!. with water.
The use of indicators and buffer solutions to determine the pH of saliva is described on page 94, to determine the pH of urine is described on page 230.
SOURCES OF ERROR IN COLORIMETRIC
DETERMINATIONS
(6) Salt Error. Place 5 ml. of phosphate buffer (pH 6.4) in each of two
tubes. Add 5 ml. saturated potassium chloride solution to one tube and 5
32
ml. of water to the other. Add 5 drops of brom cresol purple to each tube
and note the colors. Which appears to be the more acid? A more detailed
study would show that part of the effect was due to change in the apparent
hydrogen ion concentration (H ion activity) produced by the neutral salt
and part was due to the effect of the salt on the indicator.
(6) Protein and Alkaloid Error. Place 5 m!. of phosphate buffer
(pH 6.4) in each Qf two tubes. Add 5 m!. of 1% egg albumin to one tube
and 5 m!. of water to the other. Add 5 drops of brom cresol purple to each
tube and note the colors. The experiment may be' repeated using 5 m!.
of 0.1 % quinine hydrochloride instead of albumin.
(7) Effect of Temperature. Add phosphate buffer to two tubes as
described above. Introduce 5 drops brom cresol purple into each tube
and warm one. Note the colors. The H ion concentration of the phosphate solution does not change appreciably on warming. Explain the
change of color.
TITRATION CURVES
(8) Determine the titration curve of acetic acid as follows:
Get about 50 m!. of 0.1 N acetic acid from the storeroom. Measure
out by means of a pipette a 10 m!. portion into a test tube and titrate with
0.1 N sodium hydroxide. As the titration of the acid progresses determine
and record the number of m!. of 0.1 N sodium hydroxide required to bring
the solution to the following pH values:
pH
3.2
4.0
4.8
5.2
5.6
pH
Indicators used
Brom phenol blue
Brom phenol blue
Methyl red
Methyl red
Methyl red
6.0
6.4
6.8
7.2
Indicators used
Brom
Brom
Brom
Brom
cresol purple
cresol purple
thymol blue
thymol blue
To accomplish this, proceed as follows:
Prepare a set of standard color tubes by adding 5 drops of the appropriate
indicator to 10 m!. portions of buffer solutions in different test tubes.
Refer to the above list for the names of the indicators suitable for use at the
particular pH. For example, place 10 m!. of the phosphate buffer solution
having a pH of 3.2 in a test tube and add 5 drops of brom phenol blue.
Begin the titration by adding to the acetic acid solution exactly 5 drops
of the indicator first required (brom phenol blue) and then, drop by drop,
add 0.1 N sodium hydroxide from the burette until, after mixing, the color
in your tube exactly matches the color of the first (pH 3.2) standard color
tube. The color comparison must be made in tubes of similar bore and
color. Perform this titration in a good light with the color standards at
34
hand and match colors as carefully as possible. Record the number of ml.
of alkali required to produce a solution of pH 3.2 and continue the titration
in the same tube until the 4.0 tube is matched. As these points are being
reached, add 1 drop more of the indicator for each 2.0 ml. increase in volume
of the titrated solution in order to keep the concentration of indicator
constant.
Since the determination of the next three points requires the use of a
different indicator, discard the solution just titrated, measure out a new 10
ml. portion of 0.1 N acetic acid and at once run in from the burette the same
amount of standard 0.1 N sodium hydroxide that was previously required
to reach pH 4.0. Now add the next indicator (methyl red) in proportion of
1 drop per 2 ml. total volume of titrated solution and continue the titration
until the pH 4.8, 5.2 and 5.6 tubes are matched, recording the number of
ml. alkali required in each case as above.
After this, take a third portion of the 0.1 N acetic acid, titrate directly
with the amount of alkali previously required to reach pH 5.6, add the third
indicator (brom oresol purple) in the same proportion as before and titrate
to match the pH 6.0 tube. Continue the titration as described matching
the unknown against the other standard tubes.
If your sodium hydroxide is not exactly 0.1 N, calculate the number
of ml. of 0.1 N alkali required for each titration. Plot a curve of pH
against ml. of 0.1 N sodium hydroxide from the points determined above
and add to the curve the calculated points for the acetic acid before titration and for the sodium acetate when titration is complete. Acetic acid,
K = 1.8.10-5•
Repeat the above procedure by titrating 10 m!. of 0.1 M sodium aoetate
solution (obtained at the storeroom) with your 0.1 N hydrochloric acid.
Carry out the titration and use the same buffers and color standards exactly
as described for acetic acid, but in the reverse order. Plot the results on
the sheet with the other curve.
.Repeat the experiment first titrating your 0.1 N hydrochloric aCid with
your 0.1 N sodium hydroxide, then titrating 0.1 M sodium chloride with
your 0.1 N hydrochloric acid. Assuming complete dissociation of the hydrochloric acid, calculate the pH of the solutions at the beginning and the
end of the titrations, and at the point in the first titration where 1 ml.
of hydrochloric acid remains unneutralized and, in the second titration
where 1 ml. of hydrochloric acid has been added.
ACIDITY OF CARBON DIOXIDE
(9) To 5 ml. of 0.01 M sodium bicarbonate, add a drop of phenol red,
and blow air from the lungs through the solution by means of a pipette
until the color ceases to change. Compare the color with that of a buffer
standard of pH 7.4. Aerate the solution with room air (or boil) to remove
36
CO 2 and note the color change. Repeat the experiment using 0.001 M
sodium bicarbonate obtained by diluting a 0.01 M solution of the salt.
The reaction of the blood may be said to be due to the relative concentrations of carbonic acid and sodium bicarbonate.
(10) The Hydrogen Electrode Method for Determining pH. A few
students may elect to run some determinations of pH with the hydrogen
electrode. For a discussion of the method see: W. M. Clark, the Determination of Hydrogen Ions.
(11) The. Glass Electrode Method for Determining pH. will be demonstrated.
38
COLLOIDS
DIALYSIS OF COLLOIDS AND CRYSTALLOIDS
A very satisfactory dialyzing bag may be prepared from collodion, a
cellulose nitrate. A 15% solution of collodion ("Parlodion") in equal
parts of ether and alcohol is used.
(1) Carefully pour some collodion solution into a clean, dry test tube
taking care not to form air bubbles. Pour the collodion back into the bottle
after wetting the inside of the test tube completely. Allow to drain for
about a minute. Rotate the tube slowly, holding it in a horizontal position, until the ether has partly evaporated. . When the odor of ether has
diminished considerably and the mouth of the test tube appears dry but
retains a finger print, fill the tube with tap water and allow it to stand for 10
or 15 minutes. After standing the required time, remove the collodion tube
carefully after first detaching it from the mouth of the test tube. When
loose draw it out slowly and carefully and keep it from drying by immersion
in water. If the sack is not transparent it is unsatisfactory due to adding
water too soon.
Suspend the empty bag by tying it loosely with a cord to a stirring rod
laid across a 200 ml. beaker. Add water to the beaker until the end of the
bag is submerged, then fill the bag one third full of blood by means of a
pipette or funnel and finally, add water to the beaker until the levels of the
fluids inside and out are about the same. From time to time test the outer
fluid for chlorides and note whether the red pigment diffuses through
the sack.
DIFFUSION THROUGH GELATIN
The rate at which particles in solution diffuse depends upon their size. This may
be strikingly shown with solutions of colored substances whose diffusion can be
easily followed.
(2) Prepare a 5% solution of gelatin as follows: Break up one sheet
(about 5 g.) of gelatin into small pieces (or use 5 g. of powdered gelatin)
and place in a 200 ml. beaker. Cover the gelatin with 25 ml. of water and
allow it to imbibe for at least 30 minutes. In the meantime start a water
bath heating. Place the beaker containing the gelatin in the water bath
and add the remaining 75 ml. of water. Stir occasionally until the gelatin
is dissolved. Use portions of the solution for several experiments which
follow.
40
(3) Half fill three small test tubes with the warm solution and after
the liquid has cooled and gelatinized, add one of the following colored solutions to each.
Copper sulfate: methyl orange: colloidal ferric hydroxide.
At the end of several hours note the depths to which the colored substances
have penetrated the gelatin. Observe after a day or two. Can the distances penetrated be correlated with the molecular weights of the substances?
(4) Fill a test tube two-thirds full with a slightly alkaline solution of
gelatin (5%) containing a drop of phenolphthalein and a few drops of
potassium ferrocyanide. When the gelatin has set, add 2-3 ml. of ferric
chloride. Observe for several days. Explain results.
SOL AND GEL FORMS OF COLLOIDAL SOLUTIONS
Certain colloids form solutions which solidify under suitable conditions to a jellylike mass which contains a considerable amount of water.
(6) Place 10 ml. of warm 5% gelatin in a test tube and cool under the
tap. Warm and cool alternately. Is the reaction reversible?
EMULSOIDS AND SUSPENSOIDS
Colloids may be divided into two groups, emulsoids and suspensoids. Emulsoid
solutions are viscous and form a permanent foam when they are violently shaken
with air. Suspensoid solutions do not form permanent foams. Emulsoids are
precipitated by electrolytes only in high concentration: Suspensoids are precipitated
by electrolytes in much lower concentration.
(6) Examine 5% solutions of gelatin, egg albumin and colliodal ferric
hydroxide. Shake the last two solutions with air.
(7) To 5 ml. of 1 per cent gelatin add saturated ammonium sulfate
drop at a time, counting the drops until a permanent precipitate is formed.
Repeat with 1 % colloidal ferric hydroxide instead of gelatin. Repeat the
last experiment with ~aturated ammonium sulfate diluted 1 to 100.
ELECTRICAL CHARGE
Suspensoid particles in water carry electrical charges and are precipitated by ions
tba,t carry charges of opposite sign but not by ions that carry charges of the same
sign. Note however the quantitative difference between monovalent and bivalent
ions.
(8) To 5 ml. of 1% colloidal ferric hydroxide add normal sodium chloride
a drop at a time, counting the drops, and shaking after each addition until
a permanent precipitate is formed. Make similar experiments using normal
42
solutions of sodium sulfate, magnesium sulfate and magnesium chloride.
Compare colloidal arsenious sulfide with colloidal ferric hydroxide in its
conduct tmvard the electrolytes used above.
Colloids that carry charges of opposite sign mutually precipitate one another.
(9) Treat 5 ml. of colloidal arsenious sulfide with a few drops of colloidal
ferric hydroxide diluted 1: 10.
DETERMINATION OF RELATIVE SURFACE TENSIONS
OF LIQUIDS
Whe~e a series of liquids is allowed to form drops in contact with an9ther and
immiscible liquid, the size of the drops formed is a measure of the relative surface
tension of the members of the serie~. In this experiment, water and water solutions
of various substances are allowed to flow, drop by drop into kerosen!).. The number of
drops of the water solution formed per m!. is determined and fur.nisttes an inverse
measure of the relative surface tensions of the different solutions.
(10) Obtain from the stockroom about 150 to 200 ml. kerosene, 100
ml. 1% soap solution and 50 ml. each of 1% colloidal ferric hydroxide,
and 1% albumen solutions. Fill a 50 ml. burette with distilled water and
so arrange it that the tip of the burette is immersed 3 to 5 cm. in a beaker
of kerosene. Read the height of the water in the burette and allow it to
flow slowly (use a screw clamp), drop by drop, into the kerosene, counting
the drops carefully. When 100 drops have been counted read the burette
again. Divide the number of drops counted by the number of ml. used
and express the result in terms of drops per ml. Make at least three such
readings with water. The results should not vary by more than 0.5 drop
per ml.
Repeat using the following solutions in the burette in the order listed
below:
1.0% colloidal ferric hydroxide
,1.0% albumen solution .
0.1 % bile
1.0% soap solution
0.1 % soap solution
. Prepare the 0.1 % soap solution by dilution from the 1%. If any of
the solutions foam too much to permit accurate reading of the burette, place
one or two drops of ether on the surface of the solution. The kerosene may
be used repeatedly by pouring it off from the water layer in the bottom of
th~ beaker and discarding the latter.
Record the average result in each case. .What does the experiment show
44
8.8 to the relative effects of the different solutes on the surface tension
of the water?
Shake a few ml. of each of the above solutions in a test tube. Note
the amount of foam produced and list them in order of foam production.
How does this arrangement compare with the relative surface tensions of
these solutions? What does this indicate as to the reason for foam production? According to the results of this experiment how would you distinguish between emulsoids and suspensoids?
(11) Adsorption. To 20 ml. of a 0.01 % solution of methylene blue in
water add 0.1 g. of activated charcoal ("Norite"). Shake the solution
vigorously. Filter, and note the amount of blue color which comes through
the filter paper. Allow the paper and its contents to dry on the funnel and
then pour over the charcoal 10 ml. of 95% alcohol, collecting the alcohol
filtrate in a clean flask. Estimate approximately the amount of dye which
is recovered. How may the behavior of the methylene blue toward the
charcoal be explained?
46
ALCOHOLS, ALDEHYDES AND ESTERS
The following experiments are designed (a) to illustrate some 'of the
chemical properties of these organic compounds; (b) to demonstrate, using
compounds with simple structure, reactions characteristic of certain organic
groups that are present in sugars or fats.
(1) The Oxidation of Alcohols to Aldehydes. Wind the end of a piece
of copper wire around a stirring rod so that a coil is formed. Heat this in a
Bunsen burner. When it is removed, notice that it is coated with CuO.
Plunge the hot wire into a few ml. of 5% ethyl alcohol solution, in a test
tube. Note the change in color of the wire and the odor of acetaldehyde
(pungent and irritating). Write the equation for the reaction.
(2) The Oxidation of Aldehydes. (a) To a little silver nitrate solution
in a test tube add ammonia, drop by drop, until the precipitate which first
forms is redissolved. Add to this a few drops of aldehyde solution and
place in a water bath. Explain the formation of the silver mirror. 'Write
the equation for the reaction involved. (b) Prepare 5 mI. of mixed Fehling's solution (See footnote, p. 50). Heat the mixture. Is any precipitate
formed? If not, add a few drops of aldehyde solution and heat again.
The cupric hydroxide is reduced by the aldehyde to cuprous hydroxide.
Write the equation.
(3) The Formation of an Ester. To a little glacial acetic acid add.a few
drops of absolute ethyl alcohol. Add a drop of concentrated sulfuric acid
and warm gently. The esters are characterized by their fruity odor.
'Write the equation for the reaction which has occurred.
(4) The Hydrolysis (Saponification) of an Ester. With a Mohr pipette,
measure 1 ml. of ethyl acetate into a 250 ml. Erlenmeyer flask. Add 50
mI. of 0.1 N NaOH and a few drops of phenolphthalein. Attach a reflux
(air) condenser and he~t in a boiling water bath 15 minutes. Remove, cool,
and titrat~ the contents of the flask with 0.1 N HCI. Has hydrolysis of the
ester taken place? Calculate the number of ml. of 0.1 N NaOH that would
he r.equired to effect the saponification of 1 mI. of ethyl acetate (sp. gr. 0.9).
48
CARBOHYDRATES
(1) Molisch Test. Place 5 ml. of concentrated sulfuric acid in one test
tube and in another, mix 2 drops of 5% ex naphthol in alcohol with 5 ml. of
a sugar solution. Incline the tube containing the sulfuric acid and pour
the other solution into it carefully so that there is very little mixing. Thus
the two liquids are stratified. Note the color of the ring at the zone of contact of the two fluids. This is the most general test for carbohydrates.
It is due to furfuraldehyde condensing with ex naphthol.
MONOSACCHARIDES AND DISACCHARIDES
Write graphic formulas for glucose, maltose, lactose and sucrose. Obtain
0.1 M solutions of the sugars just mentioned and carry out the tests given
below. Compare the properties shown by the experiments with the
formulas.
(2) Fehling's Test. Boil 5 ml. of mixed Fehling'sl solution in a testtube. Add the sugar solution a few drops at a time and boil after each
addition. Compare the color and amount of precipitate with that obtained
in Benedict's test.
The following equation illustrates some of the chemical changes which
take place.
ROHO
+ 2 Ou(OH)2
=
ROOOH
+ 2 OuOH + H 20
Changes of similar nature occur in expo 3, 4, 5, 6, 7.
(3) Benedict's Test. Make the same test using Benedict's solution: 2
Compare the delicacy of this and Fehling's test by using more dilute
solutions of glucose.
(4) Nylander's Test. Test the sugar solution with Nylander's solution3
as was done in Fehling's test. 'The solution turns brown or black, due to the
formation of metallic bismuth. Note that the presence of sulfide may
cause confusion.
134,6 g. of copper sulfate in 500 ml. of water (blue solution). 173 g. of Rochelle
salt and 50 g. of sodium hydroxide in 500 m!. of water (colorless solution). Mix equal
volumes of the two solutions when needed.
2173 g. of sodium citrate and 100 g. of dry sodium carbonate dissolved in 800 m!. of
water, and filtered if necessary. 17.3 g. of copper sulfate dissolved in 200 m!. of warm
water. Stir the latter solution, a little at a time, into the alkaline citrate. Filter if
necessary.
3 Digest 4 g. of bismuth subnitrate on the water bath with a solution of 4 g. of Rochelle salt in 110 ml. of 2 N sodium hydroxide. Filter or decant the solution from any
undissolved bismuth subnitrate.
.
50
(5) Ferricyanide Test. Make some sugar solution alkaline with sodium carbonate and add enough of a freshly prepared solution of potassium
ferricyanide to give a faint yellow color. Boil the solution and note the
color. Acidify with acetic acid and add a drop of ferric chloride.
(6) Silver Test. To some silver nitrate add ammonia until the brmm
precipitate first formed just dissolves. Add some sugar solution and
carefully heat without boiling. A silver mirror will be deposited. This
test is so sensitive as to be impracticable.
(7) Barfoed's Test. Boil 5 ml. of Barfoed'sl solution with a little
sugar solution. Note which sugars give positive and which give negative
tests when similar amounts of sugar are heated similar lengths of time.
The test may be used to distinguish monosaccharides from disaccharides
if care is taken.
(8) Osazone Test. To 5 ml. of a sugar solution add 5 ml. of phenylhydrazine solution. 2 Immerse in boiling water and contInue heating from
30 to 45 minutes, replacing the water lost by evaporation from the test
tube. Allow the bath containing the tube to cool slowly. ExaJ;lline the
crystals microscopically. Always examine crystals in their mother liquor.
'Write complete equations. Some of the osazones usually form crystals
which are easily recognized microscopically.
.
(9) Fermentation Test. Shake violently about 15 ml. of a sugar solution with a piece of compressed yeast the size of a pea. Fill a fermentation
tube, making sure that no air bubble remains in it. Remove most of the
liquid from the bulb and allow it to stand at room temperature overnight.
Test the gas which accumulates by adding a little sodium hydroxide, filling
the bulb with water and inverting several times with the opening closed by
the thumb.
(10) Optical Test. Weigh accurately about 3 g. of pure lactose.
Grind finely if not already powdered, dissolve as quickly as possible in
about 75 ml. of cold water and make up the solution to 100 ml. in a volumetric flask. Without delay.fill a 2 dm. polarimeter tube with the lactose
1l_olution and polarize. Note the direction of the rotation. This direction
is called dextro-rotation. Make a second reading about 5 minutes later.
Determine the zero point of the instrument and calculate the specific rotations by means of the following formula.
[adD = 100'a
l·p
1 Dissolve 66 g. of cupric acetate and 10 m!. of glacial acetic acid in water and
make up to 1 liter.
2 A mixture containing 5% phenylhydrazine hydrochloride, 20% sodium acetate
and 10% acetic acid in water.
52
[aJD = specific rotation.
= observed angle in degrees and fractions of a degree (not minutes).
I = length of the tube in decimeters.
p = sugar percentage.
a
A constant reading is obtained only after allowing the solution to stand
a considerable time. Equilibrium may be obtained more quickly by boiling
the solution. Heat part of the solution barely to boiling in a small flask
covered with a watch glass. Cool, polarize again and calculate the specific
rotation.
Place 50 m!. of the lactose solution in a flask with 10 m!. of dilute hydrochloric acid. Immerse in a boiling water bath and heat for about 20 minutes. Nearly neutralize ,vith solid sodium carbonate, make up the solution to 100 m!. and polarize. Calculate the specific rotation and compare
with the theoretical value.
(11) Mucic Acid Test. Place 5 g. of lactose in a 100 m!. flask with 15
m!. of concentrated nitric acid and 15 m!. of dilute nitric acid. Heat
carefully with a free flame under the hood until the reaction begins. Remove the flame, allow the reaction to complete itself and the products to
stand over night. Decant the fluid from the crystals of mucic acid and
wash by decantation with a little cold water. Suspend the mucic acid in a
little hot water and bring into solution by the addition of ammonia drop
at a time until the solution is alkaline to litmus. On cooling, the solution
will deposit crystals of ammonium mucate.
For the following test use either the crystals of ammonium mucate or
the dry residue obtained by carefully evaporating the mother liquor.
Heat in a dry test tube and hold in the fumes a smooth pine splinter
that has been dipped into concentrated hydrochloric acid. Ammonium
mucate gives the pyrrol test.
(12) Seliwanoff's Reaction. Add 2-3 drops of 0.1 M solutions of
fructose, glucose, sucrose and lactose respectively to test tubes containing
2-3 m!. of Seliwanoff's reagent,! Place the tubes in a boiling water bath
and note the time. Ooserve the colors after 3, 5, and 10 minutes heating.
(13)' Orcin Test. Treat a 0.01 M arabinose solution with an equal
volume of concentrated hydrochloric acid and a knife point of orcin. The
solution when heated becomes bluish-red and gives an absorption band
bebveen the D and E lines of the spectr:um.
(14) Phloroglucin Test. Repeat the last test, using phloroglucin
instead of orcin. The solution becomes cherry red and shows an absorption
band between D and E. The colors (but not the absorption bands) are
given, by galactose. Glucuronic acid responds to the entire test and cannot
I
Dissolve 0,1 g. of resorcinol in 100 m!. of 1 N HCI.
54
in this connection be distinguished from pentose. When the tests are made
with concentrated pentose solutions the pigment is precipitated and can be
extracted for spectroscopic examination with amyl alcohol or some other
solvent.
Add a knife point of phloroglucin to 5 ml. of a concentrated solution
of gum arabic and an equal volume of concentrated hydrochloric acid, heat
gently and allow to stand until a precipitate forms. Then shake with 5
ml. of ethyl acetate to extract the pigment. Examine spectroscopically a
sufficiently dilute solution of the pigment in ethyl acetate. .
(15) Inversion of Sucrose. To 25 ml. of sucrose solution add 1 ml.
of glacial acetic acid and heat on a boiling water bath for 10 minutes. Cool
and neutralize with sodium hydroxide and carry out a reduction test, and
the osazone and fermentation tests. The reason for the use of the term
"inversion" is shown by polarimeter examination of sucrose before and
'after treatment as described above. (See Optical Test.)
POLYSACCHARIDES
STARCH
(16) Starch Grains. Scrape off a little of the inside of a raw potato,
mix with cold water and examine a drop of the finer suspension under
the microscope. Place a drop of iodine solution at the edge of the cover
and draw it under the cover glass by touching the opposite edge with a
piece of filter paper. Note the color of the starch grains in contact with
the iodine solution.
(17) Starch Paste. Powder as finely as possible about 1 g. of starch
and grind into a fine suspension with 10 ml. of cold water added a few
drops at a time as the grinding is continued. Finally, pour into 90 ml. of
boiling water. Examine a drop of the material under the microscope.
Are the starch grains visible? Test the starch paste by Fehling's test and
the fermentation test.
(18) Iodine Test. Treat some very dilute starch paste with iodinepotassium iodide soluJion. '(The iodine solution should be diluted before
use until it is light yellow in color.)
(19) Hydrolysis. Treat 25 ml. of starch paste with 1 ml. of dilute
hydtochloric acid and immerse in a boiling water bath. As the heating
continues test small portions of the solution with the iodine test and with
Fehling's test.
When the starch has completely disappeared, nearly neutralize with
sodium hydroxide, add about half a gram of sodium acetate and prepare an
osazone as directed above.
56
GLYCOGEN
(20) Grind about 10 g. of fresh oysters or scallops in a meat gr~nder.
Place in an evaporating dish with 10 ml. of water. Heat, to boiling and
acidify slightly with acetic acid to precipitate the protein. After boiling
several minutes, filter. Note the appearance of the solution. Precipitate
the glycogen by adding 2 volumes of 95% alcohol. Wash by decantation
with 50% alcohol, 95% alcohol and finally ether, and allow to dry. Dissolve a portion in water and note the appearance of the ~olution.
(21) Iodine Test. To an aqueous solution of glycogen add a drop of
iodine solution. Make a parallel test using water instead of the glycogen
solution. Compare the color with that produced by starch and iodine.
(22) Fehling's Test. Test 5 ml. of an aqueous solution of glycogen
with Fehling's solution.
(23) Hydrolysis of Glycogen. Boil a solution of glycogen (5 ml.)
for a few minutes with one-fourth its volume of concentrated hydrochloric
acid. Note the -appearance of the solution as the boiling is continued.
When clear, cool and neutralize the solution. Examine it by means of
the iodine test and Fehling's test. Identify the sugar present by the
various procedures which you have used.
CELLULOSE
Cellulose is characterized by its insolubility in all common solvents.
(24) Solubility in Schweitzer's Reagent. Mix finely picked cotton
wool with Schweitzer's reagentl in a test tube. Acidify a portion of the
cellulose solution and observe the viscous precipitate.
(26) Iodine Test. Treat cotton wool with diluted iodine-potassium
iodide solution. How does the test differ from that with starch or glycogen?
1 To 100 m!. of 0.2 M copper sulfate, add 1 M sodium hydroxide with stirring until
tbe precipitat"ion is complete. The blue precipitate becomes black if too much
alkali is added. Filter off the blue precipitate and wash free from sulfates. Transfer the moist precipitate to'a mortar and triturate with 150 m!. of 28% ammonium
hydroxide.
58
PROTEINS
ELEMENTARY COMPOSITION
(1) Heat about half a gram of dry protein (casein) in a dry test tube.
(Hood.) The odor indicates nitrogen, charring indicates carbon, while
the water which condenses on the colder parts of the tube suggests hydrogen.
(2) Test casein for nitrogen, phosphorus, sulfur and unoxidized sulfur.
See pp. 8-14.
COLOR REACTIONS
Proteins possess groups that react with certain reagents to form colored compounds.
Make the following tests with egg albumin, gelatin, casein and peptone
solutions, and with solid casein. For every color test run a negative
control, i.e., carry out the test with the reagents alone.
(3) Biuret Reaction. Treat 1 m]; of protein solution with 5 ml. of 2 N
sodium hydroxide and add a few drops of very dilute copper sulfate.
(Dilute some copper sulfate solution until the 'blue color is barely apparent.)
The color produced varies with different proteins.
(4) Make the biuret test with biuret! instead of protein.
(5) Xanthoproteic Reaction. To a small portion of protein solution
add an equal volume of concentrated nitric acid. Note the color.
Yellow nitro compounds are formed with tyrosine. and tryptophane.
(6) The Mercuric Nitrite Test. (Millon's Test2 modified by Cole.)
1 Heat half a gram of urea in a dry test tube over a small free flame.
It melts, then
boils with evolution of ammonia (odor) and finally solidifies, when the heating must
be immediately discontinued. Biuret .is formed.
NH
NH2
NH2
I
I
O=C
NH2
+
/A"
I
I
O=C
O=C
O=C
I
I
NH2
NH2
+
NH3
NH2
When cool, digest the product with warm water, filter off the solution of biuret from
the undissolved cyanuric acid and use a few m!. for the test as described.
2 Millon's reagent was previously prepared by digesting 50 g. of mercury with 100
g. of concentrated mtric acid. After the mercury dissolved, the solution was diluted
to 400m!. with water. The solution apparently contained mercury and nitrous acid
and an excess of nitric acid. The color produced with protein was rapidly destroyed
by the excess acid. Cole has proposed a much more sensitive modification of the original test.
60
To about 1 ml. of the solution, add an equal volume of 10% mercuric
sulfate in 10% sulfuric acid. Boil gently for at least one-half minute.
Usually a precipitate is formed which clings to the side of the tube and
becomes yellow. Cool, add one drop of 1% sodium nitrite solution and
warm. Note the c()lor of the precipitate or solution. Repeat the test with
0.1 % phenol; with dry casein.
The color forms in the presence of hydroxy phenyl &roups. What amino
acid found in proteins should give this test?
(7) The Aldehyde Reaction for Tryptophane. 1 To about 1 mI. of the
solution add 1 drop of a 1 to 500 dilution of 40% formaldehyde. Then
add 1 drop of 10% mercuric sulfate in sulfuric acid. Mix and add 1 ml.
(or slightly more) of concentrated sulfuric acid. Mix gently. Note the
color of the solution. Among protein hydrolytic products, tryptophane is
the only amino acid that will give the test.
(8) Pauly's Diazo Reaction. Treat 1 ml. of a 0.5% solution of sulfanilic
acid (in 2% hydrochloric acid) with an equal volume of 0.5% sodium nitrite
and allow to stand for a few minutes. Diazobenzene sulfonic acid is
formed. Add 1 ml. of protein solution and make alkaline with sodium
carbonate. A red color develops due to the presence of the histidine and
tyrosine groups in the protein.
COAGULATION BY HEAT
Proteins are coagulated with heat when in solution near the isoelectric point.
When a protein solution on the acid or alkaline side of the isoelectric point is heated
a change occurs which renders the protein insoluble at. the isoelectric point. The
changed protein is called acid or alkali metaprotein.
(9) Add 2 drops of 0.2 N acetic acid to 5 ml. of egg albumin solution
and heat to boiling.
(10) Mix about 5 drops of 0.2 N hydrochloric acid with 5 ml. of egg
albumin solution. Heat to boiling, and cool. Does a precipitate form?
Add 0.2 N sodium hydroxid~ drop by drop. Does the precipitate dissolve
in an excess?
(11) Add 2 drops of 0.2 N sodium hydroxide to 5 ml. of egg albumin
solution and heat to boiling. -Cool and add 0.2 N acetic acid drop by drop
until an excess is present.
(12) Temperature of Coagulation. Place 5 ml. of egg albumin solution
1 This is Cole's modification of an old test.
The original test was proposed by
Adanl.kiewicz who used glacial acetic acid with the sulfuric acid. Hopkins and Cole
showed that the acetic acid contained glyoxylic acid as an impurity and proposed the
use qf glyoxylic acid. Cole showed that various aldehydes could be used with mild
oxidizing agents. If the sulfuric acid contains too large an amount of oxidizing
agents, the test will fail.
62
and 1 drop of 0.2 N acetic acid in a test tube and 5 ml. water in another.
Place a thermometer in the tube containing the water and immerse both
tubes in a beaker of water that is slowly heated and stirred continually
while the temperature rises. Read the temperature at which increased
opalescence is first apparent.
/'
ADSORPTION
(13) Dilute 1% egg albumin solution with two volumes of water and
see that the solution gives a decided biuret test.
Mix 5 mI. of this solution with 1 m!. of colloidal ferric hydroxide and
add 2 drops of 1 M sodium sulfate. Filter off the precipitated ferric hydroxide and test the filtrate for protein with the biuret test.
PRECIPITATION WITH VARIOUS REAGENTS
,
(14) Strong Acids. To separate small portions of egg albumin solution
add concentrated hydrochloric acid and concentrated nitric acid. The
precipitate decomposes when the solutions are heated and the decomposition products pass into solution.
(16) Stratify some concentrated nitric acid with a very dilute protein
solution and note the ring formed near the surface of contact. This is
Heller's sensitive protein test.
(16) Heavy Metals. To separate 5 m!. portions of protein solution
add the following reagents drop at a time.
Lead acetate: silver nitrate: mercuric chloride.
(17) Alkaloidal Reagents. To separate 5 m!. portions of p~otein solution add the following reagents.
Saturated picric acid: trichloracetic acid: tannic acid:
phosphotungstic acid: sulfosalicylic acid.
Repeat the last three tests with acidified protein solution.
The formation of salts with both acids and metals illustrates the amphoteric character of the 'proteins.
(18) Alcohol. Add 10 m!. of alcohol to 1 m!. of albumin solution.
Most of the reagents used above are of value in different connections for
removmg protein from solutions.
THE "SALTING OUT" OF PROTEINS
When protein solutions are saturated with certain inorganic salts, the unaltered
proteins are precipitated. The salts commonly used for this purpose are ammonium
sulfate, zinc sulfate, magnesium sulfate and sodium chloride; but these salts differ
from'one another in their conduct toward a given protein and proteins may differ from
one another in their conduct toward a given salt. Two obvious methods are therefore suggested for separating proteins from one another.
64
(19) Saturate 10 ml. of albumin solution with solid ammonium sulfate.
Filter and test both filtrate and precipitate for coagulable protein.
(20) Saturate 10 ml. of albumin solution with magnesium sulfate or
sodium chloride (?).
SEPARATION OF GLOBULINS FROM ALBUMINS
Blood serum contains two types of protein, albumin and globulin, both of which
arc coagulable by heat under proper conditions and respond to the protein color
reactions. When blood serum is satumted with magnesium sulfate or half saturated
with ammonium sulfate, the globulins are precipitated while the albumins remain in
solution.
(21) Saturate 10 ml. of blood serum with magnesium sulfate. Filter
and test both filtrate and precipitate for coagulable protein.
(22) Treat 20 ml. of blood serum with an equal volume of saturated
ammonium sulfate. (The solution is now half saturated.) Filter. Dissolve some of the precipitate in water and test for coagulable protein.
Test a small portion of the filtrate for coagulable protein. Saturate the
remainder of the filtrate with ammonium sulfate. Filter and test the
precipitate and filtrate for coagulable protein.
DIALYSIS
(23) Place 30 or 40 ml. of clear blood serum in a collodion or parchment
bag and after the addition of a few ml. of toluene as a preservative, suspend
in a beaker of distilled water. Allow to dialyze over night or longer.
Note the precipitation of globulin when the salts have been removed
sufficiently. Filter off the precipitate and test the filtrate for globulin and
albumin (see previous experiment).
,
Test the dialysate for protein and for chlorides.
PREPARATION OF A CRYSTALLINE PROTEIN
(Cuc~rbit Seed Globulin)1
Grind air-dried pumpkin seeds in a meat grinder, first with the coarsest
cutting plate, then with the finest to obtain a fine meal. Extract the meal
with approximately two times its volume of benzene or gasoline to remove
most of the fat. Dry the material in air.
(24) Extract 50 g. of the defatted meal for an hour in a 500 ml. flask
with 200 ml. of 10% NaCI at 60° (heat in a water bath warmed to 60°-65°).
Filter the extract through a fluted paper into 3-4 volumes of water, maintained at 60°. Permit the solution to cool very gradually, and allow to
1
Vickery, H. B., et aI., J. B. C., 140,613 (1941).
66
stand overnight. Regular octahedral crystals are deposited. Examine
microscopically. Test its solubility in water and in 5% sodium chloride.
(25) Prepare a solution of this globulin and test its coagulability by heat.
(26) Treat 10 ml. of this solution with an equal volume of saturated
ammonium sulfate.
(27) Test the protein for oxyphenyl, histidine and tryptophane groups.
PREPARATION OF CYSTINE AND TYROSINE
Human hair is the best and most convenient source of cystine and tyrosine. With
careful work 5% cystine and about 0.5% tyrosine can be prepared from it. The yield
of cystine can be raised to 8 or 10% by the use of alcohol but the present method employs only isoelectric precipitation. The hair should be separated from any adhering
debris by hand removal of the latter and should then be washed with 1% cold sodium
carbonate solution. It should then be thoroughly washed with water and dried
completely before being either used or stored for future use. The hair furnished for
this preparation in the student laboratory is already prepared as described above so
that the acid hydrolysis may be proceeded with at once.
ACID HYDROLYSIS
(28) Weigh roughly 500 g. of dried hair and transfer it to a 2 liter
pyrex flask. Add 1 liter of hydrochloric acid solution made by diluting
2 volumes of concentrated hydrochloric acid with 1 volume of tap water.
Fit the flask with a reflux condenser and boil gently for 8 hours. The
requisite amount of boiling may take place on two separate days. There
is no objection to allowing the flask to stand in the cold over night. When
boiling is complete, allow the contents of the flask to cool, dilute to 1.5 liters
with tap water and filter through a Buchner funnel with suction.
Discard the insoluble residue, transfer the brown solution to a precipitating jar of at least 2 liter capacity and neutralize with small portions of solid
sodium carbonate until the solution is approximately neutral to Congo red
paper. Nearly 300 g. anhydrous sodium carbonate may be required.
At this point pour the solutiop. carefully into another jar or beaker in order
to remove any undissdlved lumps of carbonate. Then continue neutralizing with concentrated sodium hydroxide solution (a 50% filtered solution
made from technical caustic soda) until the solution is definitely alkaline
to Congo red and barely acid to litmus (a very dull red). In all cases, care
mu~t be taken not to overrun a pH of 7.0, particularly if the solution has
become warm as a result of the neutralization. The alkali must at all
times be added drop by drop with constant stirring. While sodium carbonate is used, care must be taken to prevent too much foaming. A few drops
of alcohol on the foam will cause it to subside quickly. When neutralization is complete, allow the jar and contents to stand at. least over night
68
(preferably two nights or more). The complete .precipitation of cystine
and tyrosine is, at this point, slow. Examine a sample of the precipitate
under the microscope. Regardless of its appearance, filter the precipitate
on a Buchner funnel with suction. Discard the filtrate.
Transfer the precipitate from the isoelectric precipitation (containing
both cystine and tyrosine) to a large beaker and dissolve it in the least
possible amount of 1: 1 hydrochloric acid (about 150 to 200 ml.) and dilute
to 1 liter. To the brown solution add about 10 g. decolorizing carbon, boil
the mixture for 5 minutes, allow to cool for at least an hour, filter through
a Buchner funnel, wash the residue with 3 washings of 25 ml. water each,
and add the washings to the filtrate. Should the solution still be brown
repeat the decolorization using fresh charcoal. Care should be taken
(why?) to keep the solution strongly acid to Congo red by addition of small
amounts of hydrochloric acid as the latter is lost by boiling.
When colorless, neuti'alize the filtrate, slowly and carefully with a concentrated solution of sodium hydroxide. As soon as a permanent precipitate appears examine a sample under the microscope and follow the course
of the precipitation by this means. When the reaction towards Congo
red paper is barely alkaline, and if no tyrosine needles are seen under the
microscope, filter at once through a Buchner funnel, wash slightly and continue neutralizing the filtrate and washings until neutrality to litmus has
nearly been reached. Examine this precipitate under the microscope.
What is the principle of this separation between cystine an.d tyrosine? Filter on a separate paper and wash with water. Lay aside the sample'of crude
tyrosine for future purification. ]f the cystine precipitate is not perfectly
white, dissolve once more in 1 : 1 hydrochloric acid and reprecipitate as before
(without using decolorizing carbon). Filter as before and wash with distilled
water until the washings show no test for chlorides with silver nitrate after
acidification with nitric acid. Dry the sample on a watch glass in a desiccator and weigh. Calculate the percentage yield of the purified cystine.
In case difficulty. is experienced in separating the last mixture of cystine
and tyrosine, hot water extraction of the tyrosine may be resorted to.
The solubility of tyrosin~ increases with rising temperature far more
r'apidly than does that of cystine. For this reason the mixture of the two
'may be brought to boiling temp~rature in 100 m!. of water and filtered at
once (hot) through a Buchner funnel. On cooling, the solution will deposit
large tyrosine crystals. The solid residue may be extracted again in the
same way as long as it contains any tyrosine and the cystine finally remaining may be recrystallized and added to the pure cystine previously
obtained.
70
The tyrosine obtained by this method contains, however, small amounts
of cystine and shows an abnormally high optical activity unless further
recrystallized once or twice as described below.
When the purified cystine is thoroughly dry, weigh a 0.500 g. sample,
dissolve in 1.0 N hydrochloric acid, made by rough dilution of one volume
concentrated hydrochloric acid up to 10 volumes with water, and dilute
with the same in a 50 ml. volumetric flask to the mark. Determine its
optical activity in a polariscope. Calculate the [aln value. Test the
sample for complete removal of tyrosine.
The crude tyrosine should be purified by a procedure similar to that
used with cystine. Dissolve in the smallest possible amount of 1: 1
hydrochloric acid, dilute about 10 times, decolorize if necessary and
reprecipitate by neutralizing with sodium hydroxide solution to bare
alkalinity towards Congo red (purple). Allow the solution to stand at
least over night in order to precipitate any small amounts of cystine
which may yet remain. Filter and precipitate the tyrosine as before.
Filter, wash free from chlorides, dry and weigh. Determine the optical
activity of the tyrosine by the same procedure as that used for cystine.
Test the sample for complete removal of cystine.
Hand in both samples in stoppered dry, clean test tubes with labels
giving your name, the name of the sample, its [aln value and its weight.
(29)
SORENSEN'S FORMALDEHYDE TITRATION
A neutral solution of a substance having amino, as well as acidic groups, is treated
with a neutral solution of formaldehyde, a considerable excess being used. The
solution becomes acid and may be titrated. For amino acids the probable mechanism
is as follows: In a neutral solution the amino acids are predominantly in the zwitterion form, with a small amount of the cationic and the anionic; forms. Reaction
(A) represents the equilibrium between the zwitterion and the anion.
(A)
+H 3N·R·COO+HCHO
(B)
~
H 2N·R·COO- + H+
+HCHO
CH 2 (OH)·NH·R· COO- ==(C=)==
'The reaction m!l-y be shifted to the right by removal of the hydrogen ion, as by
adding base, but the concentration of hydroxyl ions is so great by the time one
equivalent of hydrogen ions has been removed to form water that a sharp titration
end-point is impossible. In the formaldehyde titration the equilibrium (A) is shifted
to the right by removal of the anion to form the methylol derivative of the amino acid
72
anion according to reaction (B). An excess of formaldehyde may cause the formation of the dimethylol derivative (reaction (C» which, in strongly alkaline solution,
may lose water and formaldehyde, irreversibly, to give the methylene derivative
according to reaction (D). By employing a sufficient excess of formaldehyde, equilibrium (A) is shifted BQ far to the right as to make the yield of hydrogen ions from the
-NHt groups virtually quantitative. The amount of hydrogen ions thus formed
may be determined by titration, the end-point coming in a pH region where the use
of phenolphthalein is possible.
•
Formaldehyde Solution. Dilute commercial formaldehyde (40%) with
an equal volume of distilled water. Add 5 drops of 1% phenolphthalein
for every 100 ml. of the solution, and titrate with 0.1 N sodium hydroxide
until a faint pink tinge is obtained. It may be necessary to add a few more
drops of the alkali from time to time owing to the oxidation of formaldehyde to formic acid.
The Determination. Transfer 25 ml. of the protein solution to a large
test tube, and neutralize! it to about pH 7.0, using red and blue litmus
paper as an indicator. Add 10 m!. of neutral 20% formaldehyde and 6
drops of 1 % phenolphthalein, and then titrate the solution with 0.1 N
sodium hydroxide. Use as an end point the color that is produced by
mixing 25 ml. of neutral boiled water, 10 m!. neutralized 20% formaldehyde, and 0.3 m]. of 0.1 N sodium hydroxide with 6 drops of the phenolphthalein (about pH 10).
If the original protein solution is colored or slightly turbid, the standard
should be placed in a color comparator behind some of the protein solution,
similarly diluted, and the resulting color used as the end point for the
titration. If the titration is greater than 5 m!. the standards should be
diluted by this additional quantity of liquid.
.
Since the reaction of one amino group with formaldehyde liberates one
hydrogen ion, one can calculate from the ml. sodium hydroxide neutralized
(after substracting the 0.3 m!. blank) the amount of amino nitrogen in the
original solution. If the t9ta( nitrogen is known the percentage of free
amino nitrogen to total nitrogen may be calculated.
Deteqnine the percentage of free amino nitrogen to total nitrogen in
egg albumin, peptone and alanine.
(30) THE ISOELECTRIC POINT OF CASEINZ
The insolubility of casein at its isoelectric point may be used as a convenient means
for determining that point. A series of tubes, each containing a standard amount of
'1 It is important to neutralize the solution only when thc solution is far from neutra.! as when gastric or pancreatic digests are studied.
2 Method of Michaelis and Pechstein (Biochem. Zeit., 47, 260 (1912» as modified
~C~.
.
casein solution (with sodium acetate) is treated with "varying amounts of standard
acetic acid. The resulting pH of each tube is then calculated by means of the standard buffer formula, neglecting the buffer effect of the casein itself, and the relative
amounts of casein precipitated in the different tubes are judged by inspection. The
pH of the tube showing maximum precipitation is taken as the isoelectric point.
Obtain from the stockroom the following:
10 ml. standard casein solutionl (each ml. contains 0.006 g. casein
held in solution by 0.1 N sodium acetate)
5 ml. 1.0 N acetic acid
Prepare 20 m!. of 0.1 Nand 10 m!. of 0.01 N acetic acid.
Make up the following series of solutions, using clean test tubes of
uniform diameter.
Tube number
1
2
3
4
5
6
7
8
9
-- -- -- -- -- -- -- -- -mi.
mi.
mi.
ml.
mi.
mi.
mi.
mi.
1
7.75
1.25
1
8.75
1
8.5
1
8.0
1
7.0
1
5.0
1
1.0
1
7.4
0.25
0.5
1.0
2.0
4.0
8.0
mi.
Casein solution .............. 1
Distilled water .............. 8.38
0.01 N acetic acid ........... 0.62
0.1 N acetic acid ............
1.0 N acetic acid ............
1.6
-
Measure the various solutions with either a Mohr pipette or a burette,
the former being more convenient. Place the casein solution in the tubes
first, then the water and mix. N ow add the acetic acid to the first tube and
shake immediately; then add the necessary acid to the second tube and
shake, etc.
Examine the tubes at intervals of 10 and 20 minutes and record for each
examination, the relative amounts of precipitated casein in each tube.
Cal~ulate the pH of each tube from the concentration of acetic acid
and sodium acetate present. Tabulate the results as follows:
o = no change in tube.
+
= opalescence.
x = precipitate.
Tube number
4
----------- -- -- -- -- -- -
6
9
-- ----
pH .... , ... •.................... .
10 minutes .................... .
20 minutes .................... .
What is the isoelectric point of casein? Explain fully.
1 Casein Solution.
Weigh out 12.00 g. best quality casein, add 1 liter H 20 at 40°C.
and 200.0 ml. 1.00 N sodium hydroxide. Stir until the casein dissolves and theu add
200.0 mI. 1.00 N acetic acid as rapidly as possible. Mix, cool and make up to 2000 mI.
76
FATS
PREPARATION OF NEUTRAL FAT
Hydrolysis of fat occurs easily so that fats arc usually contaminated with fatty
acid.
(1) To 25 ml. of alcohol in a 100 ml. flask add a drop of phenolphthalein
and then 0.1 M sodium carbonate drop at a time until the indicator has a
pink tinge. Introduce 5 ml. of cotton seed oil. Add 0.1 1\1 sodi'um carbonate drop at a time until the pink color is re~tored.
Pour the product into a large test tube and after the fat has separated,
pipette off the aqueous layer and wash the fat by agitating with successive
portions of warm water. Reservf3 most of the material for experiments on
"emulsification" (see below).
PROPERTIES OF FATS
A drop of fat leaves a translucent spot on paper which does not disappear
on warming.
(2) Solubility. Test the solubility of fat in water, hot water, alcohol,
hot alcohol, ether, chloroform and benzene. Make each test with a drop
of fat and if uncertain of the solubility, see if a drop of the filtered solvent
leaves a translucent spot on paper.
(3) Crystallization of Fat. Dissolve a piece 9f lard the size of a small
pea in 1 ml. of ether with shaking. Add alcohol until the solution is
turbid, then stopper with a cork. Allow to stand until crystals form.
Withdraw a little of the liquid containing crystals in the tip of a pipette,
place on a slide and cover at once with a cover glass. Examine the crystals
with a microscope.
(4) Formation tif AcroleiI!.. To a small amount of potassium bisulfate
in' a dry test tube add a drop or two of cotton seed oil and heat cautiously
over a small free flame. ' Acrolein is formed whicn may be recognized
,by its irritating odor, Repeat the experiment with glycerol. Write the
equation.
(6) Emulsification. Prepare two series of 3 test tubes each, Each
series consists of tubes containing, respectively, 5 ml. of the following:
water, 0.1 M sodium carbonate, 0.5% soap solution. To each tube of one
series add 4 dmps of neutral fat (prepared above from cotton seed oil)
and to each tube of the other series add 4 drops of rancid cotton seed oil.
78
Shake all of the tubes violently.
a:r;l.d after half an hour. Explain.
Note their appearance immediately
SAPONIFICATION
Hydrolysis of fat can be brought about easily by heating with sodium hydroxide,
in which case the sodium salt of the fatty acid is formed. The alkaline salt is called
a soap and the process is known as saponification.
(6) Warm 50 ml. of alcohol with 10 g. of sodium hydroxide until
no more of the alkali is dissolved and pour the clear solution on 25 g. of
lard placed in a 200 m!. flask which is provided with a reflux condenser.
The solubility of both fat and alkali in hot alcohol is a favorable condition
for hydrolysis.
Heat the solution on a water bath, until no turbidity is produced (by
fat globules) when a drop or two are added to 5 ml. of distilled water.
When saponification of the fat is thus shown to be complete, pour the mixture (beware of fire) into an evaporating dish containing 200 ml. of distilled
water and acidify the hot solution with dilute sulfuric acid. Upon cooling,
the insoluble fatty acids will form a crust on the surface of the liquid.
Wash them with hot water. Write the equation for the reaction.
FATTY ACIDS
(7) Repeat with fatty acids the experiments described under "Properties of Fats."
(8) Dissolve a small portion of fatty acids iI). chloroform and add Htibl's
solution l a drop at a time with shaking. Explain the decolorization of the
iodine.
•
.
(9) Repeat the experiment with fat. Make a control with chloroform
alone.
. SOAPS
(10) Suspend about a gram of fatty acids in 100 ml. of warm water
and 'bring most of the material (but not all) into solution by the careful
addition of sodium hydroxide. Filter off the solution and use it for the
following tests.
(11) Acidify a portion of the soap solution with sulfuric acid.
(12) Treat separate portions of the soap solution with calcium chloride,
magnesium sulfate and lead acetate.
1 Hiibl's iodine solution contains 26 g. of iodine and 30 g. of mercuric chloride in 1
liter of 95% alcohol. It has been used in the determination of the "iodine number"
of fats.
80
(13) Shake violently a drop or two of soap solution with distilled water.
Note the permanence of the foam and the presence or absence of a precipitate.
(14) Repeat the experiment with a drop of soap solution in tap water.
Continue to add soap solution drop at a time, shaking violently after each
addition until a persistent foam is finally produced. This reaction is the
basis of a method for the quantitative determination of "hardness" of
water.
(15) Saturate some soap solution with sodium chloride.
'(16)
THE SAPONIFICATION NUMBER
The saponification number is the number of milligrams of potassium hydroxide
neutralized in the complete saponification of one gram of fat. This number is a
mcasure of the mcan molecular weight of thc fatty acids making up the fat.
Reagents. Prepare 500 ml. of approximately 0.5 N hydrochloric acid
and standardize it against your standard sodium hydroxide.
Obtain 150 to 200 ml. of an alcoholic solution of potassium hydroxide
from the special reagent shelf, and standardize it by titration against the
0.5 N hydrochloric acid. Note: The alcoholic potassium hydroxide solution contains about 40 mg. potassium hydroxide for each ml. of 95%
alcohol.
The Determination. Weight 4-5 gm. (accurate to the second decimal
place) of fat into a 250 ml. Erlenmeyer flask, add a definite volume (50
m!.) of the standard alcoholic potassium hydroxide solution, and connect
with a reflux condenser. The solution should be made to boil gently, on
a water bath, for at least thirty minutes. When saponification is complete
(indicated by a homogeneous solution on cooling), cool, add about 100 m!.
of water and titrate the residual potassium hydroxide solution with the 0.5
N hydrochloric acid (place acid in burette), using phenolphthalein as an
indicator.
Calculate the saponification number.
(17)
THE IODINE NUMBER (ROSENMUND METHOD)
l'he iodine number is the grams of iodine absorbed by 100 g. of fat. This nllmber
is a measure of the unsaturated fatty acids in a fat or oil. Ho\vever, in the procedure
described below, bromine rather than iodine is employed for the saturation of double
bonds in unsaturated fats because it is more satisfactory. For the purpose of obtaining a mild and smooth reaction, the bromine is used in the form of pyridine sulfate
dibromide.
82
The excess of bromine reagent remaining after the reaction is complete is treated
with KI, liberating free iodine in amount equivalent to the bromine present. The
iodine is then titrated with standard Na 2S 203 solution.
Potassium Iodide Solution. Dissolve 150 grams of potassium iodide in
water and make up to 1 liter.
Starch Solution. Soluble starch may be prepared by Small's method.
In a flask equipped with a reflux condenser place 100 grarp.s of dry potato
starch, 500 ml. of 95 per cent ethyl alcohol to which 4 ml. of concentrated
hydrochloric acid have been added, and mix. Heat in a boiling water bath
for 15 minutes. Filter, using a Buchner funnel, and wash the precipitate
with several changes of distilled ,Yater. Dry the starch in the air.
A 0.5% or a 1% solution of this starch may be made up by bringing to
boiling weighed quantities of the starch in an appropriate quantity of
distilled water. Instead of water, 20% sodium chloride solution may be
used. Starch solution in sodium chloride does not have quite as sharp an
endpoint as an aqueous solution of starch, but mold formation is retarded
to a marked degree.
0.1 N Potassium Dichromate. Dissolve 4.903 grams of potassium dichromate in water and make up to 1 liter.
.
Approximately 0.1 N Sodium Thiosulfate (accurately standardized).
Dissolve 24.8 grams of sodium thiosulfate (N a2S20a' 5H 20) in water and
dilute to 1 liter. Bring the solution to boiling, transfer hot to a clean,
dark-colored bottle equipped with a syphon, and cover with a layer of
toluene. l Standardize as follows: Into a 500 ml. Erlenmeyer flask, introduce 20 ml. of the potassium dichromate solution together with 10 ml. of
potassium iodide and 5 ml. of concentrated hydrochloric acid. Dilute
to 300-400 ml. with water and titrate with the sodium thiosulfate solution
until the solution is faintly yellow, when a few drops of starch solution
should be added and the titration continued to the point of the disappearance of the blue color. The reactions taking place are as follows:
K 2Cr 207 +'14HCI
+ 6KI.= 2CrCh +
8KCI + 31 2 + 7H 20
i . I It is now recognized that the deterioration of thiosulfate is due to bacterial
action. Work in our laboratory has confirmed the fact that sterilization is a very
effective means of preserving thiosulfate solutions, sterilization plus toluene being
particularly effective. Even 0.005 N solutions may be preserved for months by these
means.
~(ilpatrick, M., Jr., and Kilpatrick, 1\1. L., J. Amer. Chern. Soc., 45, 2132 (1923).
Mayr, C., and Kerschbaum, E., Z. anal. Chern., 73, 321 (1928).
84
Rosenmund ReagenU Add 40 g. of pyridine (40.4 ml.) to 100 ml. of
ice cold glacial acetic acid; mix 50 g. of concentrated sulfuric acid with
:wother 100 ml. of ice cold glacial acetic acid. Mix the two solutions and
and add a solution of 40 g. of bromine in 100 ml. of glacial acetic acid.
Then add glacial acetic acid to make 5 liters. The resulting solution is
approximately 0.1 N and will keep indefinitely.
The Determination. In a clean, dry 250 ml. Erlenmeyer flask, weigh
accurately 0.1 to 0.2 g. of fat (4 to 8 drops). Dissolve in 20 ml. of chloroform and add 25 ml. of the Rosenmund reagent. The bottle of Rosenmund
reagent will be found connected to a burette, so that measurement may be
made directly into your flask, which contains the fat sample. Close the
flask with a stopper and allow to stand 15 minutes. Add 10 ml. of the
15% potassium iodide solution, mix thoroughly, and allow to stand for
several minutes. Titrate carefully with standardized sodium thiosulfate
until colorless, shaking thoroughly after each addition of thiosulfate.
(Starch does not improve the sharpness of the end point.)
To ascertain the strength of the bromine solution, a blank determination
must also be carried out using the same quantities of reagents with the fat
omitted. An instructor will determine the "blank" value and report it.
Calculate the grams of iodine absorbed by 100 grams of fat.
Calculation: A = m!. thiosulfate required in blank titration. B = ml.
thiosulfate required in the determination. C = normality of the thiosulfate.
(A-B) X C X 0.127 X 100
. d'
b
= 10 Ine num er
g.o f f at.
1 Peters, J. P. and Van Slyke, D. D., Quantitative Clinical Chemistry.
Vol. 2,
1st Ed., p. 923. Page, 1. n., Pasternack, L., and Burt, M. L., Biochem. Zeitschr.
223,445 (1930).
86
PART
II. BODY TISSUES AND FLUIDS
SALIVA
(1) Acidify a small amount of saliva with a drop of 2 N hydrochloric
acid and add a drop of ferric chloride: The red color is produced by thiocyanate in saliva which is usually present only in traces but is often found
increased in the saliva of smokers.
(2) Make protein color tests with saliva.
INORGANIC TESTS ON SALIVA
(3) Oxidation of organic material (Ashing). Collect 25 ml. of saliva
stimulated through paraffin chewing. Do not filter. Evaporate to dryness in a small porcelain dish over a water bath. Push any material which
solidifies on the sides to the bottom of the dish with a stirring rod, so that
the solid is concentrated in a small area.
Moisten the dry material with a few drops of concentrated HNO a and
heat the dish over a small free flame in the hood. After charring takes
place, increase the heat until the dish becomes red hot and all black material
disappears. After cooling, add 5 ml. of 2 N acetic acid stir to dissolve the
ash, and filter through a small filter paper. Wash the dish and paper with
5 ml. of distilled water.
Test the filtrate for Ca++, PO;, SO'4, and Cl- as follows:
a. Calcium. Add 1 ml. of ammonium oxalate to a 2 ml. portion of the
filtrate.
b. Phosphate. Add 4 ml. of NH 4NO a in HNO a and 1 ml. of ammonium
molybdate to a 2 ml. portion of the filtrate. Place tube in a hot water
bath and allow to stand a few minutes.
c. Sulfate. Add 1 'drop of concentrated HCl and 1 ml. of BaCh to a
2 m!: portion of the filtrate.
d. Chloride. Add 1 drop 'of concentrated HNOa and 1 ml. of AgNO a
to,a 2 ml. portion of the filtrate.
MUCIN
(4) Place 100 ml. of alcohol in a dry 250 ml. Erlenmeyer flask, add 30
ml. of saliva in small portions at a time, mixing well after each addition,
close the flask with a cork and allow to remain on its side until the precipitated mucin has settled.
88
Decant as much as possible of the aqueous-alcoholic fluid from the muc~n,
add 50 ml. of alcohol, mix thoroughly taking care to wash down the sides
of the flask, and allow the mucin to settle again. Decant the fluid, bring
the mucin on a small filter with alcohol, wash with alcohol and allow to
dry in a desiccator. The mucin thus obtained should be a white powder
that does not adhere to the filter paper.
(5) Make the biuret test with a little mucin. (Dissolve in 2 N sodium
hydroxide and add a trace of copper sulfate. See p. GO.)
(6) Place the rest of the mucin with 3 ml. of 2 N hydrochloric acid in
a wide test tube. Heat to the boiling point and keep hot for 15 minutes.
Cool, make alkaline with sodium hydroxide, add a little mixed Fehling's
solution and boil. To what class of proteins does mucin belong?
SALIVARY DIGESTION
There is present in saliva an active agent called "amylase" in whose presence
starch is changed through the dextrins into maltose with considerable rapidity.
The decomposition can be easily followed because some of the dextrins yield colors
with iodine and do not reduce Fehling's solution, while maltose reduces Fehling's
solution and does not yield a color with iodine. Maltose ferments with yeast forming
carbon dioxide and alcohol which latter may be identified by conversion into iodoform.
(7) Prepare some 1% starch paste as directed on page 56. Place 10
ml. of the starch paste in a test tube with 1 ml. of saliva, mix thoroughly,
and place in a beaker of water kept at 37°. At intervals of one-half minute
test a drop with a drop of diluted iodine solution (diluted until only light
.yellow) on a porcelain test plate. From time to time test the digestion
mixture with Fehling's test. Explain the results.
(8) From this preliminary experiment make such dilution of your
saliva that it requires not less than 3-5 minutes to carry out the digestion
just described. Note the time required for the digestion to proceed to a
point where no color is given with iodine. Using this "diluted saliva",
study quantitatively the effects of the following.
These experiments .demonstrate how enzyme action may be studied.
The time required for a definite amount of substrate to disappear is inversely proportional to the a~tivity of the enzyme .
. (Sa) Temperature. Study digestion at the temperatures, 37°, 20°
(or· room temp.), 100°, 10°. Have the saliva and starch at the desired
temperature before mixing. Use 10 ml. of starch solution and 1 ml. of
"diluted" saliva and test with iodine every half minute. Note the time
required for the disappearance of the color. Determine the temperature
optimum by plotting the reciprocals of the digestion times against their
corresponding temperatures. Are the decreases in velocity of starch
hydrolysis the result of enzyme destruction or inhibition?"
(8b) Acid and alkali. In each of four test tubes a, b, c, and d, place
10 m!. of starch paste. To a, add 1 ml. of 0.1 N hydrochloric acid, to b,
.
90
1 ml. of 0.005 N acetic acid, to c, 1 ml. of 0.01 M sodium carbonate and to d,
1 ml. of water. Mix intimately 1. ml. of "diluted" saliva with the contents
of each tube, immerse the four tubes in water at 37° and test every half
minute with iodine. Plot a time-digestion curve and determine experimentally whether enzyme destruction or inhibition has occurred.
(8c)
Dilution of enzyme.
Tube a. 9 ml. of water and 1 ml. of "diluted" saliva
Tube b. 9 ml. of 0.2 M NaCI and 1 ml. of "diluted" saliva
Place 10 ml. of starch paste in each of three tubes. To the first add 1 ml.
of the contents of Tube a, to the second, 1 ml. of the contents of Tube b,
and to the third, 1 ml. of "diluted" saliva to be a control. Mix each tube
carefully, keep at 37° and note when the color with iodine just disappears.
Compare the times of digestion of these three solutions. Observe the result of dilution and the coenzyme effect of NaCl.
(8d) Antiseptics. Add 5 drops of the antiseptics mentioned below separately to 10 ml. portions of starch paste, and mix each with 1 ml. of
"diluted" saliva. Set up a control with "diluted" saliva and digest all
the solutions at 37°.
Test chloroform, toluene, ether, 5% HgCI 2 , and 10% CUS04.
CONVERSION OF MALTOSE INTO ALCOHOL
(9) Treat 100 ml. of 2% starch paste with about 25 ml. of saliva and
digest at 40° until the starch has been converted into maltose.
Shake up the solution with some compressed yeast, fill into a flask closed
with a cork and outlet tube bent twice at right angles so as to dip into water
contained in a second flask. Place the apparatus in a warm place and note
the continual slow bubbling of carbon dioxide through the water in the
second flask for several days. When bubbling ceases, distill about 25 ml.
of fluid from the products of fermentation and test for alcohol as follows:
Heat the solution to boiling, add enough iodine-potassium iodide solution
to give a permanent yellow color, then drop at a time enough sodium carbonate solution to remove this yellow color, but avoiding an excess of sodium .carbonate. Iodoform is'produced which can be detected by its odor,
Th~ following equations show the reactions concerned.
+
+
+
CH 3CH 20H
12 = CH 3CHO
2 HI
CHaCHO
3 12 = CI 3 CHO
3 HI
CI 3CHO + NaOH = CHI 3 + HCOONa
+
Allow the material to cool undisturbed. Iodoform crystals will be deposited: No matter how these crystals are clustered there is always a hexagonal arrangement.
.
92
COLORIMETRIC DETERMINATION OF THE HYDROGEN ION
CONCENTRATION OF NORMAL AND STIMULATED
SALIVA
(10) In order to collect normal mixed saliva without loss of carbon dioxide proceed as follows: about 10 minutes previous to the period of collection, rinse the mouth well with tap water. Prevent stimulation of saliva
flow by having the mouth closed and empty and the jaws quiet. Place 2
or 3 ml. of paraffin oil in a clean test tube and introduce, under the oil,
one end of a glass tube which extends well above the mouth of the test tube.
Eject the saliva at intervals through the tube and allow it to accumulate
under the layer of oil. Do not blow air through the collected saliva.
Determination of pH: To a clean test tube having the same bore as the
as the standard tubes,l add 9 ml. carbon dioxide-free water,2 5 drops of
brom thymol blue indicator, cover with a layer of oil and mix. The color
should match a standard tube between pH 6 and pH 7. Be sure that this
is the case before proceeding further. Now add 1 cc. of the saliva from a
Mohr pipette the tip of which is below the surface of the oil. Mix gently
with a clean stirring rod. Compare the coloI' with the standards. As the
solution containing the saliva is turbid and the standards are clear, a better
estimation can be made by comparing the saliva in a color comparator with
the standard, behind which is placed saliva similarly diluted but containing
no indicator. Record the pH on a chart, preserve the tube and compare
the color with those obtained in the following determinations.
Stimulated Saliva; Procure a cube of paraffin and chew it vigorously for
15 minutes. Then, while still chewing, collect a portion of saliva under oil
as directed above and determine the pH. 10-12 minutes after discontinuing the chewing, collect another sample of saliva and determine the pH.
_\1ake a graph, plotting pH of saliva against time.
Experiments designed to show the effect of smoking, overbreathing, and
ingestion of sodium bicarbonate may be carried out.
1 Standard cQlor tubes cQntaining the indicatQr and buffer sQlutiQns ranging frQm
pH 6.0 tQ pH 8.0 in steps Qf 0.2 pH wili be fQund Qn the shelves Qf special reagents.
See p. 30.
.
. ': CarbQn diQxide-free water may be prepared by bQiling distilled water in a pyrex
flask and allQwing it tQ coQl withQut shaking.
!H
GASTRIC JUICE
METHODS FOR DETECTING THE ACIDITY OF STOMACH CONTENTS
IDENTIFICATION OF HYDROCHLORIC ACID
Make up 40 ml. quantities of the following:
0.1 N HCI: 0.01 N HCI: 0.01 N
lactic acid: equal volumes of 0.01 N HCI and 1 % pept one.
(1) Topfer's Reagent. pK = 3.5. To 10 ml. of each solution add ta
drop of Topfer's reagent.1
(2) Giinzberg's Test. Evaporate very carefully a drop or two of the
Giinzberg's reagent2 on a clean white porcelain surface on a water bath.
Add a drop of the solution to be tested and evaporate again. A purplishred color is produced by free hydrochloric acid but the color will be obscured
by a brown if the material is over-heated. This is said to be the best test
for free hydrochloric acid.
IDENTIFICATION OF LACTIC ACID
(3) UffeImann's Reagent. 3 Treat 5 m!. portions of the reagent with
each of the following solutions,
0.1 N HCI: 0.01 N HCI: 0.1 N lactic acid
Note the change in color in each case. The color yielded by lactic acid is
given by any a-hydroxy acid.
(4) Hopkin's Thiophene Test. To 3 drops of a one per cent alcoholic
solution of lactic acid in a dry test tube add 5 ml. of concentrated sulfuric
acid and 3 drops of saturated copper sulfate. Heat the tube in boiling
water for about 15 minutes, cool under the tap, add 2 drops of a 0.2%
alcoholic solution of thiophene and replace the tube in boiling water in a
beaker. A temporary cherry red color develops. 'The presence of too
much water interferes with the test.
Half a gram of dimethylaminoazobenzene in 100 ml. of 95% alcohol.
Two g. of phloroglucin and 1 g. of vanillin in 100 ml. of alcohol.
. 3 1'0 a 2% solution of phenol add a few drops of ferric chloride. The amethyst
colored fluid is known as Uffelmann's reagent.
1
2
96
GASTRIC DIGESTION
The proteolytic enzyme of the stomach is present in the mucosa and is secreted
in an inactive form called pepsinogen. It is activated by hydrochloric acid which
is also secreted by specialized groups of cells. The pepsinogen may be extracted
fro~ the mucosa by glycerol or extracted and activated by dilute acid. The relative
stabilities of the enzyme and proenzyme are demonstrated in one of the experiments
described below.
(7) E:xtracts of gastric mucosa. Dissect the mucous membrane from a
pig's stomach. Hash the mucous membrane in a meat chopper; Place
about! of the material in a beaker and add 3 times its weight of 0.1 N
hydrochloric acid (prepared roughly from desk reagents). Place the
remainder in another beaker and cover with glycerol (about 75 mI.).
Stir occasionally and allow the glycerol extraction to proceed at room
temperature. over night.
Acid extract. As soon as the hashed tissue can be suspended in the
fluid, transfer to a flask, add chloroform, stopper the flask and keep at 40°
over night or longer. (Label your flask and hand it in at the storeroom so
that it may be placed in an incubator.) The protein of the mucosa is
digested and the characteristic products are formed. When digestion
is sufficiently complete, strain off the liquid through muslin and then
filter through a fluted paper. The filtrate contains products of gastric
digestion.
Titrate portions of the filtrate for free and total acidity as described
below.
Glycerol extract. Strain through cloth and use the extract for the following experiments.
(8) Optimum reaction for peptic digestion. Add 0.3 mI. of the glycerol
extract to each of a series of test tubes containing:
a.
b.
c:
d.
e.
f.
g.
10 mI. O.lO M sodium carbonate
10 mI. water
10 mI. 0.01 N hyd:r;ochloric acid
10 ml. 0.05 N hydrochloric acid
10 m!: 0.10 N hydrochloric acid
10 ml. 0.50 N hydrochloric acid
10 ml. 2.0 N hydrochloric acid
Place the tubes in a water bath kept at 40° and add to each a shred
of fibrin. Note the changes in the fibrin for a period of an hour.
(9) Stability of pepsinogen and pepsin. Mix 1 ml. of the glycerol
extract with 5 mI. of 0.1 N hydrochloric acid and place in a water bath
kept at 40° for 15 minutes. Neutralize to litmus with 0.5 M sodium
98
carbonate and add 1 m!. in excess. In another tube, mix 1 ml. of glycerol
extract with 6 m!. of water and add 1 m!. 0.5 M sodium carbonate. Place
both tubes in a water bath at 40° for 30 minutes. Then acidify with 10 m!.
of 0.1 N hydrochloric acid, add fibrin and digest at 40°.
QUANTITATIVE GASTRIC ANALYSIS!
(10) Free Hydrochloric Acid. Titrate 10 m!. of strained gastric
contents 2 in a large test tube with 0.1 N sodium hydroxide using 3 drops
of dimethylamino-azobenzene (Topfer's reagent) as the indicator. In
order to gain acquaintance with the so-called "salmon-pink" end-point
. for free hydrochloric acid with Topfer's reagent, 10 m!. of a buffer solution
of pH 2.8 with 3 drops of the indicator should be used for comparison.
Calculate the m!. of 0.1 N sodium hydroxide required to neutralize the
hydrochloric acid in 100 m!. of gastric contents.
(11) Total Acidity. Titrate 10 m!. of strained gastric contents using
2 drops of phenolphthalein as the indicator. The phenolthalein may be
added to the sample which has just been titrated and the titration continued for total acidity. Calculate the m!. of 0.1 N sodium hydroxide
required to neutralize 100 m!. of gastric contents.
1 For a critical discussion see L. Michaelis, Some Problems concerning Gastric
Juice, The Harvey Lectures, 1926-2i, p. 59.
2 Artificial gastric contents may be prepared by adding 2.6 m!. of 2 N hydrochloric
acid to 100 m!. of 1% peptone solution; or the filtrate from the digest of the acid
extract of gastric mucosa may be used.
100
PANCREATIC JUICE
PANCREATIC EXTRACT
(1) Preparation. Dissect a fresh pig's pancreas free from most of the
fat and other tissue. Grind the pancreas in a meat chopper and mix with
four times its weight of 25% alcohol. After standing several hours (or
any time up to 1 or 2 days) strain through cloth. Filter, nearly neutralize
with sodium hydroxide (desk reagent) and finally neutralize exactly with
sodium carbonate solution. Use this neutral extract for the following
experiments.
(2) Trypsin. Prepare a series of test tubes containing, respectively,
, 3 m]. of the following (a second series is needed for the next experiment):
water: 0.05 M Na 2CO a : 0.1 M Na2C03: 0.1 N HCI:
0.01 N HCI: equal volumes 0.01 N Hel and 1 % peptone;
0.01 N acetic acid.
To each tube add 3 m!. of neutral pancreatic extract, introduce a shred
of fibrin and allow to digest at 40°. Note the change in the fibrin after 1,
2 and 3 hours.
(3) Pancreatic Amylase. Prepare a series of tubes containing the
reagents used in the last experiment and mix the contents of each tube with
2 m!. of 1 % starch paste (p. 56). Introduce 2 ml. of neutral pancreatic
extract as rapidly as possible and place the tubes in a beaker of water at 40°.
From time to time test a drop from each tube with a drop of diluted
iodine solution on a white porcelain surface. The test must be carried out
in neutral or faintly acidic solution, so a drop of 0.1 N acid should be added
to the iodine which is to receive the test drops of alkaline solution. After
30 minutes, test with Fehling's solution.
(4) Pancreatic Lipase. Place 10 ml. of milk in each of two test tubes
and add several drops of brom cresol purple solution. Introduce 3 m!.
of neutral or faintly alkaline pancreatic extract into one tube and 3 ml. of
boiled extract into the other. Both tubes should be definitely blue. Allow
to digest at 40°. Explain any changes in color of the indicator. Add more
indicator solution if necessary to bring out the color.
(5) Place 5 ml. of water and 0.5 m!. of neutral ethyl butyrate in each
of three test tubes a, b, c, and color each with brom cresol purple. To a
and ~ add 3 ml. of faintly alkaline pancreatic extract and to c, 3 m!. of boiled
extract. Allow a and c to digest at 40°; b, in cracked ice. If the color of
102
the indicator changes, restore the blue with 0.1 N sodium hydroxide and
allow the digestion to continue.
TYROSINE
(6) 250 g. of finely ground pancreas, 500 m!. of water and 10 mI. of
chloroform are allowed to digest at the room temperature for 24 hours in a
tightly closed vessel. The mixture is violently agitated from time to time
in order that the tissue may become permeated with chloroform and
putrefaction be prevented. The mixture is then digested at 40° for 24-48
hours, cooled and filtered. This filtration is slow but continuous and may
proceed over night without danger of putrefaction.
To 500 mI. of the clear extract are added 25 g. of casein; the mixture
is made faintly alkaline with ammonia, more chloroform is added and the
digestion of the material at 40° is allowed to continue for any convenient
period from 5 to 10 days.
The digested product is evaporated to one-third of its volume and
allowed to cool, when impure tyrosine will be deposited. Remove the
fluid and extract the crude tyrosine with three successive portions of boiling
water (250-100-50 mI.), boiling hard with each extraction and allow the
united extracts to cool. Crusts of needles of very pure tyrosine will be
deposited. Examine microscopically. Yield 1.5 g.
(7) Mercuric Nitrite Test.· As described under "Proteins." Use a
very small amount of tyrosine, as the test is extremely sensitive.
(8) Piria's Test. Heat a little tyrosine for 15 minutes with a drop or
two of concentrated sulfuric acid in a test tube submerged in boiling water.
Dilute with 15 or 20 ml. of water, trallsfer to a beaker and neutralize the
sulfuric acid by boiling with barium carbonate (about 1 g.). Filter, and
evaporate to a small volume on a water bath. The concentrated fluid
will give a characteristic violet color with a drop or two of ferric chloride.
FORMALDEHYDE TITRATION
Digestion of protein/with the formation of increasing quantities of free amino
and carboxyl groups, may be followed quantitatively by means of the formaldehyde
titration. (See page 72.)
.
. .(9) Warm 80 m!. of casein solution l to 37°, add 5 m!. of pancreatic
extract, shake the solution and immediately pipette out 10 ml. To this
1 The casein solution may be prepared as follows: Triturate 10 g. of purified casein
with 5 m!. of cold water, add 90 m!. of cold water and 5.5 m!. of 2 N sodium hydroxide.
The resulting solution should have a pH of about 8.1 and therefore test portions
sho,uld be colorless with phenolphthalein and red with phenol red. Adjust if necessary with dilute hydrochloric acid or sodium hydroxide.
104
portion add 10 m!. neutralized formaldehyde. Place the remainder of the
solution in a bath at 37°. Complete the formaldehyde titration after
adding 4 drops of phenolphthalein. Use as a standard, 10 ml. of boiled
water mixed with 10 m!. of formaldehyde solution, 0.2 ml. of 0.1 N NaOH
and 4 drops of phenolphthalein. Run additional determinations on 10 ml.
portions after intervals of i hr., ! hr., 1 hr., 3 hrs., and longer from the beginning of the e~periment. Place 2 m!. of toluene in the portion which is
to stand over night.
Subtract the blank of 0.2 ml. from each titration and calculate the
amount of free amino nitrogen in 10 m!. Plot the free amino nitrogen
against time.
106
MILK
Milk contains a characteristic compound protein, simple protein, carbohydrate,
fat and inorganic salts. It is not only an excellent food but serves well for a chemical
examination since its constituents admit of a very simple and easy separation from
one another.
GENERAL
(1) Examine a drop of milk under the microscope.
(2) Test the reaction of milk with sensitive litmus paper, both red
and blue.
(3) Place 5 ml. of milk and several drops of brom cresol purple in
each two test tubes. Plug one with cotton and boil for several minutes
without wetting the cotton. Allow both tubes to stand for several ho~rs
or over night and note the color of the indicator.
(4) To 1 ml. of milk add 6 drops of tincture of guaiac. Mix and note
the color. Now add 2 drops of dilute hydroge~ peroxide and do not mix.
What is the cause of the color?
SEPARATION OF THE CONSTITUENTS OF MILK
(6) Place 150 ml. of milk in a beaker and put it in a warmed water
bath until the temperature of the milk is 40°. Add acetic acid a few drops
at a time, stirring gently, until clotting occurs. The amount of acetic acid
required will depend upon the state of preservation of the milk and may
be any amount between 3 ml. and 10 ml. Ail excess of acetic acid is to
be avoided.
Allow to stand until a cohesive clot forms, from which the supernatant
cloudy liquid may be sharply decanted.! . (Slowly and carefully here.)
By gently pressing the clot with a rod a large amount of liquid may be
squeezed out and decanted. The cloudy liquid is to be reserved.
Casein. Pour upon the co_agulum 100 ml. of warm water, break up with
a rod, acidify with acetic acid and when thus washed allow to settle.
Decant the wash liquid.
The clot is now to be defatted and dried as follows: Press out as much
water as possible between folds of dry muslin and grind in a mortar with
1 Place 10 m!. of the liquid in a test tube and add 5 drops of methyl red.
Compare
the. color produced with the standard buffers containing methyl red and estimate
as well as possible the pH of the solution. Record the result and compare it with
the isoelectric point of casein determined in an earlier experiment (p. 74).
108
100 ml. of alcohol. After remaining in contact with the alcohol for half
an hour, filter off the colorless solid, press between folds of dry muslin and
cork in a dry 200 m!. flask ,vith 100 ml. of ether. (Avoid.the neighborhood of
flames when using ether.) Allow it to remain in contact with ether for two
hours or longer, filter through a dry filter and wash with a little ether.
Spread out on dry filter paper or grind gently in a mortar as the etl).er is
fanned away. Test as described below.
Fat. Allow the ether to evaporate spontaneously and drive off the
alcohol by heating on the water bath (Danger !). Test some of the yellow
drops for fat, and with the remainder run Exp. 14.
Coagulated Protein. (Albumin and Globulin.) The cloudy liquid from
which casein was precipitated will be found difficult or impossible to filter.
Strain through muslin to remove the coarse particles, and heat to boiling.
Filter the liquid on a fluted filter and dry the small precipitate as directed
above for casein, using correspondingly small quantities of alcohol and
ether to dry it. Test as described under Casein (below).
Lactose. Remove 10 m!. of the filtrate l for use as described below.
Then treat the remainder of the warm solution with a slight excess of lead
acetate and when cool, filter into a flask. Remove the excess of lead from
the filtrate with hydrogen sulfide. (See note on precautions to be observed
when using hydrogen sulfide, p. 7.) Evaporate the filtrate from lead
sulfide over a small flame to about one-half its volume and then on a water
bath to about 5 m!. Filter again if necessary and pour into twice its volume
of glacial acetic acid (hood) in an Erlenmeyer flask. Crystals will be deposited either immediately or after standing over night. Filter on a small
filter under the hood, wash with about 10 m!. of glacial acetic acid, then
with a little alcohol and allow to dry in the air. Test with protein and
carbohydrate color tests. Try the osazone test.
Calcium Phosphate. Evaporate to dryness on a water bath the 10 ml.
of the solution obtained above, extract with faintly alkaline warm water,
filter off the precipitate ·and wash it with hot water. Suspend the
pre.cipitate in hot water, bring into solution with a little nitric acid and
test for calcium and phosphate.
CASEIN
(7) Solubility. Grind a small portion of casein with 5 m!. of water
and add 0.02 N sodium hydroxide drop at a time. When most of the
solid has dissolved, pour off some of the solution and add to it acetic acid.
1 If it is necessary to allow this solution to stand over night, it should be kept in
a stoppered flask with 2-3 m!. of chloroform added.
110
(8) Repeat the experiment using hydrochloric acid instead of sodium
hydroxide. Explain the solubility.
Make the following tests as described in the earlier chapters.
(9) Test for Sulfur.
(10) Test for Unoxidized Sulfur.
(11) Test for Phosphorus.
(12) Test for Nitrogen.
(13) Protein Color Tests.
BUTTER FAT
(14) Dissolve 5 g. of sodium hydroxide in 5 m!. of water (100 m!.
flask). Pour in the melted butter fat, add 90 ml. of alcohol and heat
on the water bath until a drop of the liquid is found completely soluble
in water. When saponification is complete, evaporate in a dish on the
water bath until alcohol can no longer pe detected by its odor. Add 30 m!.
of dilute sulfuric acid and note the odor of butyric acid. Occasionally the
pleasant odor of ethyl butyrate can be detected.
(15)
LACTIC ACID FERMENTATION OF CANE SUGAR
When milk is exposed in a warm place, its sugar is fermented by bacteria to form
lactic acid. The milk thus becomes sour and the acidification causes precipitation
of casein. The lactic acid may be isolated as the zinc salt. Zinc lactate obtained
by fermentation is optically inactive while the zinc salt of muscle lactic acid (sarcolactic acid) is levo-rotatory. As milk contains a relatively small amount of lactose
and a relatively large amount of substances whose presence makes difficult the isolation of lactic acid the reaction is more easily studied by the following arrangement.
Dissolve 25 g. of molasses (cane sugar) in 200 m!. of tap water, add 10
g. of zinc carbonate, 20 m!. of sour milk and allow the mixture to remain
in the thermostat at 40° with frequent stirring for 8 or 10 days. The cane
sugar is slowly converted into lactic acid which, as it is produced, acts
upon the zinc carbonate forrp.ing zinc lactate.
Heat the liquid to "boiling and filter hot. As the filtrate cools, crystals
of ziIic lactate wiLl be deposited. (If not, evaporate.) Recrystallize from
hot water and examine under the microscope.
. Pissolve the zinc lactate in a small amount of water that has been acidified markedly with sulfuric acid and shake the acid solution in a separatory
funnel with successive portions of ether (when using ether avoid the neighborhood of flames and always distil the ether on a water bath that has been
previously heated and the light extinguished). Filter the united ether
extracts through a dry filter paper into a dry flask, distil off most of the
ether, transfer the material to an evaporating dish and. allow the rest of
112
the ether to evaporate at the room temperature. There will remain a few
drops of an oily liquid (lactic acid) soluble in all proportions in water,
alcohol and ether.
(16) Uffelmann's Test. Treat 5 ml. of Uffelmann's reagent (See
p. 96) with a drop of lactic acid.
(17) Hopkins'Test. Prepare a dilute alcoholic solution of lactic acid
and test as described on p. 96.
(18)
THE PROTEINS OF MILK
Saturate 10 ml. of milk with magnesium sulfate (about 5 grams).
Place on a fluted filter and allow to filter over night.
Faintly acidify the filtrate with acetic acid and boil.
Remove the precipitate from th~ filter and grind in a mortar with a little
water. Filter and acidify with acetic acid. Filter off the precipitate and
heat the filtrate to boiling.
What proteins are precipitated at various points in the experiment?
(19)
THE CLOTTING OF MILK BY RENNIN
Prepare a solution of rennin by dissolving 0.1 g. of the commercial preparation in 100 ml. of water. If solution is not complete, filter. Treat
100 ml. of milk with 5 ml. of the rennin solution and allow to remain at 40°,
Wash a portion of the clot and dry with alcohol and ether as directed under
"casein." Make protein tests 'with the material. (Include test for phosphorus.) Test the whey for the permddase reaction and for coagulable
protein.
114
BLOOD: GENERAL
RED CORPUSCLES
The membrane of the red corpuscle, like most cell membranes, is semipermeable.
Water and some anions pass through the corpuscular membrane easily whereas it is
practically impermeable to cations such as Na+ and K+. When corpuscles are
suspended in such a salt solution having an osmotic pressure equal to that of the solution inside the corpuscles the rates of passage of water in and out of the membrane
are the same and no change in volume of the corpuscle occurs. The solution is said
to be isotonic. When the outer solution has a higher osmotic pressure than the inner
it is said to be hypertonic and water is lost from the corpuscle and it becomes shrunken
or crenated. When the outer solution has a lower osmotic pressure than the inner
it is hypotonic and the corpuscle gains water. If the outer solution is sufficiently
hypotonic enough water may enter the corpuscle to cause it to swell and burst,
releasing the red pigment, hemoglobin, contained within. This is known as hemolysis or laking. After hemolysis the blood appears darker and is transparent.
(1) Hemolysis by Hypotonic Solutions. Prepare test tubes containing
the following dilutions of 1% NaCl solution.
1.
2.
3.
4.
5.
6.
7.
3 m!.
4 m!.
5 m!.
6 m!.
7 m!.
8 m!.
9 m!.
1% NaCI
1% NaCI
1% NaCI
1% NaCI
1% NaCI
1% NaCI
1% NaCI
+
7 m!. water = 0.30%
= 0.40%
5 m!. water = 0.50%
4 m!. water = 0.60%
3 m!. water = 0.70%
2 m!. water = 0.80%
1 m!. water = 0.90%
+ 6 m!. water
+
+
+
+
+
NaCI
NaCI
NaCI
NaCI
NaCI
NaCI
NaCI
or 0.05M
or 0.07M
or 0.08M
or O.1OM
or 0.12M
or 0.14M
or 0.15M
To each tube add 3 drops of defibrinated blood, mix well and note the
rate and degree of hemulysis. Observe for several hours.
(2) Add a drop of blood to 2 or 3 ml. of 10% sodium chloride. Examine the corpuscles under the microscope and compare with those from
tube 7 above.
(3) Hemolysis by Various Reagents. Place 1 ml. portions of blood in
4 test -tubes, add the following reagents and mix thoroughly.
a. Two volumes of water. _
- b. A few drops of ether.
c: An equal volume 0.8% sodium chloride containing 0.2% saponin.
Compare the color of each tube with the 4th (control) tube. Half fill
all four tubes with 0.8% sodium chloride and note which are transparent.
HEMOGLOBIN
The following tests are used for detecting the presence of blood.
(4) Guaiac Test. Add 1 or 2 drops of defibrinated blood to 5 ml. of
116
water. Introduce several drops of a freshly prepared alcoholic solution of
guaiac (1:60) and a little hydrogen peroxide and shake. The color may
be extracted with chloroform,
(5) Benzidine Test. To 5 ml. of a solution of benzidine1 in glacial
acetic acid, add 2 ml. of hydrogen peroxide and a few drops of greatly
diluted blood.
(6) Hemin. Evaporate carefully a small drop of 10% sodium chloride
upon a slide. Place some fine particles of dried blood (particles of suspected material powdered or teased as finely as possible) upon the thin
layer of crystallized salt. Cover with a cover glass and bring enough
glacial acetic acid on the slide and in contact with the edge of the slip to
fill the space between the glasses. Heat carefully until bubbles of gas begin
to form between the glasses. H~min crystals will be formed immediately.
These crystals appear under the microscope as rhombic plates which are
brown by transmitted light, but steel blue by reflected light. They also
show a very characteristic metallic lustre.
(7) Crystallized Oxyhemoglobin. Shake a few milliliters of defibrinated dog's blood with air and place 1 drop on a microscope slide. Add
1 drop of water, mix with a small pointed rod and allow to evaporate
at room temperature until the edge of the drop is dry, cover carefully
with a cover glass and examine under the microscope. Long red needles
of oxyhemoglobin will be observed. If it is possible to obtain the material
do the same experiment with the blood of the rat.
(8) Shake. 5 ml. of defibrinated dog's blood with ether until hemolyzed
and add a few crystals of ammonium oxalate. Place in the ice box for a
few hours. The tube now contains a mass of crystals. Examine microscopically.
.
SPECTROSCOPIC EXAMINATIOW
The absorption spectra of hemoglobin and its derivatives are characteristic and readily ,distinguishable under proper conditions. Of the
common derivatives of hemoglobin, carboxyhemoglobin (HbCO), m~t­
hemoglobin (MHb), sulfhemoglobin (SHb) and hematoporphyrin have
special clinical interest. Such pigments are present in the blood in detectable quantities only under abnormal circumstances.
0
1 Heat to 50 4.3 m!. of glacial acetic acid in a small Erlenmeyer flask and add 0.5
g. benzidine. Continue heating at 50 0 for 8-10 minutes. Add 19 mI. distilled water.
This solution will keep several days without deterioration.
2 These directions are based upon quantitative spectrophotometric studies.
Drabkin, D. L. and Austin, J. H., J. BioI. Chem., 98, 719 (1932); 112,51,67,89,105
(1935-36).
.
118
In qualitative spectroscopy, analysis is based upon:
(a) The number of bands, their relative sharpness and intensity and
their location in the spectrum.
(b) Change or absence of change from one characteristic spectrum to
another after the addition of suitable reagents to the pigment solution.
Apparatus
(a) Simple spectroscopes. The instruments may be conveniently provided with small accessory prisms in front of the slit opening. This
arrangement permits the viewing of two superimposed spectra from separate sources. It is particularly helpful in the direct comparison of such
relatively similar pigments as oxyhemoglobin (Hb0 2 ) and carboxyhemo~lobin (HbCO).
(b) The source of the continuous spectrum is a Welsbach burner furnishing a bright heterogeneous source of light.
(c) A simple guide as to wave-length is furnished by heating sodium·
to incandescence. The monochromatic sodium flame is interposed between
the Welsbach source and the slit of the instrument, so that when one views
the field in the spectroscope a bright yellow line is seen superimposed upon
a continuous spectrum. This line is the so-called D line and is located at
lambda (>') 589 m/).. The visible portion of the spectrum is said to lie
between >'800 m/). and >'400 m/). respectively, the red and violet ends of
the visible spectrum. Actually, visual sensitivity is poor beyond the limits
of >'700 and >.470 m/).. The wave-length values given below are for the
optical center or "peak" of each absorption band.
(d) Cells with parallel ends or cells similar to polariscopic tubes are
often employed. In most instances, however, the students will use test
tubes. Proper qualitative spectroscopic analysis may be attained with
test tubes provided it is recalled that, when filled with liquids, they behave
somewhat like astigmatic convex lenses. To overcome this difficulty the
tube should be m.ounte& (by clamp and stand) parallel and rather close to
the slit of the spectroscope and in such a position that the light passing
through the tube will be sharply focused along the length of the slit.
Pigment Solutions
A stock solution of oxyhemoglobin will be furnished·. This solution is
approximately 1: 10 with reference to the normal concentration of hemoglobin in the blood. It has been prepared by the hemolysis of thoroughly
washed blood cells.
120
Experiments:
(9) Oxyhemoglobin (Hb0 2 ). In certain concentrations oxyhemoglobin has a typical two-banded spectrum; the bands being located at
}'578 and }'542 mIL.
(a) Examine the stock solution.
(b) Dilute (a) 1: 10 and examine.
(c) Divide (b) into two parts; dilute one part 1:3 and the other 1:10.
Examine each.
Your experiments should yield a qualitative answer as to the relative
strength of the two bands. Which band is sharper?
(10) Reduced Hemoglobin (Hb). Hemoglobin has a typical onebanded spectrum, the center of the band being at }'555 mIL. To 10 m!.
of solution (b) of experiment Q, or a solution similarly prepared, add a
"knife-point" of sodium hydrosulfite (Na 2S204) and examine. What is
the probable mechani1)m of the action of sodium hydrosulfite upon oxyhemoglobin? By what other means may hemoglobin be prepared?
(11) Carboxyhemoglobin (HbCO). Carboxyhemoglobin may be readily prepared by bubbling illuminating gas through a solution of oxyhemoglobin. Why does carbon monoxide replace oxygen under these conditions?
(a) The spectrum of carboxyhemoglobin does not differ qualitatively
very materially from that of oxyhemoglobin. There is a displacement of
bands towards the blue end of the spectrum, the centers being at }'569
and M38 mIL. This displacement can be verified by the direct visual
comparison of the spectra of oxyhemoglobin and carboxyhemoglobin, using
the comparison prism (mentioned above) and the D line of sodium as a
guide. Such an arrangement will be demonstrated. . Qualitative spectroscopy is valueless for concentrations of carboxyhemoglobin of less than
5 per cent of the total pigment.
(b) Bubble illuminating gas through a solution of 1: 100 oxyhemoglobin.
Complete conversion to carboxyhemoglobin is usually attained when the
foam is distinctly pin~ and when the solution has taken on a pink-purple
tint .. Filter the solution if necessary. To 10 m!. of this solution add a
"knife-point" of sodium hydrosulfite and examine. Has the spectrum of
hemoglobin been produced? .
. (12) Methemoglobin (MHb). With reference to its color and spectrum methemoglobin behaves very much like an indicator. Quantitative
spectroscopic studies have shown that methemoglobin behaves like a weak
monobasic acid (pI( = 8.12). At pH 6.0 we are dealing with the freecacid
form, while at pH 9.4 we have complete conversion to a salt of the acid.
These two forms of methemoglobin have different spectra. At pH values
122
between 6.0 and 9.4 various mixtures of acid and salt exist. The spectrum
of methemoglobin at pH 6.0 consists mainly of 2 bands with centers at
1-.630 and A500 mJ.L respectively. There is a slight suggestion of some
absorption also at A578 mJ.L. The spectrum of methemoglobin at pH 9.4 is
three banded-a very slight band at A600, somewhat stronger bands at
A578 and A542 mJ.L. The two latter bands are not as well defined as in
the case of oxyhemoglobin. Perform the following experiments:
To 50 ml. of a 1: 50 solution of oxyhemoglobin add 1 ml. of 40 mM
potassium ferricyanide. (Describe the reaction of oxyhemoglobin and
ferricyanide.) Divide the solution into 4 equal portions.
(a) To portion 1 add 2 ml. of 50 mM phosphate buffer, pH 6.0, and
examine spectroscopically.
(b) To portion 2 add 2 ml. of 50 mM borate buffer, pH 9.2, and examine
spectroscopically.
(c) Titrate solution (a) with 0.01 N sodium hydroxide and examine the
solution spectroscopically after eacp- 0.5 ml. of alkali has been added.
Note the number of cc. of alkali needed to completely convert solution (a)
to a solution yielding a spectrum similar to that of solution (b). Verify
by experiment that oxyhemoglobin, unlike methemoglobin, exhibits no
change in spectrum between the pH limits of 6.0 and 9.0.
(d) To portion 3 add 2 ml. of 0.1 per cent potassium cyanide (from
burette) and examine spectroscopically. The spectrum is. that of cyanhemoglobin.
.
(e) To portion 4 add a" knife-point" of sodium hydrosulfite and examine
spectroscopically.
The various experiments outlined under (12) should suggest the necessary spectroscopic data, which one must have to prove definitely the presence of methemoglobin in blood.
(13) Sulfhemoglobin (SHb). By the addition of hydrogen sulfide to a
solution containing oxyhemoglobin a pigment is produced which has a
2 banded spectrum, the bands being located at A620 and at A560 mJ.L.
This pigment cannot be produced from hemoglobin, if oxygen is excluded
during the reaction. with hydrogen sulfide. Due to absorption in the red
region of the spectrum this pigment must be differentiated from the acid
form of methemoglobin.
Bubble hydrogen sulfide for approximately 4 minutes through 50 ml.
of a 1: 50 solution of oxyhemoglobin. Filter the solution and divide into
3 portions.
(a) Examine portion 1 spectroscopically. Now add 2 ml. of borate
buffer, pH 9, and re-examine. Is there any change in spectrum?
124
(b) To portion 2 add 2 ml. of 0.1 per cent potassium cyanide and examine. Has the spectrum of cyanhemoglobin been produced?
(c) To portion 3 add a "knife-point" of sodium hydrosulfite and examine.
These tests distinguish between methemoglobin and sulfhemoglobin.
The mere presence of a band in the red region of the spectrum is insufficient
evidence of the presence of either pigment.
(14) Acid Hematin. Acid hematin is commonly employed in the
colorimetric estimation of hemoglobin concentration. Mix 1 ml. of the
1: 10 stock solution of oxyhemoglobin with 10 ml. of 0.1 N hydrochloric
acid. Examine spectroscopically at 5 and 40 minutes after the preparation
of this mixture. The reaction of hemoglobin with dilute acid is relatively
slow. The completely developed spectrum of acid hematin shows a band
in the far red at A660 mt-! and diffuse, increasingly strong absorption from
A560 mt-! on.
(16) Hematoporphyrin. Add 1 ml. of the 1: 10 stock solution of oxyhemoglobin to 5 ml. of concentrated sulfuric acid. Stir thoroughly. A narrow band to the left of the sodium line at about X600 mt-! and a compound
band to the right of the sodium line are characteristic of hematoporphyrin
in sulfuric acid.
(16) Oxidized and reduced hemochromogens. These pigments are
of particular interest, since they (or related compounds with similar
spectra) are thought to be important catalysts of cellular oxidation-reduction processes. Both cytochrome and the respiratory ferment (Warburg) are
thought to contain hemochromogens in their makeup.
Mix 1 ml. of the 1: 10 stock solution of oxyhemoglobin with 10 ml. of
0.01 N sodium hydroxide. The spectrum yielded by this solution, a light
band at X578 mt-! and a darker, more distinct band at X545 mt-!, is qualitatively difficult to distinguish from the spectrum of alkaline methemoglobin.
The nature of the pigment in the solution (oxidized hemochromogen) is
readily brought out by the addition of a "knife-point" of sodium hydrosulfite. After the addition of 'the reducing agent, the very characteristic
spectrum of reduced hemo'chromogen is yielded by the solution: a very
intense, narrow band at A560 mt-! and a broader, lighter band at A530 mt-!.
The ease with which reduced hemochromogen is oxidized may be demon. strated by a re-examination of the spectrum after the solution has been
gently shaken for a short time.
BLOOD SERUM
Dilute blood serum with 25 times its volume of water and carry out the
fQllowing tests as described in the chapter on proteins.
(17) Stratify with nitric acid.
126
(18) Boil with a few drops of acetic acid.
(19) Carry out the protein color reactions.
(20) Test with lead acetate, mercuric chloride, potassium ferrocyanide
and acetic acid.
(21) Treat with saturated picric acid, trichloracetic acid, and phosphotungstic acid (acidify with hydrochloric acid).
(22) Proteins. To 20 m!. of undiluted serum add 20 m!. of a saturated
solution of ammonium sulfate. Filter off the precipitate (?). Saturate
the filtrate with ammonium sulfate. Filter or decant (?). Dissolve each
precipitate in water and test for coagulability. Test the coagulum with
the mercuric nitrite test.
(23) Saturate 20 m!. of blood serum with magnesium sulfate (?).
Filter, acidify and heat the filtrate to boiling (?).
(24) Non-protein Constituents. To 50 m!. of blood add 400 m!. of
water and heat to boiling in a porcelain dish. Add drop at a time 0.2 N
acetic acid until a brown coagulum is thrown down leaving a perfectly
colorless fluid. This coagulation requires great care. When too much
acid is added the coloring matter passes again into solution and one must
then use sodium hydroxide, a procedure which is highly undesirable. It
is best to filter off a little of the fluid from time to time during the addition
of acid. As,soon as a colorless filtrate can be obtained, discontinue the
addition of the acid, heat to violent boiling and filter while hot through a
fluted filter paper. Evaporate the filtrate on the water bath to about 10 m!.
and make the following tests:
Fehling's test for sugar.
Test a portion of the liquid for chloride.
Evaporate a m!. of the fluid on a watch glass bver the water bath and
examine the residue under the microscope for cubes of sodium chloride.
Test for phosphate with molybdic solution.
Test for phosphate with magnesia mixture.
128
BLOOD: QUANTITATIVE METHODS 1
DUBOSCQ COMPARATOR
Many "colorimetric" methods have been devised based upon a comparison of the intensity of color produced in an unknown solution with that
produced in a standard solution when treated with the same reagents. A
Duboscq type comparator has been extensively used for the color comparisons and for this reason is commonly called a Duboscq colorimeter. This
instrument permits a comparison of two beams of light one ·of which has
passed through a standard and the other through an unknown solution,
the two beams being brought side by side in the eye piece. By means of a
plunger and movable cup the depth of liquid through which the light
passes may be varied until the intensities in the two fields are the same and
the colors are said to match. When such adjustment has been made the·
depths of the fluids through which the light passes are inversely proportional to the concentration of the color. A scale on the instrument gives
a measure of the depth of fluid in each cup through which the light passes.
Thus from the scale readings and the strength of the standard solution the
concentration in the unknown may be calculated by the following formula
reading of standard
reading of unknown
concentration of unknown
concentration of standard
When using a Duboscq comparator first focus the eyepiece until the
line demarking the two fields stands out sharply, then adjust the instrument
in front of a window or artificial light until the two fields are equally illuminated. The dull reflector is usually used instead of the mirror. Ordinarily, daylight or artificial "daylight" light is used although filters were
originally furnished to give narrow spectral regions.
Check the accuracy of your readings by placing the standard solution
in both cups (filling the cups a little more than half full so that they will
not overflow when the plungers are immersed) setting the right hand cup
so that a convenient depth of liquid is obtained (usually 20 mm.) and then
adjusting the left hand cup until the two fields match. Readings should
be made until variations not greater than 0.3 mm. are obtained, and the
depths of liquids in the two cups do not vary by more than 0.3 mm. The
1 Excellent critical discussions of quantitative methods used in biochemical
work may be found in Peters and Van Slyke's, Quantitative Clinical Chemistry,
vol. ii, Baltimore, 1932.
130
color from the left hand cup now matches the color produced by a depth of
20 mm. in the right hand cup. When suitable readings have been obtained
remove the right hand cup (without disturbing the setting of the other),
rinse the cup and plunger twice with portions of the unknown solution then
introduce a portion of the unknown and compare it with the standard.
In case the reading of the unknown is less than two-thirds or more than
one and one-half times the reading of the standard, the determination
should be repeated using enough of the solution being analyzed to get a
suitable depth of color when the reagents are added.
When the 'determination is finished, rinse the plungers and cups by filling
the cups half full of distilled water and immersing the plungers as in a determination. Care should be taken not to spill fluid on the reflector and
not to chip the plungers.
It might be pointed out that these and the following instruments, commonly called colorimeters, are photometers which measure the amount of
light transmitted.
PHOTOELECTRIC PHOTOMETER
Quantitative analysis by "colorimetric" methods has been greatly advanced by the introduction of the photoelectric photometer which is sometimes called a photoelectric colorimeter'. By its use greater accuracy has
been attained by the elimination of the subjective factor which often influences visual readings. Smaller amounts of material may be determined
because lighter colors may be used.
In a photoelectric photometer, light passing through a colored solution
is caused to fall upon a photoelectric cell and the electric current thus generated is measured by means of a galvanometer or potentiometer. The
absorption of light by the solution is a measure of the amount of color.
This is proportional to the unknown concentration of the material studied.
The sensitiveness is increased by interposing a light filter which permits
light to pass from only a narrow spectral region.
There are two types of photoelectric photometers: those with one photoelectric cell and those with two. When one cell is used, the machine is
calibrated by determining the readings with known concentrations of the
substance being studied. When two cells are used, a tube containing the
substance in known concentration is compared with a tube containing an
unknown concentration.
The photoelectric photometer that we use may be described briefly as an
instrument furnishing relatively monochromatic light, which passes through
a colored solution. Some of the light is absorbed by the solution and the
remainder is received by a photoelectric cell and generates an electric cur131
rent assumed to be proportional to the intensity. Comparison of a standard concentration with an unknown concentration of absorbing material
permits the calculation of the concentration in the unknown.
The figure shows the essential elements of a photoelectric photometer in
which measurements are by the substitution technique, i.e. where the blank
solution is adjusted to read 100% transmittancy, or zero optical density.
T%-+
/),or
9 Ig __ ~
_:e _~e _:e_~E_ _7J__ ~P__ :P~_'3°~1
9"-----0;'-- -ol--o~
-!ogmT
@
or -~5-~6-,-or8~~oetc. r
This figure has been kindly furnished by Dr. D. L. Drabkin.
(1) Source of light (or radiation).
(2) Power supply and provision for controlling intensity and steadiness
of radiation.
(3) Provision for collimating lens or other device.
(4) Aperture diaphragms to cut out stray radiation ..
(5) Provision for securing monochromatic radiatIon-filters or monochromator.
(6) Liquid being examined, in a test tube or, preferably, in a calibrated
cuvette of known depth. Provision for rapid substitution of blank by
solution.
(7) Photocell receiver.
(8) Device for measuring electric current from photocell.
(9) Scale of measuring device. T is transmittancy in % of total, D is
the density or the negative logarithm of the transmission. The concentrations in two solutions are directly proportional to the densities
within the range of applicability of Beer's law.
DIRECTIONS FOR USING THE KLETT-SUMMERSON
PHOTOELECTRIC PHOTOMETER
Before turning the photometer lamp on. Be sure that a light filter is in
place in the space provided for it between the lamp housing and the instru132
ment proper. Examine the pointer to make sure that it coincides exactly
with the line on the blank pointer scale. If it does not, it should be
adjusted by the instructor. Get into the habit of noting that the pointer
I
is at its proper setting before turning the lamp on.
To use the instrument. Place a clean photometer tube containing distilled water in the instrument. Turn the scale by means of the large knob
on the front of the instrument until the scale reading is O. Switch on the
photometer lamp by means of the lamp switch located on the front of the
lamp housing.
Switching the lamp on will in general cause the pointer to move somewhat away from the line on the pointer scale. Find the zero adjustment
knob, which is located in the top of the photometer to the left of the test
. tube, and turn this knob one way or the other until the pointer is brought
back to the line on the pointer scale. This operation is called setting the zero.
Allow the lamp to burn for a few minutes to permit the instrument to
reach equilibrium. Again check the position of the pointer to be sure that
it is on the line. If it is not, turn the zero adjustment knob carefully until
the pointer is exactly on the line. The instrument is now ready for use.
To read an unknown solution: remove the distilled water tube and place
the photometer tube containing the unknown solution in the instrument.
The pointer will be deflected from its position at the zero line. Turn the
scale knob until the pointer has been brought back exactly to the zero line.
The reading on the scale at this point is the reading of the unknown solution.
The concentration of the unknown solution is ordinarily then obtained
from the scale reading (corrected for the reagent blank if necessary, see
below) by multiplying the scale reading by the proper factor. The value
of the factor depends on the particular photom~tric procedure being employed. This is fully discussed below, and also in connection with the
detailed procedures for the individual methods.
The light jilters. The light filter is inserted in the filter holder and placed
in the instrument in the space provided for it just in front of the lamp
housing. The frame containing the filter should always be inserted with
the round opening facing the operator. As mentioned above the photometer lamp should not be turned on unless a filter is in place in the instrument.
Keep the filter clean and free from dust and grease, and do not handle the
glass part with the fingers. The filter may be cleaned with a damp cloth
or piece of tissue paper.
The choice of light filters depends on the particular analytical procedure
being used. There is a particular filter specified for each analytical procedure and the specified filter should be used unless there is good reason for
changing.
133
When changing from one filter to another, the change in the intensity
of light striking the photoelectric cell rpquires restabilization, as discussed
in paragraph 3. It is necessary of course to re-set the zero with distilled
water when a new filter is placed in the photometer, since the cell adjusted
for one filter is not necessarily adjusted for another.
The proper filter for a ·particular photometric procedure is usually specified in the directions for the procedure. Ordinarily the filter selected is the
one whose spectral transmission is opposite to that of the solution being
measured, i.e. the filter which transmits most light over the range where the
solution absorbs most light. In this way ma..'<imum sensitivity is usually
obtained.
There are a number of exceptions to this rule, however. In some cases,
as for instance with the blue color obtained in the Folin-Wu blood sugar
method, if a red filter were used the increase in sensitivity would be so great
as to make it impossible to read accurately even the moderate color obtained with a normal sample, let alone a high blood sugar, without such a'
dilution of the color as to impair the accuracy of the procedure. Under
these conditions a green or blue filter is used. In this way the most satisfactory relationship between scale reading and a wide range of concentration is obtained.
The zero adjustment knob. It cannot be too strongly emphasized that all
photometric measurements with the instrument are based on the pointer
being at its zero position when the distilled water tube or reagent blank
tube·is in place in the photometer and the photometer scale reads O. This
adjustment is made possible by the use of the zero adjustment knob. The
accuracy of the instrument depends to a large extent on how carefully the
zero adjustment is made.
The photometer tubes. The tubes have been carefully standardized for
use in the photometer. They must be kept scrupulously clean, both inside
and outside. Before inserting a tube in the photometer, it is good practice
to wipe off finger marks etc. from· the outer surface of the tube by means of a
clean cloth or piece of, cleansing tissue and to hold the tube up to the light
and l<;>ok through it, to be sure that the glass surfaces are perfectly clean.
Scratched or marred tubes should be discarded.
The smallest volume that can be safely read in a tube is a little under
.5 m!. For this reason it is well to have the liquid level at least to the 5 m!.
mark, if calibrated tubes are used. More fluid than this in the tube has no
influence on the readings. Bubb.les formed on the sides of the tube in certain photometric procedures may be dislodged by tapping the tube smartly
on a wooden block or table top. The tube should be seated firmly and as
deeply as possible in the opening provided for it in the photometer. Always place the tube so that the orientation mark faces the operator.
134
The scale. The scale is a most important part of the photometer. It
has been designed so that for the majority of the common phot,ometric
procedures and under certain specified conditions the scale reading is directly
proportional to the concentration of the substance being determined. The scale
reading is a measure of and is proportional to the optical density of the
colored solution as determined by the photoelectric cell, and since the
optical density is theoretically proportional to the concentration of colored
substance (Beer's law), the scale readings are likewise proportional to the
concentration under the same conditions. In reading the scale, note that
it is logarithmically spaced and not linearly spaced.
For good photometric measurements, readings should fall between
approximately 150 and 400. Readings above 500 or 600 should not be
used as a basis for calculating results, since such readings represent relatively dense solutions for which a small change in concentration produces
an almost undetectable change in color. From 0 to 100 other errors enter
since transmissions are over 90%.
Compensating for absorption owing to color in the blank. If the reagents
alone give a color, correction for this color must be made. The corrected.
reading for the unknown is obviously the observed reading minus the blank
reading, and if both the unknown and the blank are read against distilled
water set at zero, the corrected reading must be used in calculating the concentration of the unknown.
There is a simple way to avoid this subtraction process, and that is to
read the unknown against the blank set at O. This is done by placing a
photometer tube containing the reagent blank solution in the instrument,
and adjusting the pointer to its zero position by means of the zero adjustment knob, with the photometer scale turned to O. The unknown solution
is then placed in the instrument and the reading made as usual. Since the
unknown solution ~s read against the blank at 0, the reading of the unknown must be due solely to absorption above that given by the blank.
In this way the absorption of the blank is subtracted from the total absorption and the reading of the unknown is a direc~ measure of the concentration of substance present.
The blank tube is usually prepared by running through a complete photometric procedure on a sample of distilled water which has been t~eated in ...
such a way as to make it as nearly identical as possible with the unknown
solution except fol!.. the presence of the substance being determined.
Calculation of results using a calibration factor. It has been pointed out
that under the conditions which have been specified for the common photometric procedures the scale reading of the unknown (corrected for a blank
if present, as described above) is directly proportional to the concentration.
135
Hence it is merely necessary to multiply the scale reading by the proper
factor to obtain concentration directly.
reading of unknown X factor = concentration of unknown
The value of the factor is of course different for each photometric procedure,
since but one scale is used for all procedures. The value of the factor is
obtained from the scale reading for a solution of known concentration.
Then using the equation above,
concentration of standard
fac t or = -----;-;-.-----=----,----0-----0-readmg of standard
This operation is obviously analogous to the "preparation of the calibration curve" that is necessary when a photoelectric photometer with a linear
scale rather than a logarithmic scale is used, only in the case of instruments
with a linear scale it is 'necessary to plot the reading of the standard on semilogarithmic paper against the concentration, whereas in the above procedure it is simply a question of dividing one value by another.
In many cases the standard solution used to determine the calibration
factor may be the ordinary standard of visual photometric procedures that
. is usually already available in the laboratory, or the standard lllay be
prepared by suitable dilution of an available stock standard solution. It is
well to run the calibration determination in duplicate or triplicate and take
the average value for the calibration factor, and greater precision is doubtless obtained if the factor is determined for two or more concentrations of
standard and the average value used in the calculation of unknown concentrations.
It is well to recognize that the amount of color yielded by a certain known
concentration of substance is influenced by a large number of variable factors, such as the nature of the chemicals used in preparing the reagents,
the age of the reagents, the rate and duration of heating and cooling if such
are necessary, the rate of addition of the reagents, the time of standing
before reading in the photometer, the temperature at which the color is
developed, etc., etc.. Furthermore, slight variations in presumably identical light filters, or differences between even carefully chosen photoelectric
cells, will aid in changing t~e value of the calibration factor for a given procedure. For this reason a calibration factor must be regarded as valid
'only under the particular conditions of calibration, and these conditions
must be held as constant as possible in order to use such a factor in the
future. If there is any change in these conditions, such as the preparation
of a new lot of reagents, or the purchase of a new filter to replace a broken
one, it is well to redetermine the calibration factor to be sure that no correction is necessary. It is of course essential that unknown solutions be
run in exactly the same way that the standard solution"s were run when the
calibration factor was determined.
136
Running (1, standard solution along with the unknown. The well-known
procedure of running a standard solution along with the unknown solution
and determining the value of the unknown in terms of the absorption of the
standard eliminates most of the sources of error mentioned in the above
discussion, and is undoubtedly to be preferred over the use of a calibration
curve or factor if precise results are desired.
This procedure is carried out exactly as with visual photometry. The
standard and unknown are run simultaneously, and each is read in the
photoelectric photometer, against distilled water or the reagent blank if
there is one. Since the readings for both the standard and unknown are
proportional to the concentration, the results are calculated by the use of
the following formula:
concentration of standard
X reading of unknown
reading of standard
=
concentration of unknown
It will be noted that this formula is similar to that used in visual photometry except that the readings are directly proportional to the concentration instead of inversely proportional.
The use of the photoelectric photometer with any standard photometric procedure. Since it is possible as described above to determine the concentration ,of an unknown solution in terms of a similarly prepared standard
solution, the photoelectric photometer may be used for many photometric
procedures which have been devised for the visual photometer, and in
general without any modification. The unknown and standard are read
in the photometer and the calculations made as described above.
The only point to which attention should be directed is the desirability
of using the proper light filter. The same problem of proportionality between readings' and concentration ("deviation from Beer's law") that
arises in visual photometry is of course of significance in photoelectric
photometry, since calculations are made on the assumption of true proportionality. It will usually be found that the proper light filter in the photometer will considerably extend the range of concentration over which
true proportionality exists.
Many of the photometric procedures that have been developed for visual
photometry produce colored solutions whose transmissions are too low for
accurate reading in the photoelectric photometer. Here again by the
use of the proper light filter or greater dilution it will frequently be possible to make such colors readable in the photoelectric photometer with
an accuracy which is usually greater than that obtainable by visual
photometry.
137
The fact of the increased sensitivity of the photoelectric photometer to
weak colors, as compared to the visual photometer, together with the possibility of further increasing the sensitivity by the use of specific light filters,
should bring to mind the possibility of running an analysis on a much
smaller portion of sample than is ordinarily required. It may frequently
be found that the entire photometric procedure (including centrifuging,
heating, etc.) can be carried out directly in the photometer tube itself, and
on a portion of sample just a fraction of the usual amount. It is quite
probable that the future development of photoelectric photometry will be
largely directed towards the perfection of micro methods of analysis.
(1) PREPARATION OF PROTEIN-FREE BLOOD FILTRATE
Folin and Wu1 proposed in 1919 a system of analysis which requires only about
10 m!. of blood for the quantitative determination of a series of constituents including
non-protein nitrogen, urea, c~eatine, creatinine and uric acid. Many other micro
methods have been devised by Folin and his collaborators a~d by others 80 that.
now many constituents of blood may be determined with suitable accuracy on small
amounts of blood. The proteins of the blood are removed most frequently by tungstic acid or trichloroacetic acid, and the materials studied in the filtrates.
Transfer 1 volume (this and the following volumes are to be accurately
measured) of blood to a dry flask and add, with stirring, 8 volumes of 0.083
N sulfuric acid. 2 Then add slowly, with stirring, 1 volume of 10% sodium
tungstate 3 (0.33 M, Na2W04·2H20). Mix well and, after standing a few
minutes, filter or centrifuge. If the solution is filtered, use a dry filter
paper, a dry funnel and a dry flask because the final dilution of the blood
has already been made. Start the filtration of the coagulated blood by
pouring a few ml. of fluid on the double thickness of a dry flat filter that is
large enough to hold the entire amount of fluid, withholding the remainder
until the entire filter has become wet. The filtrate obtained in this way
is almost invariably as clear' as water from the first drop .
Folin, 0., and Wu, H., J. B. C., 88, 81 (1919).
This modification has been introduced by Haden, J. B. C., 56, 469 (1923). The
original and more commonly used procedure is: To 1 volume of blood add 7 volumes
of water and 1 volume of 0.33 M sodium tungstate. Then add slowly with stirring,
1 volume of 0.33 1\1 sulfuric acid.
3 Commercial sodium tungstate is sometimes too acid or too alkaline.
If alkaline
to phenolphthalein, not more than 0.4 m!. of 0.1 N hydrochloric acid should be required to neutralize 10 m!. of the 10% solution. If more hydrochloric acid is required, the remainder of the'10% tungstate solution should be neutralized appro,priately. Similarly, if the tungstate solution is acid it should be made neutral
to phenolphthalein with sodium hydroxide.
.
I
2
138
(2)
NON-PROTEIN NITROGEN
(Method of Folin and Denis 1 modified by Wong. 2)
Fifty percent sulfuric acid by volume. Gradually pour 50 m!. of nitrogenfree concentrated sulfuric acid into a 300 m!. flask containing 50 m!. of
distilled water, keeping it cool under the tap.
Saturated Potassium Persulfate. The persulfate used should be nitrogenfree as shown by a blank test. Shake about 7 g. of potassium persulfate
with 100 m!. of water in a bottle. The undissolved crystals are left in the
bottle and serve to keep the solution saturated even though some of the
persulfate decomposes.
Nessler's Solution. 3 Dissolve 22.5 g. of iodine in 20 m!. of water containing 30 g. of potassium iodide. After the solution is complete add 30 g.
of pure metallic mercury, and shake the mixture well, keeping it from becoming hot by immersing in tap water from time to time. Continue this
until the supernatant liquid has lost all of the yellow color due to iodine.
Decant the supernatant aqueous solution and test a portion by adding a
few drops thereof to 1 m!. of a 1% soluble starch solution. Unless the
starch test for iodine is obtained the solution may contain mercurous compounds. To the remaining solution add a few drops of an iodine solution
of the same concentration as employed above, until a faint excess of free
iodine can be-detected by adding a few drops thereof to 1 m!. of the starch
solution. Dilute to 200 m!. and mix well.
To 975 m!. of an accurately prepared 10% sodium l)ydroxide solution now
add the entire solution of potassium mercuric iodide prepared above.
Mix thoroughly an<;l allow to clear by standing.
10 m!. of this solution should be present in 100 m!. of Nesslerized solution.
As the amount of alkali influences the color development the amount It!ed
in the standard and unknown must be the same. Be sure that the solutions
are cooled to 20° before Nessler's Solution is added.
Nessler's solution with extra alkali. 4 One volume of the above Nessler's
solution is mixed with 2 volumes of 10% sodium hydroxide. The extra
alkali serves in. this analysi_s to neutralize the sulfuric acid of the digest.
Standard Ammonium Sulfate Solution. Prepare a stock solution by dissolving 4.716 g. of purified ammonium sulfate in distilled water and make
the volume up to I liter. For use, dilute 100 m!. of the stock ·solution to
I liter. This makes a standard solution, each ml. of which contains O.Img.
of nitrogen.
Pyrex test tube (200 x 25 mm.) graduated at 35 m!. and 50 m!.
Folin, O. and Denis, W., J. B. C., 26, 473 (1916).
Wong, S. Y., J. B. C., 55, 431 (1923).
3 Prepared according to the directions of Koch, F. C. and McMeekin, T. L., J.
Am. Chern. Soc., 4.6, 2066 (1924).
4 Peters and Van Slyke, Quantitative Clinical Ghemistry.
Methods, p. 533.
1
2
140
The Determination. Place 5 m!. of the protein-free blood filtrate (containing 0.2-0.3 mg. N) in a dry test tube marked at 35 mI. and at 50 mI.,
add 1 m!. of 50% sulfuric acid, introduce a quartz pebble to prevent bumping and boil vigorously over a inicro burner until the characteristic dense
fumes begin to fill the tube. This lfsually occurs in 3 to 7 minutes. When
the fumes nearly fill the tube turn down the flame so that the material is
just kept boiling, close the mouth of the tube with a watch glass and
continue boiling very gently for 2 minutes.
Remove the burner and allow to cool for 1 minute. Take off the watch
glass and add 2 drops of saturated potassium persulfate with a fine pipette
or dropper. Replace the burner and continue the boiling until the digestion
mixture becomes colorless. Stop the boiling about 15 seconds after the
re-appearance of the white fumes, the test tube being covered with a watch
glass during this period. Allow to cool 70-90 seconds, then add 20-25 mI.
of distilled water. Cool to room temperature under the tap and dilute·witl?distilled water to the 35 mI. mark.
Into another similar Pyrex test tube, measure exactly 2 m!. of standard
ammonium sulfate solution containing 0.1 mg. of nitrogen per m!. Add 1
ml. of the 50% sulfuric acid and dilute to the 35 m!. mark with distilled
water. Now add to both the standard and unknown, 15 m!. of "Nessler's
solution with extra alkali." Let the Nessler's solution fall directly into
the acid solution. Insert a clean rubber stopper, mix and compare in a
Duboscq comparator. Calculate mg. Of total N.P.N. per 100 mI. of blood.
The method may be used for nitrogen determinations in various biological materials. The time of boiling and the amount of persulfate to be J
added vary depending upon the type of material which is being digested.
Procedure for photoelectric photometer. Dilute the digest with 1-2 m!. of
distilled water, cool, add 1 drop of phenolphthalein and make the solution
alkaline (add 10 mI. of 10% sodium hydroxide). Stopper quickly and
distill or aerate into 5 mi. of 0.1 N HC!. Add 2 drops of gum ghatti (see
Exp. 4, below) to the distillate, make the volume up to about 35 or 40 mI.,
add 5 m!. of regular Nessler's solution and make up to 50 mi. The Standard "is prepared by taking 2 m!. of standard ammonium sulfate solution,
adding 5 m!. of 0.1 N HCl and 2 drops of gum ghatti. Water and regular
"N:essler's solution are added as described above. The solutions are read
in the photometer with a blank at 0 prepared by adding 5 m!. of Nessler's
solution to 45 m!. of distilled water containing 2 drops of gum ghatti.
Filter 54 (green) is used.
(3)
UREA
The urea is changed to ammonium carbonate by means of. the enzyme urease.
The ammonia is aerated into standard acid solution. By titrating the excess acid,
the amount neutralized by the ammonia can be determined and the quantity of urea
calculated.
142
Urease solution.! Wash about 3 g. of permutit powder in a 200 ml. flask
by decantation first with 0.2 M acetic acid, then with water. To the moist
powder add 100 ml. of 15% alcohol. Introduce 10 g. of jack bean meal,
shake vigorously for 10 minutes and filter. The filtration proceeds more
rapidly if the fluid is shaken vigorously just before pouring it on the filter.
The alcoholic filtrate will be free from ammonia (which was absorbed by
the permutit) and will contain substantially all the urease of the bean powder. The extract will remain active for a week at room temperature if
protected from direct sunlight and may be preserved on ice for 3-5 weeks.
When about to prepare urease solution, it is worth while to rinse all containers with nitric acid and then water to eliminate possible traces of mercury which are extremely harmful to the urease activity.
Phosphate Buffer. Dissolv.e 14 g. of sodium pyrophosphate in enough
0.5 M phosphoric acid to make a volume of 100 m!. The solution is about
neutral and is used to neutralize the ammonium carbonate formed when
urease acts upon urea and to catalyze the urease activity.
0.01 N HCl and 0.01 N NaOH. Prepare by diluting 0.1 N solutions.
Titrate 15 ml. of the acid with the alkali in order to restandardize the alkali. Use the red brown color of alizarine red as the end point.
Potassium carbonate, saturated solution. 900 g. of potassium carbonate
dissolved in 1 liter of water.
Caprylic alcohol.
Aeration apparatus.
The Determination. Carefully introduce 2 m!. of blood into the bottom
of a large test tube of an aeration apparatus and dilute with 2 ml. of water.
Add 2 drops of phosphate buffer solution, and. 1 m!. of urease solution.
Stopper and place the tube in a water bath heated to 45-50°C. At the end
of 15 minutes remove the tube, add 3 drops of caprylic alcohol and 5 m!. of
saturated potassium carbonate and connect it at once with a receiving tube
containing 15 m!. of 0.01 N hydrochloric acid, 1 drop of caprylic alcohol
and 1 drop of al~zarine -red indicator. Pass a moderate stream of air,
washed free of ammonia, through the tubes for the first 5 minutes, and a
rapid stream of air for 30 minutes longer. Rinse the connecting tube and
titrate the excess acid with 0.01 N sodium hydroxide.
Calculate mg. of urea and urea nitrogen per 100 m!. of blood and millimols
per liter.
1 Urease solution may also be prepared from urease powder.
A 10% solution is
made by rubbing up to a paste a suitable amoUl,t in a few drops of water and then
diluting with the full amount of water. This solution is usually more active than the
"jack bean extract so that, by using it, the time or temperature of digestion may be
diminished.
144
(4)
UREA
(Method of Karrl )
Protein-free filtrate is incubated with urease and the ammonia produced (from
urea) is Nesslerized directly.
Urease Solution. See p. 152.
Nessler's Solution. See p. 148.
Buffer Solution. Add 20 g. of sodium acetate to 2.2 m!. of 10% acetic
acid and make up to 100 ml.
Stock Standard Urea Solution. Dissolve 0.3215 g. of urea in water and
make up to 500 ml.
Standard Urea Solution. Place 5 ml. of the stock standard solution in a
100 ml. volumetric flask and make up to the mark.
Gum Ghatti. Tie about 3 g. of gum ghatti (tears, not powder) in a double
gauze bag and place in 100 m!. of water overnight. Remove the bag,
squeezing it gently. Filter the water solution through cotton if dirty.
Refilter if precipitate appears on standing.
Important. If Nessler's solution has been placed in the digestion tubes,
they must first be rinsed with concentrated nitric acid to remove any
mercury, then ",ith water. Mercury destroys the activity of urease.
The Determination. Into separate test tubes, pipette 5 m!. of standard
urea solution and 5 m!. of protein-free blood filtrate. Add to each tube
0.5 m!. of buffer solution and 5 drops of urease solution. Incubate the
tubes 10-15 minutes in a water bath at 50° C. Transfer the contents of
the tubes quantitatively to special test tubes graduated at 22.5 m!. and
25 m!. Add 2 drops of gum ghatti solution to each tube, dilute to 22.5 m!.
then add Nessler's solution to the 25 m!. mark. Mix and read in a color
comparator.
Calculate mg. of urea and of urea N per 100 m!. of blood and millimols
per liter.
.
Procedure jor Photpelectric Photometer. The method may be carried out
as d~scribed above. Readings with filter 54 (green) are made of the final
colored solutions and of a b_1ank prepared with all of the reagents except
. the blood filtrate. The readings are made against distilled water at o.
The blank reading is subtracted from the readings of each of the colored
solutions. From the corrected readings, calculate mg. of urea and of
urea N per 100 m!. of blood and millimols per liter.
Note. Slight turbidities often form in solutions containing Folin-Wu
filtrates, and these turbidities may interfere. much more with photoelectric
1
Karr, W. G., J. Lab. & Clin. Med., B, 329 (1924).
146
readings than with visual comparator readings. Aeration of the ammonia.
into dilute acid yields a solution which may be read satisfactorily.
.
(5)
CREATININE
There are compounds in blood which react with alkaline picric acid to give a red
color. Nearly all of this material in normal plasma and 50% or less in normal erythrocytes is creatinine. l
Standard Creatinine Solution. In a liter flask place 6 mr. of the standard
creatinine solution used for urine analysis (p. 252), add 10 mr. of normal
hydrochloric acid and dilute to the mark with water. Transfer the well
mixed solution to a bottle and preserve with a few drops of toluene.
Five ml. of this solution contains 0.03 mg. of creatinine, and this amount
plus 15 ml. of water is the standard required for the vast majority of human
blood specimens as it covers the range from 1 to 2 mg. per 100 ml. Other
dilutions can of course be made when blood specimens are encountered
which are richer in creatinine.
Alkaline Picrate. Treat 25 ml. of saturated solution of purified picric
acid with 5 ml. of 2 N sodium hydroxide. The solution should be prepared
fresh each day.
The Determination.-Procedure for Visual Comparison. In one of two
small dry flasks mix 10 ml. of protein-free blood fi,ltrate with 5 ml. of freshly
prepared alkaline picrate and in the other mix 5 ml. of standard creatinine
solution with 15 ml. of water and 10 ml. of alkaline picrate. After standing
10 minutes (but not more than 20 minutci3) compare the colors of the two
solutions with a comparator in the usual way.
It is especially important on account of the small amount of creatinine
present for the standard and unknown to contain similar amounts of color.
Calculate mg. of creatinine per 100 ml. of blood.
Procedure for Photoelectric Photometer. The solutions described above
may be read in a photoelectric photometer using filter 54 (green). Use to
obtain the zero setting, 5 ml. 'Qf distilled water mixed with 2.5 ml. of alkaline
picrate.
(6) . "TOTAL CREATININE" (CREATININE AND CREATINE)
,There is some creatine in blood and its estimation may be made as described
below. Subtracting the amount of "creatinine" from "total creatinine" should
give the amount of creatine (in terms of creatinine) present in blood, Possibly
compounds other than creatine and creatinine yield a red color however.
Place 5 ml. of protein-free blood filtrate and 1 ml. of normal hydrochloric
acid in a test tube marked at 25 ml. Cover the mouth of the test tube with
1
Miller, B. F., and Dubos, R., Jour. BioI. Chem., 121,447,457 (1937).
148
tin foil and heat in the autoclave either for 10 minutes at 155° or for 20
minutes at 130°. Allow to cool.
Prepare a standard in a 50 ml. volumetric flask by treating 10 m!. of
creatinine solution with 2 m!. of normal hydrochloric acid.
Both the unknown and the standard solutions are treated with alkaline
picrate (5 m!. for the unknown and 10 m!. for the standard) and after
standing 10 minutes, both are diluted with water to the mark (25 and 50
m!.) and their colors compared in the ordinary way.
Calculate mg. of total creatinine per 100 m!. of blood, subtract the value
for creatinine and the result is mg. of creatine calculated as creatinine.
Procedure for Photoelectric Photometer. Carry out the method as described above but use a photoelectric photometer. See Creatinine for the
necessary suggestions.
(7)
URIC ACID
(Method of Benedict and Behre1)
When uric acid is treated with arsenotungstic acid, in the presence of sodium
cyanide, a blue color develops the intensity of which is proportional to the amount
of uric acid present. Thioneine also gives a blue color. Thioneine may be removed
when silver chloride is precipitated from a strongly acidified solution while uric acid
remains in solution.
Arsenotungstic Acid Color Reagent. 100 g. of pure sodium tungstate are
placed in a liter flask and dissolved in about 600 m!. of water. 50 g. of pure
arsenic pentoxide are now added, followed by 25 m!. of 85 per cent phosphoric acid, and 20 m!. of concentrated hydrochloric acid. The mixture
is boiled for 20 minutes, cooled, and diluted to 1 liter. The reagent appearS
to keep indefinitely.
Sodium Cyanide Solution. A 5 per cent solution of sodium cyanide, containing 2 m!. of concentrated ammonia per liter. This solution improves
during the first 2 to 3 weeks after its preparation, but should not be used
after 6-7 weeks. .
. Lithium Chloride. 3 g. of lithium chloride and 20 m!. of concentrated
hydrochloric acid per liter.
Silver Nitrate Solution. 11.6 gm. of silver nitrate per liter.
Stock Standard Uric Acid Solution. 2 9 grams of pure crystallized disodium hydrogen phosphate (Na2HP04·12H20), together with 1 gram of
crystallized sodium dihydrogen phosphate (NaH 2P0 4·H 20) are dissolved
;n 200-300 ml. of hot water and the solution is filtered, if not perfectly clear.
rhe filtrate is made up to about 500 m!. with hot water and this warm
1
2
Benedict, S. R. and Behre, J. A., J. B. C., 92, 161 (1931).
Benedict, S. R. and Hitchcock, E. H., J. B. C., 20, 619 (1915).
150
solution is poured upon exactly 200 mg. of uric acid suspended in a few ml.
of water in a liter volumetric flask. The mixture is agitated a moment or
two until the uric acid dissolves and is then cooled. Exactly 1.4 m!. of
glacial acetic acid are added, and the contents of the flask are diluted to the
mark and mixed. About 5 m!. of chloroform are then added to prevent the
growth of bacteria or moulds. The solution will keep for 2 months.
Standard Uric Acid Solution. 10 m!. of the stock standard (containing
2 mg. of uric acid) are measured into a 500 m!. volumetric flask and diluted
to about 400 m!. with water. 5 m!. of concentrated hydrochloric acid are
then added and the solution is diluted to 500 m!. and mixed. This solution
should be prepared fresh about once a week. 5 m!. contains 0.02 mg. of
uric acid.
Note. The solutions containing arsenic and cyanide are very poisonous.
They should be measured in burettes and not pipettes. Do not breathe
the hydrocyanic acid liberated when the sodium cyanide is added to the
acidified blood filtrate.
The Determination. Transfer 5 m!. of the 1: 10 blood filtrate l to a 15 m!.
centrifuge tube. Add 2.5 m!. of the acid lithium chloride solution and mix
by inversion of the tube. Add 2.5 m!. of the silver nitrate solution, and
shake the contents of the tube thoroughly (using a tight rubber stopper).
Centrifuge for about t minute or longer and pour off the clear (or opalescent) supernatant liquid into a test-tube. Transfer 5 m!. of the standard
uric acid solution to another tube, and add 5 m!. of water. Add 4 m!. of
the sodium cyanide solution to each tube followed by 1 m!. of the color
reagent. Each tube is inverted once immediately after addition of the
reagent and placed at once in a boiling water bath, where the tubes are left
for 3 minutes after immersion of the last tube. The tubes are then removed
from the water bath and allowed to stand at 'room temperature for about
2 minutes, after which they are read in a c~parator· while still warm or
even hot.
,(8)
SUGAR
"Total" Reducing Substances
(Method of Shaffer and Hartmann)2
The cuprous oxide which is formed when a cupric salt is heated in alkaline solution
with reducing sugars may be determined by iodometric titration. The cuprous oxide
is reoxidized to a cupric salt by iodine and the excess of iodine determined by titration with thiosulfate llsing starch as the indicator.
1 Folin-Wu blood filtrate may be used though Benedict recommends a tungstomolybdic acid precipitating reagent. Benedict, S. R., J. B. C., 92, 135 (1931).
2 Shaffer, P. A" and Hartmann, A. F., J. B. C., 45, 349, 365 (1920-21).
de Long,
W. A., ibid., 72, 731 (1927).
152
The chemistry involved is of interest in showing how a reversible reaction may
be caused to proceed in one direction or another depending upon the concentrations
of the constituents in solution. The reaction involved may be written
or
The amount of Cu++ in solution may be determined by using an excess of iodide
thereby driving the reaction to the right and permitting the titration of the free
iodine formed. This determination may be carried out by using a ratio of CuS04:KI
not greater than 1:5 with an optimum concentration of 0.25-{}.50 M of KI.
On the other hand, the amount of cuprous copper may be determined by keeping
the concentrations of Cu++ and 1- at a minimum and thereby causing the reaction to
proceed to the left. This may be accomplished by keeping the ratio of CuS04:KI
about 1: 1 and the final concentrations of Cu++ and 1- below 0.005 M each. In this
way the free iodine in excess of the amount necessary to oxidize all of the Cu+ may be
titrated by thiosulfate. If dilution were the only method for maintaining the low
concentration of cuprie and iodide ions necessary to insure complete oxidation of the
euprous copper by iodine the method would be l!mited necessarily to the determination of small amounts of copper. This difficulty, however, has been overcome
by the use of oxalate which apparently combines with cupric ions forming a complex
which yields cupric ions to only a very slight extent. The diminution of the cupric
ion concentration permits the reaction to proceed to the left so completely that the
reaction of free iodine with cuprous copper practically goes to completion and the
excess iodine may be titrated.
The following reactions involved in this method also may be called to mind.
When a solution of iodate and iodide is acidified, free iodine is liberated.
In practically all determinations involving iodine, the iodine is titrated by means
of thiosulfate according to the equation
.
12
+ 2 S 20,- =
2 1-
+ S.08-
The Shaffer-Hartmann Reagent for Cuprous Titration (as modified by
SomogYt1 )
g.
per I.
Copper Sulfate (crys.) ............................ . 6.5
. Rochelle salt ................................... , .. 12.0
Sodium carbonate (anhydrous) .................... . 20.0
Sodium bicarbonate .............................. . 25.0
Potassium iodide .... , ............................ . 10.0
Potassium iodate ................................. . 0.8
Potassium oxalate ................................ . 18.0
Final Concentration
0.026
0.060
0.020
0.030
M
M
M
M.
0.023 N 12
0.1 M
1 Somogyi, M., J. B. C., 70, 599 (1926).
West, E. S., Scharles, F. H. and Peterson, V. L., ibid., 82, 137 (1929). (This paper
describes a second modification by Somogyi, previously unpublished. It differs
from the above in that it contains 7 g. of copper salt and 5 g. of oxalate per liter
instead of 6.5 g. and 18 g. respectively of these two constituents.)
154
Dissolve the Rochelle salt, sodium carbonate and sodium bicarbonate in
about 500 ml. of warm water. In another container dissolve the copper
sulfate in about 100 ml. of warm water. Pour the copper solution into the
Rochelle salt-carbonate solution with stirring. Dissolve 'the iodide, iodate
and oxalate (neutral) in about 300 ml. of warm water and add the solution
to the other- constituents. Cool and dilute to 1 liter. (Of the reagents
used, only the iodate has to be weighed accurately to cgm.)
Soluble Starch Solution, 1 %. See p. 84.
Standard Sodium Thiosulfate, 0.1 N. The preparation, standardization
and preservation of this solution are described on p. 84.
Standard Sodium Thiosulfate, 0.005 N. Transfer 25 ml. of the 0.1 N
thiosulfate solution to a 500 ml. volumetric flask and make up to the mark.
The solution keeps for only one day.
Sulfuric Acid, 1 N. This may be made up roughly from the desk
reagent.
The Determination. Transfer 5 ml. of the protein-free blood filtrate,
prepared by the Folin-Wu procedure on p. 138 (representing 0.5 ml. of
blood) and 5 ml. of the Shaffer-Hartmann reagent to a large test tube and
heat in a boiling water bath for 15 minutes. The tube must be covered with
a filter paper cap to diminish the reoxidation of the cuprous copper by convection currents of air. At the end of the heating, which should be carefully
timed, cool the tube to 35°0. by immersing it in a beaker of cool tap water
for several minutes. Add 5 ml. of 1 N sulfuric acid, mix and allow to
stand for 2 minutes. The acid permits the iodate and iodide to react to
form free iodine which oxidizes the cuprous copper. Titrate the excess
iodine with 0.005 N thiosulfate solution. When the brown color of the
iodine solution becomes a pale, yellow-green, add 6 drops of soluble starch
solution. The complete titration of the free iodine is indicated by ·the
sharp change of color from the characteristic blue of the starch iodide to
the pale, green-blue of the cupric salts.
A "blank" titration must be ca~ried out upon the reagents, using water
instead of blood filtrate, in order to determine the amount 6f iodine that
can be-liberated. Th~ blank titration will require about 22.3 ml. of 0.005
N thiosulfate and should be redetermined periodically. It will, of course,
be found that more thiosulfate is needed in the blank than in a sugar deternlination. In the blank there is no reducing substance and, therefore,
none of the liberated iodine is used to reoxidize cuprous to cupric copper.
On the other hand the more reducing sugar present in the blood, the more
cuprous copper will be formed, the more free iodine will be used to reoxidize
it. all,d the less thiosulfate will be required for" ation. It follows, ac156
cordingly, that the difference between the blank titration and the titration
of a determination represents the amount of iodine which has been used up
in reoxidizing the cuprous copper and may be related, therefore, to the
amount of sugar present.
The following empirical table may be employed without significant error,
provided that the determination has been carried out as described above,
i.e., 5 m!. of reagent + 5 m!. blood filtrate or sugar solution, heated for 15
minutes in a boiling water bath. (Values obtained by Somogyi.)
Mg. of Glucose Equivalent to Differences Between Blank and Final Thiosulfate Titration
1\11. of 0.005 N
. Na,S,O,
Mg. of glucose per
100 ml. of blood
Ml.ofO.OOSN
Na.S.o.
Mg. of glucose per
100 mI. of blood
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
00
29
41
53
65
77
89
101
113
124
135
146
157
168
179
190
201
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
212
223
234
245
256
267
279
290
301
312
323
334
345
356
367
378
390
400
"TRUE" BLOOD SUGAR
It has been recognized for some time that glucose is not the only substance in the
blood which has the power of reducing alkaline copper solutions. Glucose is fer·mented by yeast. If a suspension of yeast is added to blood, glucose will be destroyed. This blood, however, will still have the power to reduce alkaline copper
solutions. This "residual reduction" obviously is due to substances other than
glucose. Glutathione and ergothioneine have been definitely identified as the main
non-sugar copper-reducing substances present in blood. Benedictl has suggested
the term "sacchatoids" for this group of substances, which are mainly though not
wholly limited to the cells of the blood.
1
Benedict, S. R., J. B. C., 92, 135, 141 (1931).
158
Somogyi 1 has found that the quantity of "saccharoids!! is of the order of 20-30%
of the total reducing substances in normal blood. This value is derived by two
"sugar" determinations upon the same normal human blood by the Shaffer-Hartmann
method. The first determination is carried out in the usual way. The second determination i~ upon the same blood which has been exposed to the action of yeast.
The difference between the two determinations represents the reduction due to "true"
or fermentable sugar.
(9) The Fermentation Procedure. Fresh ye~st, relatively free of wort
and unmixed with tapioca, should be used. The yeast should be washed
free of reducing substances by means of water. This may be accomplished
readily by about four separate washings, 50 ml. of water being used in each
for a 10 gram sample of yeast. The water should be decanted after centrifuging. With the washed yeast, prepare a 10% suspension in water.
Prepare the Folin-Wu blood filtrate in the usual manner, except for the
substitution of 7 volumes of yeast suspension for the 7 volumes of water.
Allow to stand for 15 minutes before the addition of the sulfuric acid ~nd
tungstate. It is now recognized that this procedure is entirely adequate
for the complete fermentation of the "trUE~" sugar.
(10) Preparation of Blood Filtrate According to Somogyi.
Somogyi 2 has found also that the so-called saccharoids may be removed from
blood by employing special reagents for deproteination. Thus, when the proteinfree filtrate is obtained by the special technique, the determination approximates
the value for "true" sugar. If two determinations by the Shaffer-Hartmann method
are carried out upon the same blood, one upon a filtrate prepared according to Folin
and Wu, the other upon a Somogyi filtrate, the difference between the two determinations will approximate the saccharoid content of the blood.
Reagent 1. 12.5 g. of zinc.sulfate (ZnS04·7H 20) are dissolved in water,
125 ml. of 0.25 N sulfuric acid are added, and the whole made up to 1 liter
with water.
Reagent 2. 0.75 N sodium hydroxide solution. The solutions should
be so adjusted to each other that the addition of 6.7-6.8 ml. of reagent 2
to 50 m!. of reagent 1 will just produce a permanent pink color, using
phenolphthalein as an indicator.
A 1: 10 blood filtrate, is prepared by laking 1 volume of blood with 8
volumes of reagent 1, adding 1 volume of reagent 2, stoppering tightly,
shaking well, and filtering after a few minutes through a dry filter paper.
(11)
SUGAR
(Method of Folin and Wu3)
Cuprous copper is formed by heating glucose with a cupric salt in alkaline solution.
The reduced copper reduces phosphomolybdic acid and forms a deep blue color.
Somogyi, M., J. B. C., 75, 33 (1927): 78, 117 (1928):
Somogyi, M., J. B. C., 86, 655 (1930).
3 Folin, O. and Wu, H., J. B. C., 41,367 (1920).
1
2
160
Standard Sugar Solutions. Three standard solutions should be on hand:
(1) a stock solution of 1% glucose preserved with toluene; (2) a solution
containing 1 mg. of glucose per 10 m!. (5 ml. of stock-solution diluted to
500 mI.); (3) a solution containing 2 mg. of glucose per 10 m!. (5 m!. of stock
solution diluted to 250 mI.).
Alkaline Copper Solution. Dissolve 40 g. of pure anhydrous sodium
carbonate in about 400 m!. of water and transfer to a liter flask. Add7.5
g. of tartaric acid and when the latter is dissolved add 4.5 g. of crystallized
copper sulfate. Mix and make up to 1 liter.
Phosphomolybdic Acid Solution. Transfer to a liter beaker 35 g. of
molybdic acid and 5 g. of sodium tungstate. Add 200 m!. of 10% sodium
hydroxide and 200 m!. of water. Boil vigorously for 20 to 40 minutes so
as to remove nearly the whole of the ammonia present in the molybdic acid.
Cool, dilute to about 350 m!. and add 125 m!. of concentrated (85%) phosphoric acid. Dilute to 500 m!.
.
The Determination.-Procedure for visual comparison. Transfer 2 m!.
of the tungstic acid blood filtrate to a Folin blood sugar tube and to two
other similar tubes (graduated at 25 m!.) add 2 m!. of standard sugar solution containing respectively 0.2 and 0.4 mg. of glucose. To each tube add
2 m!. of alkaline copper solution. The surface of the mixtures must now
have reached the constricted part of the tube. Place the tubes in a boiling
water bath and heat for 6 minutes. Then transfer them to a cold water
bath and allow to cool, without shaking, for 2 or 3 minutes. Add to each
tube 2 m!. of phosphomolybdic acid solution. When the cuprous oxide
has dissolved, dilute the resulting blue solutions to the 25 m!. mark, and
mix. Allow to stand 10-15 minutes and then Compare the colors of the
unknown ana the nearer standard in a color comparator within the next
15 minutes.
Procedure for photoelectric photometer. Proceed as described above.
Read in the photometer using, to obtain the zero setting, a solution prepared as described _above but with 2 m!. of distilled water in place of the
blood filtrate.
(12) CHLORIDE IN BLOOD OR SERUM
(A Modification of Van Slyke's Method)1
Blood serum is digested with silver nitrate and nitric acid. The chloride is
precipitated as silver chloride and can be determined by titrating the excess of silver
nitrate with standard ammonium thiocyanate solution.
1
Van Slyke, D. D., J. B. C., 58, 523 (1923).
Wilson, D. W. and Ball, E. G., J. B. C., 79,221 (1928).·
162
Standard Silver Nitrate Solution 0.15 N. Dissolve 25.5 g. of silver nitrate
in about 400 ml. of water, transfer to a 1 liter volumetric flask and make up
to the mark.
Standard Ammonium Thiocyanate. Dissolve about 8 grams of ammonium thiocyanate in water and make up to one liter. Standardize
against standard silver nitrate, and dilute to make it exactly 0.1 N. Dilute
this solution 5 times with distilled water to prepare a 0.02 N solution.
Ferric Alum Indicator. Prepare a 0.05 M solution of ferric alum (48 g.
per liter).
The Determination. Transfer 1 m!. of serum or whole blood to a large
test tube (29 x 200 mm.), add 1 ml. of standard silver nitrate solution and
mix by shaking. After standing 2 or 3 minutes, add 3 ml. of concentrated
nitric acid, washing down the sides of the tube. Cover with a small funnel
to act as a condenser and heat over a small flame until frothing ceases and
then boil gently for 10 or 15.minutes. The solution should be water .clear
and light yellow. Cool thoroughly, add 6 m!. of ferric alum solution and
titrate with 0.02 N ammonium thiocyanate solution. 0.04 ml. of the thiocyanate solution is necessary to produce a strong red color in the volume
used. Therefore subtract the 0.04 ml. excess from the amount of thiocyanate used in the titration.
Calculate mg. of chloride and mg. of sodium chloride per 100 ml. of blood
or serum and millimols per liter.
~(13)
INORGANIC PHOSPHATE
(Method of Fiske and Subbarow Modified)l
Phosphomolybdic acid and phosphotungstic acid are easily reduced forming
solutions which are colored intensely blue. Various reducing agents may be used
and, as a result, a number of colorimetric methods have been developed. Photometric methods for sugar, uric acid and phenols are based upon their common property of reducing phosphotungstic .or phosphomolybdic acid. Phosphate may be
determined after it reacts with molybdic acid to form phosphomolybdic acid which
is then reduced by soms reducing agent. In the method described below, the reducing agent, elon, is oxidized to quinone.
10% Trichloracetic Acid ..
.10 N Sulfuric Acid. Pour 282 ml. of cone. H 2S04 into 600 ml. of water.
Cool and make up to 1 1.
Molybdate-Sulfuric Reagent. Mix 1 part of a 7.5% solution of sodium
molybdate (Na 2Mo0 4 ·2H 20), 1 part of 10 N sulfuric acid and 2 parts of
water.
1
Fiske, C. H., and Subbarow, Y., J. B. C., 66, 375 (1925).
Gomori, G., J. Lab. and Clin. ]\Ied., 27, 955 (1942).
Joncs, J. H., Personal communication.
164
Reducing Solution.
Dissolve 1 g. of methyl-p-amino-phenol sulfate (elon)
in 100 m!. of a 3% solution of sodium bisulfite.
Stock Standard Phosphate Solution (5 m!. = 0.4 mg. P). Dissolve 0.351
gm. of pure monopotassium phosphate in water. Transfer quantitatively
to a liter volumetric fla3k, add 10 m!. of 10 N sulfuric acid, dilute to the
mark and mix. The etandard keeps indefinitely.
The Determination.-Procedure jor visual comparison. Transfer to a dry
Erlenmeyer flask 20 ml. of 10% trichloracetic acid. Add, while rotating
the flask gently, 5 m!. of blood. l Close the flask with a clean, dry rubber
stopper and shake vigorously aJew times. Filter through an ashless paper.
Measure 5 m!. of the filtrate into a tube graduated at. 10 m!. Add 2 m!.
of molybdate-suJfuric reagent and after mixing, 1 m!. of the reducing solution. Dilute to the mark and mix. At the same time prepare a standard
by transferring 5 m!. of standard phosphate solution (containing 0.4 mg.
of phosphorus) to a 100 m!. volumetric flask and add about GO m!. of water,
20 m!. of molybdate-sulfuric reagent and 10 m!. of reducing solution.
Dilute the contents of the flask to the mark, mix, and compare \rith the
unknown after 15 min. Set the standard at 20 mm. Calculate the mg.
of P per 100 m!. of blood and millimoles per liter.
Procedure for photoelectric photometer. See p. 182.
'(14)
CALCIUM IN SERUM
(Method of Clark and Collip2)
This is an estimation of total calcium of serum. There is no calcium in the corpuscles. The total calcium may be fractionated with suitable methods into three
fractions. They are (1) non-diffusible calcium .(thought to be bound to protein)
and diffusible calcium which may be separated into (2) ionizable and (3) non-ionizable
fractions.
In the method described below, calcium oxalate is ~recipitated. After washing
out the exce~s of the reagent, the oxalate· of the precipitate is titrated by oxidizing
it with permanganate in acid solution.
Place 2 m!. of serum, 2 mI. of distilled water and 1 m!. of 4% ammoniur.l
oxalate (measure serum and oxalate accurately) in a clean 15 m!. centrifuge
tube. Mix thoroughly with a fine stirring rod. Allow the mixture to
stand at least! hr. (While waiting for the complete precipitation, proceed
with the standardization of the permanganate solution (see below).) After
30 min., centrifuge the solution until the the precipitate is well packed in the
bottom of the tube. Pour off the supernatant liquid carefully and while
1
2
Oxalated blood or plasma, or serum may be used.
Clark, E. P. and Collip, .J. B., J. B. C., 63, 461 (1925).
1,66
the tube is still inverted place it in a test tube rack for 5 minutes to drain.
The mouth of the tube should rest on a filter paper.
Prepare some dilute ammonium hydroxide by taking 15 m!. of the desk
reagent and diluting to 100 ml. Wipe the mouth of the centrifuge tube
with a piece of filter paper to remove the liquid still adhering to it. Then
add 3 m!. of the diluted ammonium hydroxide from a pipette allowing the
first portion to flow directly onto the precipitate so as to break up the mat
and the remainder to flow down the sides of the tube. Shake the tube until
the precipitate is well suspended, centrifuge and drain as before. Add
2 m!. of approximately normal sulfuric acid, shake until the mat is broken
and heat in a boiling water bath for about a minute. Titrate while hot
with standardized (approximately 0.01 N) potassium permanganate solution. As the total titration is usually not much more than 1 m!. the regular
burettes are not suitable. Attach a burette tip to a 5 m!. Mohr pipette
and use as a burette. The first appearance of a pink color which persists
for 1 minute is taken as the end point. Express the amount of calcium in
mgm. per 100 m!. and in milliequivalents per liter of serum.
Standardization of permanganate: During the time allowed for the complete precipitation of the calcium oxalate from the serum, standardize the
permanganate solution as follows: To 5 m!. of a 0.05 N soln. of oxalic acid
in a small flask add about 5 ml. of dilute sulfuric acid. Heat to boiling and
tit~ate, while hot, with the permanganate solution from a 50 ml. burette.
Use the same end point as that described above. Calculate the normality
of the permanganate solution.
(15)
CARBON DIOXIDE COMBINING CAPACITY OF PLASMAl
(Alkali Reserve)
A known quantity of plasma which has been equilibrated with air having a carbon
dioxide tension approximating that of normal arterial blood, is acidified within
a Van Slyke carbon dioxide apparatus, the carbon dioxide liberated by means of a
partial vacuum, and its volume meas'ured under atmospheric pressure.
Separatory Funnel (300 ml.) with small bottle containing glass beads.
Van Slyke Carbon Dioxide Apparatus.
Drawing the Blood. The blood is drawn without stasis from an arm vein
dir.ectly into a centrifuge tube containing 2 m!. of mineral oil and enough
powdered potassium oxalate to make about 0.5% solution in the amount of
blood drawn. The tube should be filled with blood and stoppered or the
oil replaced by a layer of melted paraffin. When the paraffin has solidified,
1
Van Slyke, D. D. and Cullen, G. E., J. B. C., 30, 289 (1917).
Van Slyke, D. D., J. B. C., 30, 347 (1917).
Van Slyke, D. D. and Stadie, W. C., J. B. C., 49, 1 (1921).
168
the tube is centrifuged and the plasma removed. If freshly drawn blood is
not available, any specimen of plasma or serum may be used to study the
procedure.
Saturating the Serum with Normal Alveolar Air. Transfer about 3 m!. of
plasma to the separatory funnel. Incline the separatory funnel and fill it
with alveolar air or air containing about 5.5% carbon dioxide. First attach
to the outlet tube of the funnel a bottle containing glass beads which will
serve to cool the expired air and condense the excess moisture. Air containing approximately 5.5% carbon dioxide may be obtained as follows.
After a normal inspiration, expire quickly and as completely as possible
through the bottle of glass beads and the separatory funnel. Insert the
stopper in the funnel just before the expiration is finished. After closing
the stopcock, rotate the funnel so that the plasma forms a thin film over the
inside surface. After 2 minutes rotation, fill the funnel again with alveolar
air and repeat. Then a]]ow the funnel to stand upright in order to collect
.
the plasma at the bottom.
The Determination of the Carbon Dioxide Content of the Plasma. A Van
Slyke apparatus is used for the determination. Before the analysis is
begun see that the stopcocks are we]] greased and do not leak and that the
air has been removed from the mercury in the rubber tubing. To test the
apparatus: With the upper stopcock closed, lower the mercury leveling
bulb until the apparatus is empty thereby forming a considerable vacuum.
A1Iow the mercury to rise taking care that it does not rise too rapidly in the
narrow tube at the upper part of the apparatus. If no air has entered a
metallic click may be heard when the mercury is allowed to gently strike
the upper stopcock.
Carefully rinse the cup of the apparatus and place in it 1 m!. of distilled
water. By means of a Mohr pipette introduce 1 m!. of plasma under the
layer of water. Lower the mercury leveling bulb and allow the liquid in
the cup to run into the apparatus taking care that no air enters. Before
a1l the liquid is run tn, add, ~ drop of caprylic alcohol and see that it is run
into the capillary. Place about 1 ml. of I N lactic acid in the cup and run
in enough to bring the total volume. in the machine up to 2.5 ml. Follow
. this with a globule of mercury which should fill the capillary at the stopcock
to make a more effective seal.
Lower the mercury bulb to evacuate the apparatus and, when the mercnry level reaches the 50 ml. mark, close the lower stopcock. Remove the
apparatus from the stand and invert about 15 times to insure complete
reaction. Return the apparatus to the stand, and, with the leveling bulb
lowered, open the lower stopcock and drain the liquid into the lower right
hand chamber. Reverse the stopcock and allow the mercury to flow bark
170
into the upper chamber. The bulb is raised cautiously until the mercury
levels inside and out are the same, taking care to obtain the minimum of
oscillation after the mercury reaches the calibrated portion of the tube.
Read immediately the volume of gas above the water layer.
Calculation. Part of the gas which is measured is air and carbon dioxide
extracted from the 2.5 ml. of liquid, and part of the carbon dioxide remains
dissolved in the liquid. Some carbon dioxide is redissolved in the small
amount of liquid remaining above the mercury when the reading is made.
Other factors involved in the calculation are described in the references
given. Roughly, about 0.10 ml. should be subtracted from the reading to
obtain the carbon dioxide bound as bicarbonate in the plasma. A more
exact calculation may be made by multiplying the observed volume of gas
by the barometric reading, dividing by 760 and referring the result to the
accompanying table. The temperatures given at the top of the columns
refer to the room temperature at which equilibration and determination are
carried out. Multiply the result by 1.02 to correct for the carbon dioxide
reabsorbed by the liquid in the apparatus when the reading is made. This
final figure represents ml. carbon dioxide (at 0° and 760 mm.) bound as
bicarbonate in 100 ml. of plasma when the plasma is saturated with carbon
dioxide at 20° and 41 mm. Hg. The formulas used for calculating the
data given in the table may be found in Van Slyke's article (J. B. C., 30,
347 (1917)).
(16)
CARBON DIOXIDE CONTENT OF PLASMA OR WHOLE
BLOODl
It is more accurate and sometimes more convenient to analyze whole
blood or plasma as it is removed from the animal, i.e., without loss of carbon
dioxide. Blood is collected under oil to prevent loss of carbon dioxide.
It may be stirred gently to defibrinate or to mix with oxalate. 1 ml. portions may be used for analysis. If plasma is desired, most of the oil is
removed and replaced with paraffin warmed so that it is just melted.
When the paraffin has solidified the solution may be centrifuged. A hole
is made in the paraffin with a warm cork borer and the plasma removed
with a pipette.
Analysis is carried out on the whole blood or plasma without loss of
carbon dioxide and without equilibrating. The procedure is as described
under carbon dioxide combining capacity. Whole blood gives off varying
amounts of oxygen so that a correction cannot be applied. In this case
the carbon dioxide is absorbed by sodium hydroxide. About 1 ml. of
sodium hydroxide (desk reagent) is placed in the upper cup of the Van
1
Van Slyke, D. D. and Stadie, W. C., J. B. C., 49,1 (1921).
172
TABLE 1
Table jor Calculation oj Carbon Dioxide Combining Capacity of Plasma
Ob,erved
vol. gas
B
X 760
Ml. of C02, reduced to 0° ,760 mrn.,
Observed
vol. gas
bound as bicarbonate by 100 mI.
of plasma
15'
B
X 760
15'
20'
25'
30'
47.7
48.1
48.5
48.6
1
2
3
4
5
6
7
8
9
0.70
48.7
49.7
50.7
51.0
52.6
53.6
54.5
55.5
56.5
57.4
49.0
50.0
51.0
51.9
52.8
53.S
54.8
55.7
56.7
57.6
49.4
50.4
51.3
52.2
53.2
54.1
55.1
56.0
57.0
57.9
49.5
50.4
51.4
52.3
53.2
54.1
55.1
56.0
56.9
57.9
1
2
3
4
5
6
7
8
58.6
59.5
60.5
61.4
62.4
63.3
64.3
65.3
66.2
67.2
58.9
59.8
60.7
61.7
62.6
63.6
64.5
65.5
66.4
67.3
58.8
59.7
60.6
61.6
62.5
63.4
64.3
65.3
66.2
67.1
68.1
69.1
70.0
71.0
72.0
72.9
73.9
74.S
75.8
76.7
68.3
60.2
70.2
71.1
72.1
73.0
74.0
68.0
69.0
69.9
70.8
71.8
72.7
73.6
74.5
75.4
76.4
25'
30'
9.9
10.7
l1.,S
0.60
10.9
11.8
12.8
13.7
14.7
15.7
16.6
17.6
18.5
19.5
11.'7
12.6
13.6
14.5
15.5
10.4
17.4
18.3
19.2
20.2
12.6
13.5
14.3
15.2
16.1
17.0
IS.0
18.9
19.8
20.8
20'
- - - --- - - - --0.20
9.1
I
10.1
11.0
12.0
13.0
13.9
14.9
15.9
16.8
17.S
18.8
2
3
4
5
0
7
8
9
0.30
---------
MI. of CO" reduced to 0',760 mm.,
bound as bicarbonate by 100 ml.
of plasma
--- --- -----------
--------I
Z
3
4
5
6
7
8
9
0.40
I
2
3
4
5
6
7
8
9
0.50
I
2
3
4
5
6
7
8
9
0.60
10.7
20.7
21. 7
22.6
23.6
24.6
25.5
26.5
27.5
28.4
20.4
2l.4
22.3
23.3
24.2
25.2
26.2
27.1
28.1
29.0
21.1
22.1
23.0
24.0
24.9
25.8
26.8
27.7
28.7
29.6
21.7
22.6
23.5
24.5
25.4
26.3
27.3
28.2
29.1
30.0
0.80
58.4
59.4
60.3
61.3
62.3
63.2
64.2
65.2
66.1
67.1
29.4
30.3
31.3
32.3
33.2
34.2
35.2
36.1
37.1
38.1
30.0
30.9
31.9
32.8
33.8
34.7
35.7
36.6
37.6
38.5
30.5
31.5
32.4
33.4
34.3
35.3
36.2
37.2
38.1
39.0
31.0
31.9
32.8
33.8
34.7
35.6
36.5
37.4
38.4
39.3
1
2
3
4
5
6
7
8.
9
0.90
68.1
60.0
70.0
71.0
71.9
72.9
73.9
74.S
75.8
76.8
39.5
40.4
41.4
42.4
43.3
44.3
45.3
46.2
47.1 48.1
40.0
40.9
41.9
42.8
43.8
44.7
45.7
46.6
47.5
48.5
40.3
41.2
42.1
43.0
43.9
44.9
45.8
46.7
47.6
48.6
1
2
3
4
5
6
7
8
9
1.00
\)
--- --- ------
39.1
40.0
41.0
42.0
42.9
43,9
44.9
45.8
46.8
47.7
------
173
---------
- - - - - - ----
7~.9
75.8
76.8
-------- ,
77.7
77.7
77.3
77.8
78.2
78.6
78.7
78.7
79.6
79.2
79.7
79.6
80.6
80.1
80.7
80.5
81.5
81.5
81.0
81.6
82.6
82.5
82.4
82.0
83.6
83.4
83.4
82.9
84.3
83.8
84.5
84.4
85.3
85.2
84.8
85.5
86.2
86.2
85.7
86.5
Slyke apparatus after a reading is made of the carbon dioxide and other
gases obtained from blood. With the mercury leveling bulb lowered
somewhat, the upper stopcock is opened cautiously and a few drops of
sodium hydroxide allowed to enter the apparatus. Portions of sodium
hydroxide are allowed to enter until there is no more diminution in gas
volume. A second reading of the gas mixture gives the volume of air and
Factors for Calculating Carbon Dioxide Content Determined by Volumetric Apparatu8
with Blood or Plasma
Temperature
Air in e.'ttracted gases from
plasma and water.
Subtract from observed air
CO. volume if CO, and
air are mea!!.ured together
+
Factors by which milliliters of CO: extracted from
1 rot. pJasma or blood are multiplied to give
V olwne per cen t CO2
·C.
mi.
15
0.048
16
17
18
19
48
48
47
47
99.5
98.9
98.3
97.8
20
21
22
23
24
46
46
45
45
45
97.2
96.6
96.0
95.4
94.8
25
26
27
28
29
44
44
44
43
43
94.2
93.6
93.1
92.4
91.8
30
31
32
33
34
43·
43
42
42
.42
91.2
90.6
90.0
89.4
88.8
"
B
100.2 X 760
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
Millimoles CO, per liter
B
44.9 X 760
44.7
"
44.4
"
44.2
"
43.9
"
43.7
43.4
43.1
42.9
42.6
42.3
42.1
41.8
41.5
41.3
41.0
40.7
40.4
40.2
39.9
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
bxygen. This, subtracted from the 1st reading, gives the carbon dioxide
obtained from the sample. Calculation of the carbon dioxide content of
whole blood or plasma may be made by reference to .the following table.
If the carbon dioxide has been absorbed by the sodium hydroxide, the 3rd
and 4th columns are used. If the carbon dioxide has not been absorbed
(\vhcn plasma or serum is analyzed), the correction for absorbed air given
in column 2 must also be used.
.
174
(17)
CHOLESTEROL IN WHOLE BLOOD, PLASMA OR SERUM
(Klett Modification of Method of Bloor, Pelkan and Allen 1)
The lipids are extracted from blood by means of an alcohol-ether mixture:
After evaporation of a portion of the solution, the residue is dissolved in chloroform and the total cholesterol allowed to react with acetic anhydride in the presence of sulfuric acid. A pink color is produced which is compared with that produced by a standard solution.
Alcohol-ether Mixture. Mix 1 part of ether with 3 parts of 95% ethyl
alcohol.
Chloroform must be anhydrous and free from alcohol. It should be redistilled and dried.
Acetic Anhydride.
Slock Standard Cholesterol Solution. Prepare 100 ml. of a solution containing 160 mg. of cholesterol in chloroform.
Working Standard Cholesterol Solution. 1 ml. of the stock standard solution of cholesterol is transferred to a 25 ml. volumetric flask and made up
to volume with chloroform. This solution contains .32 mg. of cholesterol
in 5 ml. It will keep well for a few days in the refrigerator.
Filter. Use filter 42 (blue).
Procedure for photoelectric photometer. Place about 20 ml. of alcoholether mixture in a 25 ml. glass-stoppered flask. Add slowly and with rotation of the flask 0.5 ml. of the whole blood, plasma, or serum to be analyzed.
The resulting precipitate should be finely divided and not lumpy. Immerse
the flask in boiling water, shaking gently to prevent bumping, until the
contents of the flask have boiled for a few seconds. Remove the flask and
cool to room temperature. Make up to the 25 fil. mark with alcoholether mixture, stopper, and shake well. Pour onto a dry fat-free filter
paper, collecting the filtrate in a dry flask.
Transfer 5.0 ml. of the filtrate to a small dry beaker and evJporate to
dryness on a water bath. Add' a 1 ml. portion of anhydrous chloroform to
"the dry residue, bring to boiling momentarily on the water bath, and then
carefully pour off the liquid into a clean dry photometertube. Repeat this
process with two more 1 ml.- portions of chloroform, in each case transfer'ring the extract carefully to the photometer tube. Allow the combined
extracts in the photometer tube to cool to room temperature and make up
to a final volume of 5.0 ml. with anhydrous chloroform.
Before proceeding with the analysis, prepare a blank tube as follows:
place 5.0 ml. of anhydrous chloroform in a clean dry photometer tube and
ad~ 1.0 ml. of acetic anhydride (from a burette).
Mix with a dry glass
1
Bioor, W. R., Pelkan, K. F., and Allen, D. M., J. B. C., 52; 191 (1922).
176
rod, remove the rod, place the tube in the photometer and adjust the instrument to its 0 reading.
Now add to the contents of the "unknown" tube l.0 mL of acetic 'anhydride, mix with a dry glass rod, remove the rod, and read in the photometer
against the blank tube at O. The reading of the unknown at this time is
called the RI reading, and is to correct for any color present before the
cholesterol color is developed. After obtaining this reading, put the stirring rod back in the photometer tube and set the tube aside until the
standard tube has been prepared.
To prepare the standard, place 5.0 m!. of the working standard cholesterol
solution in a clean dry photometer tube and add l.0 m!. of acetic anhydride.
Insert a glass rod and mix.
Now to both standard and unknown add 0.1 m!. of concentrated sulfuric
acid. Mix well with the glass rods and then place aside in a dark place for
15 minntes. At the end of this time remove the rods and read the tubes in
the photometer (within the next 15-20 minutes) against the blank tube set'
at o. The reading of the unknown tube at this time is called the R2
reading. The cholesterol content of the unknown is obtained from the
standard and unknown readings as follows:
mg. cholest~rol in 5.0 ml. standard
X (R. -
reading of standard
R,) = mg. cholesterol in 5.0 m!. of
alcohol-ether filtrate.
Since the 5.0 mL portion of alcohol-ether filtrate taken for analysis represents 0.1 m!. of the original sample, the value obtained by the equation
above is multiplied by 1000 to give the cholesterol content of the original
sample in milligrams per cent.
(18)
VAN DEN BERGH DETERMINATION OF
SERUM BILIRUBIN
(Method of Malloy
an~
Evelyn l )
A pink color deyelops ill blood serum on adding diazo benzene-sulfonic acid.
The qualitative Van den Bergh test is called a "direct reaction". It is carried out
as follows: Pipette 1 m!. of serum into a small test tube, add 2 m!. of water, 0.5 m!.
of diazo reagent and mix.
The reaction may be one of the following: A. Negative. Most sera with an average
concentration of bilirubin. B. Positive. If positive, it may be (1) Immediate direct
(the color change to red begins within 30 seconds and reaches a maximum within a
minute or two), (2) Delayed direct (the color change begins after 30 seconds and
gradually deepens. This may require many minutes), or (3) Biphasic direct (intermediate between the above). In the qualitative test (direct reaction), dilution is
carried out with water. In the quantitative test (indirect reaction), dilution is carried out with alcohol.
'l\fethod of Malloy and Evelyn, J. B. C., 119, 491 (1937).
]78
Absolute Methyl Alcohol.
Solution A. 1.0 g. of sulfanilic acid dissolved in 15 ml. of concentrated
HCI and diluted to 1 liter with water.
Solution B. 0.5 per cent sodium nitrite. This solution should be made
up fresh every day.
Diazo Reagent. To 10 ml. of Solution A in a 25 ml. graduated cylinder,
add 0.3 ml. of Solution B.
Diazo Blank Solution. 15 ml. conc. HCI are diluted to 1 liter with distilled water.
Bilirubin Stock Standard. Dissolve 40 mg. of pure bilirubin in chloroform and dilute to 100 ml. with chloroform.
Bilimbin Working Standard. 1 ml. of the stock standard is transferred
to a 100 Il).l. volumetric flask and diluted to the mark with methyl alcohol.
This solution contains 0.02 mg. of bilirubin in 5 ml.
The Determination. Indirect -Reaction. Three photometer tubes are
prepared as follows; Tube 1, blank, 5 ml. of methyl alcohol and 1 ml. of
diazo blank solution; Tube 2, unknown sample, 5 ml. of methyl alcohol and
1 ml. of diazo reagent; Tube 3, standard, 5 ml. of bilirubin working standard
and 1 ml. of diazo reagent.
•
1 ml. of serum (or plasma) is diluted to 10 ml. with distilled water. 4 ml.
of the diluted serum (or plasma) are added to each of tubes 1 and 2. 4 ml.
of distilled water are added to tube 3 .. The contents of each are mixed by
gentle inversion treating the tubes as uniformly as possible. Allow the
tubes to stand 30 minutes for maximum color development. Remove any
bubbles by gentle tilting or rotation of the tubes. Use the tube containing
the diazo blank solution for setting the photometer to its zero reading and
then read the unknown and the standard in the photometer. The standard as used is equivalent to 5 mg. per cent of serum bilirubin. The method
of calculating the results is given in the general description of the photoelectric method (page 137).
(19)
ACID AND ALKALINE PHOSPHATASE IN BLOOD SERUM
(Method of Shinowara, Jones and Reinhart)l
Beta glycerophosphate is digested with blood serum and the liberated phosphate
is determined photometrically. "Acid phosphatase" in serum hydrolyses glycerophosphate optimally at pH 5.0; the "alkaline phosphatase" does the same at pH 9.3.
A unit of phosphatase is defined as the number of mg. of inorganic phosphate liberated from glycerophosphate by 100 m!. of serum at 37° at the pH mentioned. Inhibition of the enzyme occurs if the inorganic phosphate is higher than 60 mg. per
100 m!. of serum.
1
Shinowara, Jones and Reinhart, J. B. C., 142, 921, 19{2.
180
Buffered Glycerophosphate Stock Solution. Measure 3 m!. of petroleum
ether and 80 m!. of distilled water into a 100 ml. volumetric flask. Add
1 gram of sodium beta glycerophosphate and 0.85 gram sodium diethyl
barbiturate. Dissolve, dilute to the mark with distilled water and mix.
Acid Phosphate Substrate. In a 100 ml. volumetric flask place 3 m!.
petroleum ether, 50 m!. of the buffered glycerophosphate stock solution and
5 m!. of 1 N acetic acid and dilute to the mark with distilled water. The
pH should be 5.0. Keep in the refrigerator.
Stock Standard Phosphate Solution. (See p. 166.)
.
Standard Phosphate Solution. Dilute 8.32 ml. of the Stock Standard
Solution to 100 ml. in a volumetric flask. 6 m!. of this solution contains
0.04 mg. of phosphorus.
Procedure for acid phosphatase.
Unknown incUbated sample. Place 9 ml. of acid phosphate substrate
solution in a stoppered test tube and allow to stand in a water bath at 37°
until the solution reaches that temperature (Test with a thermometer a
similar amount of water in a test tube).. Add 1 m!. of serum, mix and allow
to digest at 37° for 1 hour. Remove and add 2 ml. of 30% trichloracetic
acid. Stopper, shake, allow to stand a few minutes, and filter through an
ashless filter paper.
Control Sample. 'While the above digestion is being carried out prepare
a control. Place 9 ml. of acid phosphate substrate in a test tube, add 2 ml.
of 30% trichloracetic acid and then, slowly with mixing, 1 m!. of serum.
Stopper, shake, allow to stand a few minutes and filter through an ashless
filter paper.
Transfer 6 m!. of each filtrate respectively to two dry photometer tubes.
Add 2 m!. of molybdate-sulfuric reagent, 1 ml. of reducing solution and
1 m!. of water, mixing after each addition. Allow to stand 15 minutes
and read in the photoelectric photometer against the blank tube at O. Use
filter 66 (red).
Standard. Transfer 6 m!. of ,standard phosphate solution to a dry photometer tube, add,2 m!. of molybdate-sulfuric reagent, 1 m!. of reducing
solution and 1 m!. of water, mixing after each addition. Allow to stand
15 minutes and read against the blank tube at O.
Blank tube. Place 5 ml. of 2% trichloracetic acid, 2 m!. of molybdate'sulfuric reagent, 1 ml. of reducing solution and 2 m!. of water in a test tube,
mixing after each addition. Allow to stand 15 minutes and use this solution for the 0 reading.
Calculation:
~
X 0.04 X 2 X 100 = P
U = reading of unknown, S = reading of standard, P
mg. inorganic
phosphate-P per 100 m!. of serum. Subtract the mg. of P of the incubated
182
sample from the mg. of P of the control sample to obtain the units of acid
phosphatase per 100 ml. of serum.
The mg. of P0 4-P of the control sample represents the inorganic phosphate of the serum and may be thus reported.
Alkaline phosphate substrate. Place 3 ml. of petroleum ether, 50 ml. of
the buffered glycerophosphate stock solution and 2.8 ml. of 0.1 N sodium
hydroxide in a 100 m!. volumetric flask, dilute to the mark with distilled
water and mix. The pH should be 10.9. Adjust if it varies more than
0.1 pH. The substrate should be prepared daily.
Procedure for alkaline phosphatase. The procedure is identical with that
used for the acid phosphatase determination except that the serum is
allowed to act upon an alkaline substrate of glycerophosphate. Proceed
as described above, but use the alkaline phosphate solution instead of the
acid. The solution which is digested will have a pH of about 9.3.
(20)
THE DETERMINATION OF HEMOGLOBIN AS Hb0 2
Dilute ammonia solution. Dilute 4 ml. of concentrated ammonium hydroxide solution to 1 liter with distilled water.
Filter. Use filter 54 (green).
The Determination. Place 5.0 ml. of dilute ammonia solution in a photometer tube. Collect 0.02 ml. (20 c. mm.) of blood in a pipette calibrated
to contain that amount. Remove excess blood from the outside of the
pipette, place the tip of the pipette below the surface of the dilute ammonia
solution and gently blow the contents of the pipette into the solution in the
photometer tube. Rinse out the pipette several times by dra.ving up and
blowing back portions of the liquid in the tube, miXing well ,vith the tip
of the pipette at the same time. Remove the pipette, and allow the solution to stand for a few minutes. Read in the photoelectric photometer
against a distilled water O.
Calculation.
reading of unknown X oxyhemoglobin factor = gm. per cent hemoglobin.
The Hb0 2 factor, determined spectrophotometrically = 81.9.
184
BONE
OSSEIN
(1) Pour 20 ml. of 2 N hydrochloric acid on a gram or two of small
fragments of bone. Note the immediate evolution of a gas (?). On standing over night the mineral constituents will be dissolved and the residue
becomes flexible. Pour off the acid solution, add 50 ml. of water and allow
to stand half an hour. Pour off the water, wash the ossein in turn with 0.1
M s09ium carbonatc and with watcr. Place the material in a test tube
with a small quantity of water and digest in a boiling water bath for 10
minutes. Filter hot into a test tube and allow the tube to stand in cold
water. Examine.
GELATIN
(2) Warm about 5 g. of commercial gelatin on the water bath with
about 50 ml. of water until solution occurs. Cool by immersing in cold
water. Examine. 'Varm again. Remove 10 ml. of the solution for E~p. 3
and dilute the remainder with 20 ml. of water for Exp. 4 and 5.
(3) Acidify with hydrochloric acid and boil for a few minutes. Cool.
Does the solution gelatinize? The same result can be accomplished by
prolonged boiling without the addition of acid.
(4) Test the gelatin solution with the protein color reactions. Explain
the results.
(5) Test for unoxidized sulfur. See p. 14.
MINERAL CONSTITUENTS
(6) Incinerate about 5 grams of bone fragments (hood). Boil out the
residue with 5 ml. of dilute nitric acid, filter and use the solution for the
following tests.
(7) Phosphate. Treat about one-fourth of the nitric acid solution with
a· gram of ammonium nitrate and 25 m!. of 3% ammonium molybdate.
Heat to boiling, allow the yellow precipitate to settle, filter and convert the
yellow precipitate into crystalline magnesium ammonium phosphate.
See p. 8.
(8) Chloride. Test a few drops of the nitric acid solution for chloride
with silver nitrate.
(9) Iron. (Read "Separation of Phosphoric Acid, Calcium and Mag186
nesium" in chapter "Inorganic Constituents.") Make the remamder of
the nitric acid solution faintly alkaline with ammonia. A precipitate is
formed. Acidify with acetic acid. The precipitate dissolves; all but a
trace of iron phosphate. Filter, use the filtrate for calcium and magnesium
(see below). Pour on the filter a few drops of acidified potassium ferrocyanide.
(10) Calcium. Treat the filtrate from iron phosphate with one gram
of sodium acetate, and an excess of ferric chloride. Boil for a few minutes
and filter. The filtrate contains neither phosphoric acid nor iron. While
still hot add ammonium oxalate as long as a precipitate is formed. Allow
to stand until the precipitate is well separated and filter. Examine the
crystalline precipitate under the microscope.
(11) Magnesium. Make the filtrate from calcium oxalate strongly
alkaline with ammonia, heat to boiling and treat the hot fluid with sodium
phosphate a drop at a time, shaking after each addition.
(12)
QUANTITATIVE ESTIMATION OF CALCIUM AND
PHOSPHATE IN BONE ASH
Weigh accurately 250-300 mgs. of ashed bone in a small dry beaker.
Add 25 m!. of 2 N HCI and break up the ash with a glass rod until dissolved. Using the same rod, transfer the solution quantitatively to a
100 m!. volumetric flask employing distilled water for rinsing. Make up
to volume and mix thoroughly (Solution A). Use 1 ml. portions of this
solution for the calcium determination (see directions below).
For the determination of phosphate, pipette 10 ml. of Solution A into
another 100 ml. volumetric flask and make up to volume (Solution B).
Phosphate (Fiske-Subbarow Method (p. 172). Carry out the determination as described in the last paragraph (p. 174) using 1 ml. of Solution B
aJ).d 4 m!. of water in place of the 5 m!. of the filtrate called for. The
standard solution is treated just as described in the paragraph.
Calcium. Heat your water bath and have it ready for later use.
Pipette 1 m!. of Solution A into a small beaker and add 10 m!. of 4%
ammonium oxalate and 4-5 drops of thymol blue. The solution should be
clear and pink in color. If there is any turbidity add a few drops of 2 N
HCI to clear the solution. Heat to boiling over a free flame and add 2 N
NH 40H drop by drop until the indicator turns yellow. Put the beaker
immediately on the boiling water bath and heat for 30 minutes. Allow
the solution to cool slowly (10-15 minutes) and filter quantitatively using
a glass rod for the transfer. For this filtration use a specially hardened
filt~r paper (store-room) and your smallest funnel. The filter paper should
not be fluted but folded in the usual manner, carefully fitted to the funnel
and moistened with distilled water. The filtrate must be water clear!
188
After the filtration is completed, rinse the beaker with dilute NH 40H
(15 mI. 2 N H 40H diluted roughly to 100 mI.), washing 5 times successively with 10 ml. of dilute NH 40H. Rotate the funnel for each washing,
taking care to wash the upper rim of the filter paper. NOTE. Each
filtration must be complete before the next washings are added in order to
assure complete removal of ammonium oxaJate. Discard the clear filtrates.
Place a clean Erlenmeyer flask under the funnel and dissolve the precipitate by filling the funnel to the upper rim of the filter paper with hot
1 N H 2S0 4 • Do this three times. Heat the clear filtrate to boiling, remove
from the flame and titrate the hot solution with standard KMn04 solution
(O.OlN) using your burette. The first appearance of a pink color which
persists for 1 minute is taken as the end point. For standardization of the
KMn04 solution .see page 168.
Calculate the mgs. of Ca and P in the sample of ashed bone which was
weighed out. Hand in a written report giving per cent of Ca and P as
well as the molar ratio of Ca to P0 4 in your sample. Does this ratio agree
with the values reported for bone?
190
MUSCLE
Of the characteristic organic constituents of muscle, creatine, hypoxanthine and
sarcolactic acid are the most easily isolated. These three substances are treated
in the following pages.
CREATINE
(1) Place 600 g. of finely ground lean beef in a saucepan with 500 ml.
of distilled water and heat the well mixed material on a water bath for half
an hour at 30°, stirring occasionally with a thermometer which is·kept in the
mixture during the extraction. The water takes up inorganic salts, coagulable protein, coloring matter and various extractives among which are
creatine, sarcolactic acid and hypoxanthine. The temperature is kept low
during the extraction to prevent the coagulation of proteins which woukl
shut in some of the creatine.
Strain the aqueous extract through linen, press as dry as possible and
make a second extraction of the residue with 500 ml. of water, but heating
for only 10 minutes. Unite the two extracts.
Test the aqueous extract with litmus. It should be faintly but unmistakably acid. If this is not the case, make it acid with a few drops of 2 N
acetic acid. Too much acetic acid used at this point will entirely vitiate the
results: it is better not to use any unless it is found absolutely necessary.
Place the solution in a saucepan and bring it to brisk boiling but do not
continue to boil. The proteins are coagulated and drag down most of the
coloring matter, leaving an almost colorless and perfectly transparent
interstitial fluid. Filter the hot solution through the smallest convenient
fluted filter. Wash thoroughly all vessels and filter paper and make the
experiment as nearly quantitative as possible. Precipitate the phosphates,
chlorides and sulfates as well as any possible trace of protei:n by the careful
addition of basic lead acetate to the warm fluid, avoiding any great excess
. of the reagent. About 20-25 ml. of basic lead acetate will be required, but
the fluid must be tested to be sure that an excess has been added. Allow
the precipitate to subside, and bring together on a filter paper a drop of
the supernatant fluid and a drop of sodium sulfide (hood). A brown color
at the contact shows an excess of lead. The solution is filtered through a
small filter into a saucepan (best without standing over night) and heated
to boiling. CompJetely remove the lead from the solution with hydrogen
sulfide. (Read carefully note on the use of hydrogen sulfide, p. 7.) Test
the completeness of precipitation by allowing a drop of the fluid and a drop
192
of sodium sulfide to flow together on a filter paper. When the precipitation
of lead sulfide is complete, boil the solution violently until all the hydrogen
sulfide is expelled, take the material back to your desk and filter through the
smallest convenient filter. Evaporate the tiltrate on a water bath to about
15 m!. and allow to stand in a cool place. Creatine crystals will be found
deposited in a limpid pale yellow mother liquor. If the proteins were not
originally removed with sufficient care this residue will consist of a dark
red viscous paste from which it is almost impossible to remove the few
creatine crystals which may be present.
Decant the pale yellow mother liquor (give the liquid to some one who is
about to prepare hypoxanthine), wash the crystals by decantation with a
very small amount of cold water and then in turn with 50%, 75% and
absolute alcohol and allow to dry in the air.
The crystals of creatine may be purified if it is desired by recrystallization
from 7 parts of boiling water, decolorizing the hot solution with a little
animal charcoal.
(2) Conversion of Creatine into Creatinine. Dissolve 200 mg. of creatine in 400 m!. of normal hydrochloric acid and evaporate to dryness in a
porcelain dish on a water bath. Moisten the residue with alcohol and again
evaporate to dryness. Use the final residue of creatinIne for the following
tests.!
(3) Tests for Creatinine. These tests are described in the section
"Creatinine" chapter "Urine." Use a trace of creatinine residue for \VeyI's
sodium nitroprusside reaction.
(4) Dissolve the remainder of the creatinine in a. very small amount of
water, add a gram of sodium acetate in an 'equal volume of alcohol and
treat the solution with about one-third of its volume of a strong solution
of zinc chloride in alcohol. Crystals of creatinine zinc chloride will be
deposited immediately or on standing.
(6) Use these crystals'for a picric acid test.
HYPOXANTHINE
In order to separate purine derivatives including hypoxanthine from nonpurine
substances advantage is taken of the fact that the purines form compounds with
cuprOUB oxide of the general type R·Cu 20 which are insoluble in water, amI silver
compounds of the type R·Ag 20 which are insoluble in ammonia. The following
isolation of hypoxanthine from muscle serves as a good exercise in the treatment
of purine derivatives.
1 This is a peculiar reaction.
Heating with hydrochloric acid generally causes
hydrolysis; but the conversion of creatine into creatinin.e is dehydrolysis.
194
(6) Separation of Hypoxanthine from Non-purine Compounds. Secure
as many· specimens as possible of the mother liquor obtained in the preparation of creatine from muscle. These more or less viscous fluids contain
hypoxanthine. Dilute the united fluids with about ten parts of hot water
and treat at the boiling point with cuprous-purine reagent1 as long as a
precipitate is formed. Filter off the hypoxanthine cuprous oxide, wash with
boiling hot water, pierce the filter and spurt into a clean flask with as little
hot water as possible. Decompose the precipitate by the addition of
sodium sulfide, a few drops at a time, testing frequently to find when an
excess of sodium sulfide has been added by allowing a drop of the fluid and
a drop of lead acetate to run together on a piece of filter paper. When the
formation of a brown contact shows that enough of the sulfide has been
added, treat the boiling hot fluid with sulfuric acid a few drops at a time2
until acid to litmus when the emulsion breaks throwing down the copper
sulfide and leaving a perfectly transparent interstitial liquid. Filter off
the solution which contains hypoxanthine sulfate, allow to cool and ma!;:e
markedly alkaline with ammonia.
Treat the hypoxanthine solution with ammoniacal silver nitrateS as long
as a precipitate is formed. (Test a little of the fluid filtered from the
gelatinous precipitate.) Filter off the gelatinous precipitate, wash with
cold water, pierce the filter and spurt into a clean flask with as little hot
water as possible. Heat to boiling, add hydrochloric acid a drop at a time
until silver chloride is formed and settles sharply. Filter off the solution
of hypoxanthine chloride and evaporate to dryness on the water bath, carefully at the end and stir continually to avoid overheating.
Separation of Hypoxanthine from Xanthine. The procedure described
is general for purine compounds and the finai residue should be comparatively free from organic substances other than purines. It now remains to
separate the purines from one another. In the present instance the procedure is simple because the residue is composed principally of hypoxanthine
chloride with only a small amount of xanthine.
Digest the residue 'with a little water at 40°. Hypoxanthine chloride
goes into solution and the xanthine (all but a mere trace) remains undissolved. Filter and decolorize the solution with a little animal charcoal.
1 Treat 25 m!. of 0.2 M copper sulfate with 2 N ammonia until the precipitate
first formed just redissolves and decolorize the purple solution at the boiling point
with sodium bisulfite. Prepare the reagent extempore.
2 When experiments involve uric acid or xanthine, acetic acid must be used here
instead of sulfuric.
a To a solution of silver nitrate add ammonia until the brown silver oxide first,
formed redissolves.
196
The last trace of xanthine is removed with the coloring matter. Neutralize
the hypoxanthine solution with ammonia, evaporate carefully to dryness
and wash out the ammonium chloride with a little cold water. The residue
is free hypoxanthine.
(7) Hypoxanthine Nitrate. Dissolve the hypoxanthine in 10 parts' of
hot 1 N nitric acid and allow to cool. Hypoxanthine nitrate is deposited
in characteristic crystals with curved edges; the so called "whetstone"
crystals.
(8) The Xanthine Color Test. Place a speck of hypoxanthine nitrate
on a clean porcelain surface, moisten with a drop of dilute nitric acid and
evaporate most carefully to dryness over a small free flame.
a. With hypoxanthine the spot is colorless.
b. With xanthine the spot is bright canary yellow without a suggestion
of red.
c. 'Vith uric acid the spot is pink or red depending upon the amount of
substance used.
When cold, touch the spot with a drop of sodium hydroxide on a rod.
a. With hypoxanthine the spot remains colorless.
b. With xanthine the yellow spot becomes blood red.
c. With uric acid the red or pink spot becomes violet.
(9) Cuprous and Silver Compounds. Solutions of hypoxanthine naturally form insoluble compounds with copper sulfate and sodium bisulfite
at the boiling point and with ammoniacal silver nitrate in the cold.
SARCOLACTIC ACID
The lactic acid formed from cane sugar or lactose by bacterial fermentation is
a racemic form. It is called fermentation lactic acid, and has been separated into
its two optically active constituents one of which is id€'ntical with the lactic acid
obtainable from muscle and called sarcolactic' acid,
Both lactic acids are colorless syrupy liquids without striking physical properties
but they respond to Uffelmann's test (see "Fermentation of Cane Sugar" chapter
on "Milk"), form crystalline zinc salts, and arc soluble in water, ether and alcohol.
These last properties are made use of in separating the lactic acids from other substances.
"
(10) Secure from 5 to 10 specimens of the !llother liquor obtained in the
preparation of creatine. Mix the united fluids with four parts of alcohol,
. remove the aqueous-alcohol fluid, extract the insoluble residue with several
fresh portions of alcohol and evaporate the alcohol from the united extracts.
Take up the residue in about 50 m!. of 1 N sulfuric acid, place in a separatory funnel and shake out with three successive 30 ml. portions of ether
(caution when using ether). Evaporate the ether and take out a drop of
the oily residue with a stirring rod for Uffelmann's ~est.
198
(11) UffeImann's Test. Treat 2 ml. of Uffehnann's reagentl with a
drop of lactic acid. Use the remainder of the lactic acid for the preparation
of the zinc salt.
(12) Zinc Salt. Take up the lactic acid in hot water and boil with an
excess of freshly precipitated zinc carbonate (must be washed free from alkaline carbonate). Filter off the fluid, evaporate to a small volume and allow
to cool. When crystals begin to form stir into the fluid about an equal
amount of alcohol. Filter off the crystals, allow to dry and make a quantitative determination of the water of crystallization with 250 mg. of the salt by
heating to a constant weight at 115° in an air bath. The zinc salt of sarcolactic acid contains two molecules of water of crystallization; that of fermentation lactic acid (d, llactic acid) contains three.
1
See p. 96.
200
THE CELL NUCLEUS 1
The characteristic constituent of the cell nucleus is a substance callcd nucleic
acid. The chemical examination of the nucleic acids is a little tedious and cannot
be undertaken when one's time is limited. However, their more striking features
may be seen fr9m the following experiments upon a commercial preparation of
yeast nucleic acid.
GUANINE
(1) Place 5 g. of yeast nucleic acid in a 200 ml. Erlenmeyer flask, add
100 m!. of boiling hot 2 N sulfuric acid and heat carefully over a small
flame to bring the nucleic acid into solution. During this heating which
lasts only a minute, the contents of the flask should be kept constantly in
motion to avoid burning the undissolved nucleic acid. Close the flask with
a soft cork, bored with one hole into which is inserted a condensing tube,
immerse in a boiling water bath so that the surface of the liquid in the flask
is not below the level of the water in the water bath and allow to heat for
about an hour.
Disconnect the flask and while its contents are still hot, add concentrated
ammonia drop at a time until the fluid is roughly alkaline and then add 5
m!. of concentrated ammonia in excess. Guanine is thus precipitated in
granular form and can be easily filtered off, while all of the other products
including phosphoric acid and adenine remain in the ammoniacal solution.
Allow to stand several hours (best over night), filter off the pale yellow
guanine, wash it well down into the cone of the funnel with cold 0.2 N
ammonia, open the filter and allow to dry on the radiator or in a porcelain
dish on a boiling water bath. (Crude guanine = 580 mg.) Use the ammoniacal filtrate for phosphoric acid and adenine as described below.
Suspend the crude guanine in about 40 ml. of boiling water and as the
fluid boils bring the guanine into solution by the addition of sulfuric acid
drop at a tim~. Only a drop or t.wo of acid need be used in excess. Decolorize the solution with animal charcoal, filter and wash with boiling water
made acid with sulfuric acid. Treat the colorless boiling hot fluid with
concentrated ammonIa as described above but do not add the 5 ml. in
excess. Snow white guanine is precipitated. When the fluid has become
cool, filter, wash and dry.
(2) Make a nitric acid color reaction with a speck of guanine on a white
porcelain surface. The spot is muddy yellow and becomes dull brownish
red when treated with sodium hydroxide.
1
See Walter Jones, Nucleic Acids. 1920, 2d ed., London.
Levene and Bass, Nucleic Acids, 1931.
202
(3) Guanine Chloride. C.HoNoO· HCI· 2H 20. Dissolve the colorless
guanine (455 mg.) in 25 parts of hot 2 N hydrochloric acid and allow to
cool undisturbed. Beautiful slender needles of guanine chloride are deposited. Filter, wash with 0.2 N hydrochloric acid (not with water), open
the filter and allow to dry in the air (not in a desiccator). This is the most
characteristic and useful of the compounds of guanine for analytical purposes.
(4) Guanine Picrate. Make a 1% solution of guanine chloride in 0.2
N hydrochloric acid (20 mI.). From this 1% solution :rp.ake more dilute
solutions, viz.: 0.5%, 0.4%,0.3%,0.2% and 0.1 % diluting in each case with
0.2 N hydrochloric acid. Place about 4 ml. of each solution in a test tube
and to each add 1 mI. of saturated aqueous picric acid. The 0.5% solution
deposits orange needles of guanine picrate in about 30 seconds; the 0.1%
solution in about 15 minutes. The beautiful orange needle clusters of
guanine picrate contrast markedly with the pale yellow needles of adenine
picrate. (See next page.)
PHOSPHORIC ACID
(6) Heat the first ammoniacal filtrate from guanine nearly to boiling
and add magnesia mixture (drop at a time, agitating after the addition of
each drop) until the reagent produces no further precipitate; then add an
equal amount of magnesia mixture for a safe excess. Heavy crystalline
needles of magnesium ammonium phosphate fall sharply to the bottom,
leaving a perfectly clear liquid. After several hours (best over night),
filter off the crystalline precipitate, wash with 1 N ammonia and use the
filtrate for the isolation of adenine (see below).
Dissolve a trace of the magnesium ammonium phosphate in nitric acid,
add a few crystals of ammonium nitrate, treat with ammonium molybdate
and heat. The well known yellow precipitate is formed.
ADENINE
(6) Treat the ammoniacal filtrate from magnesium ammonium phosphate with sulfuric acid to. neutralize part of the ammonia. If necessary
cool, and add a slight excess of silver nitrate in ammonia. l When it becomes impossible to see whether the gelatinous precipitate increases by the
addition of silver solution filter off a few drops of the fluid and test.
Filter the adenine silver compound on a flat filter, wash with cold water,
pierce the filter paper with a sharp glass rod and spurt the gelatinous pre, 1 To 30 ml. of 0.3 M silver nitrate add enough ammonia to redissolve the brown
precipitate which forms at first.
204
cipitate back into the flask in which it was precipitated using the least
possible amount of boiling hot water (15 to 20 mI.).
Heat the suspended gelatinous adenine-silver to the boiling point and
treat with hydrochloric acid drop at a time until silver chloride falls leaving
a clear liquid. Filter hot, wash with hot water using a drop or two of
hydrochloric acid to insure the complete solution of adenine and evaporate
the solution to dryness on the water bath, stirring constantly at the end to
prevent overheating of the adenine chloride. Take up the residue in about
10 ml. of boiling hot water, add 5 or 6 drops hydrochloric acid and treat
the boiling solution with animal charcoal. Filter into a glass dish, cover
and allow to stand over night. Beautiful rosettes of adenine chloTide will
be deposited. Filter on a small paper, wash with a few drops of water,
open the filter and allow to dry in the air.
(7) Adeni,ne Sulfate. (CsH6N6)2 . H 2S04 , 2H 20. Dissolve the adenine
chloride in 10 ml. of boiling hot 2 N sulfuric acid, de colorize with charcoal
if desirable and allow to cool undisturbed. Adenine sulfate will be deposited. Recrystallize from 2 N sulfuric acid, then from water. Make a
nitric acid color test with a trace of adenine sulfate.
(8) Adenine Picrate. Treat a hot 1% solution of adenine chloride or
sulfate with saturated aqueous picric acid. Allow to cool, filter the adenine
picrate, open the filter and allow to dry in the air. Notice the appearance
of the clusters (like matted hair) and their pale yellow color. Make a
melting point determination (280°).
(9)
THE PARTITION OF PHOSPHORUS IN YEAST NUCLEIC
ACID
Place about 7 g. of yeast nucleic acid in a weighing bottle and weigh out
accurately by difference eight portions as follows. Two portions of about
500 mg. each (450 to 550 mg.) are to be used for duplicate estimations of
total phosphorus and a\'e "weighed into" 500 ml. Kjeldah_l flasks. The
other six portions of about 750 mg. each (700 to 800 mg.) are to be used for
the .determination of partial phosphorus and are "weighed into" 100, m!.
Erlenmeyer flasks.
Determination of Total Phosphorus. Digest the nucleic acid with 10
ml. of concentrated sulfuric acid, 7 g. of potassium sulfate and 5 drops of
copper sulfate (as in a Kjeldahl nitrogen determination), and boil gently
until all carbon has been oxidized.
Allow the products to cool, dilute carefully to about 50 mi., add about
10 g. of ammonium nitrate, heat to the boiling point and treat with 240 Illl.
of '3% ammonium molybdate, Heat again just to the boiling point and
allow the yellow precipitate to subside. Filter while the fluid is still hot
206
and wash with acid ammonium nitrate solution. 1 Dissolve the yellow
precipitate in boiling hot water and a little concentrated ammonia, bringing
the solution (which need not exceed 40 m!.) into a 100 m!. flask and precipitate the phosphoric acid very slowly at the boiling point with magnesia
mixture. (See phosphoric acid above.)
Allow the crystalline precipitate of magnesium ammonium phosphate
(MgNH4 P0 4 • 6H 20) to stand over night, filter and wash with 1 N ammonia.
Open the filter and allow to dry over night in the air.
Weigh the filter, dust off the precipitate and weigh the filter paper.
Calculate the weight per gram of nucleic acid.
Determination of Partial Phosphorus. Provide each of the 100 m!.
flasks containing the nucleic acid specimens with a well rolled snugly fitting
cork bored with one hole into which a beveled end condensing tube is inserted. Add to each, 20 m!. of 2 N sulfuric acid per gram of nucleic acid,
warm until the nucleic acid is dissolved, insert the cork and condensing
tube, immerse in a boiling water bath so that the level of the liquid in the
flask is not below the level of the water in the bath and note the time that
the heating is started. The six flasks are to be heated, t hour, 1 hour, 2
hours, 3 hours, 4 hours, and 5 hours respectively.
As the interval expires, remove the flask and treat its hot contents with
concentrated ammonia drop at a time until the material is roughly alkaline
to litmus, and add enough ammonia in excess to make a 0.7 N solution.
Allow to stand over night, filter off the guanine, wash with 0.3 N ammonia
and precipitate the phosphoric acid from the filtrate with magnesia mixture
as described above for total phosphoric acid. Divide the amount of ammonium magnesium phosphate obtained in each experiment by the weight
of nucleic acid from which it was produced and use the resulting values and
the time intervals as coordinates for plotting a curve. The curve beyond
the 2 hour period will be a straight line slightly inclined.
Subtract from each value 10 mg. of magnesium ammonium phosphate
for each hour of heating and with the values, plot a new curve. It will be
a straight line parallel to the base line.
, The phosphoric acid of the purine nucleotides is easily split off by acid
hydrolysis. This is half of the total phosphorus.
1200 g. ammonium nitrate and 45 m!. of 16 N nitric acid made up to 4 liters with
water.
208
BILE
(1) Pettenkofer's Test. Place 1 m!. of bile in a test tube, add 3 drops of
2% sucrose and mix. Stratify this mixture onto concentrated sulfuric
acid . Note the color.
(2) Gmelin's Test. Dilute 1 m!. of bile with 4 ml. of water. Stratify
onto concentrated nitric acid. Note the colors.
CHOLESTEROL
The isolation of cholesterol from bile is a tedious procedure but gall stonos are
composed principally of the substance and serve as a source for its easy preparation.
(3) Extract about 5 g. of powdered gall stones with 50 m!. of ether and
filter the ether extract into a 200 m!. flask. The extraction is best made in
a small flask that can be tightly corked so that the ether may be agitated
with the powder for several minutes. It is also better to make three successive extractions with 15 to 20 m!. of ether than one extraction with the
entire amount. The insoluble pigment is used for the preparation of bilirubin as directed in the next section.
The ether is distilled from the ether extract and the residue which consists of cholesterol with a little fat is treated with 50 m!. of alcoholic sodium
hydroxide and heated for 30 minutes under an inverted condenser. The
fat is thus saponified. Transfer the solution to an evaporating dish and
evaporate to dryness. Remove the last trace of water by moistening with
absolute alcohol and evaporating, and extract the residue with 10 m!. of
absolute alcohol. On cooling, the solution deposits crystals of cholesterol.
If the material is not perfectly white, recrystallize from absolute alcohol.
(4) Crystal Habit. Examine carefully under the microscope some
cholesterol that hat> been crystallized from alcohol.
.(5) Reaction with Iodine. Place a small amount of cholesterol on 3
microscope slide by the side of a drop of conc. sulfuric acid and a drop of
iodine solution. Cover ,vith a cover glass and examine. A variety of
'colors will be observed.
(6) Hesse's Reaction. Dissolve a small quantity of cholesterol in
about 5 cc. of chloroform and shake gently in a test tube with an equal
volume of concentrated sulfuric acid. The acid takes on a green fluorescence and the chloroform solution exhibits a rapid succession of colors from
red to violet. Expose a little of the violet solution to the air in a porcelain
dish and note the colors.
210
(7) Xanthine Test. Evaporate a drop of nitric acid with a trace of
cholesterol on a white porcelain surface and when cool touch the spot with
sodium hydroxide.
BILIRUBIN
(8) Wash the residue of bilirubin on the filter with 2 N hydrochloric
acid and then with water until the washings are no longer acid. Dry the
filter with its contents in a porcelain dish on the water bath, cut into pieces,
warm in a small flask with a little chloroform and filter through a dry filter.
Use this chloroform solution for the following test.
(9) Gmelin's Test. Place a few ml. of the chloroform solution in a
test tube and carefully stratify with concentrated nitric acid. At the
junction of the two liquids, colored rings appear. Note the arrangement of
the colors. This test is very sensitive, is more striking with a dilute solution and may be done in a variety of ways.
212
NOR1\lAL URINE: QUALITATIVE
GENERAL
(1) Chloride. Acidify some urine with nitric acid and add silver
nitrate. Test the solubility of the precipitate in ammonia.
(2) Preformed or Inorganic Sulfate. Acidify some urine with hydrochloric acid and add barium chloride.
(3) Ethereal or Conjugate Sulfate.
Most of the sulfur of urine is in the form of SO, ions and therefore directly precipitable with barium chloride (preformed or inorganic sulfate). On the other hand,
part of the sulfur of urine is combined in ester linkage and not directly precipitable
with barium chloride (ethereal sulfate). About one-tenth of the sulfur of urine
is present as ethereal sulfate.
Treat about 20 m!. of urine with alkaline barium chloride, avoiding too
great an excess of the reagent. The inorganic sulfate is precipitated.
Filter, treat the filtrate with half its volume of dilute hydrochloric acid and
boil for 15 min. Note the small white precipitate which forms either
immediately or on coolip.g under the tap. Explain.
(4) "Urine Crystals." Allow a liter of urine to stand a few days. The
reaction becomes alkaline (?), and a deposit occurs. Examine the sediment under the microscope, for the characteristic crystals of magnesium
ammonium phosphate. Decant the liquid, dissolve the crystals in nitric
acid and test the solution for phosphoric acid.
(5) Phosphate. To about 10 ml. of urine add a drop or two of sodium
hydroxide and boil. A flocculent precipItate of calcium phosphate is
formed which is soluble in a slight excess of acetic acid. (Important to
remember when testing urine for coagulable protein.)
(6) Acidify abc)ut 20 ml. of urine strongly with nitric acid, add 2 m!. of
molybdic solution, 2 g. of ammonium nitrate and digest at about 80° for
a few minutes. Filter off·the yellow precipitate and treat as described on
,p.8.
(7) Boil about 10 mI. of urine with one-fourth of its volume of mixed
Fehling'S solution. A bluish green bulky precipitate forms (?). Boil
about 10 ml. of mixed Fehling's solution with one-fourth its volume of
urine. No precipitation occurs. (Important to remember when testing
urine for sugar.)
214
URIC ACID
(8) Heat about a liter of urine to boiling, and add copper purine reagent!
as long as a precipitate is formed (20 ml. will be an excess for most urines).
Filter off the white precipitate (C 6ILN40 3 • Cu 20) while the liquid is still
hot and wash thoroughly with boiling water. FiiJ.ally, pierce the filter,
spurt the precipitate into a clean flask with boiling water and decompose
the suspended copper compound with a slight excess of sodium sulfide.
The point where the sodium sulfide is in slight but certain excess can be
accurately found by allowing a drop of the material and a drop of lead
acetate to run together on a filter paper. When the proper amount ,of
sodium sulfide has been added, heat the fluid to boiling and work as rapidly
as possible, otherwise the uric acid may be thrown out with the copper
sulfide and lost.
Treat the material with acetic acid a few drops at a time testing the
reaction with litmus. When the solution is slightly acid, hard boiling will
cause the copper sulfide to collect leaving a transparent interstitial liquid
which holds uric acid in solution while it is hot. Filter without delay.
Allow the filtrate to cool somewhat and acidify with hydrochloric acid.
When the deposition of uric acid begins; add 5 to 10 ml. of dilute hydrochloric acid and allow to stand. Needles of uric acid will be slowly formed.
Examine under the microscope. Filter, wash, in turn, with water and
alcohol, open the filter and allow to dry in the air.
Uric acid obtained in the manner described may have a pale yellow color
which can be removed as follows: Dissolve the uric acid (grams) by warming with 20 parts (ml.) of concentrated sulfuric acid, and stir 3 or 4 parts
of alcohol into the solution. Snow white uric acid will be precipitated.
(9) Suspend a trace of uric acid in warm water, bring into solution with
a drop or two of sodium hydroxide and acidify with acetic acid. If the
solution is sufficiently dilute, the uric acid will remain in solution. Make
alkaline with ammonia and add ammoniacal silver nitrate.
(10) The M~rexide·Test. Place a minute particle of uric acid on a
:vhite porcelain surface with a drop of nitric acid and evaporate most carefully to dryness over a small free flame. Note the color of the spot. When
cool touch the spot with a drop of ammonia. Make the same test using
sodium hydroxide instead of ammonia. Repeat the test with xanthine
and hypoxanthine. (See "hypoxanthine" under "Muscle.")
(11) Dissolve some uric acid in sodium hydroxide and boil with Fehling's solution.
(12) Suspend a small amount of uric acid in boiling water and bring
1 Treat 0.2 M copper sulfate with ammonia, until the .precipitate first formed
redissolves, avoiding an excess of ammonia. Heat the purple solution to boiling
and decolorize with saturated sodium bisulfite. Prepare the solution extempore.
216
into solution with a drop of sodium hydroxide. Acidify strongly with sulfuric acid and immediately treat with a very dilute solution of potassium
permanganate drop at a time. The uric acid reduces and decolorizes the
permanganate. The reactio~ is employed in certain methods for the quan-'
titative estimation of uric acid.
(13) Volin's Phosphotungstic Acid Test. Dissolve a little uric acid
in 2 to 3 ml. of very dilute sodium hydroxide. Add 1 ml. of Folin's uric
acid reagent l and 10 ml. of saturated sodium carbonate. Note the color.
(14) The impure highly colored crystals of uric acid which are deposited
directly from urine (as in urinary sediments) have generally a characteristic
appearance with which one should be familiar. Acidify some urine strongly
with hydrochloric acid and allow to stand over night in a cool place.
Decant the liquid and examine the sediment under the microscope.
CREATININE
(15) The Precipitation of Creatinine from Urine. 2 Treat 2 liters of'
urine with a solution of 15 to 20 g. of picric acid in 100 ml. of hot alcohoL
Stir until a yellow precipitate begins to form and allow to stand over night
in a cool place. Creatinine is precipitated as a double picrate of potassium
and creatinine. C4H7NaO· C6 H 2(N02)30H· C6 H2(N0 2 )aOK. Decant the
supernatant liquid, wash the heavy sandy precipitate several times with
small portions of cold tap water and finally filter with a Buchner funnel.
The experiment is repeated until the desired amount of picrate is obtained.
(16) Preparation of Creatinine Zinc Chloride from the Double Picrate.
Treat 50 g. of the dry or nearly dry picrate with 10 g. of anhydrous potassium carbonate and 75 ml. of tap water. Stir for about 10 minutes and
allow to stand an hour or two with occasional stirring. Filter on a Buchner
funnel and wash the sediment two or three times with small quantities of
95% alcohol. Transfer the filtrate to a 400 ml. beaker and cautiously add
10 ml. of glacial acetic acid.. (Let the acetic acid drop in the center of the
liquid without stirring ~nd it will dissipate the foam.) To the acidified
wine red fluid add about one-fourth of its volume of a strong alcoholic solution of zinc chloride. An abundant crystalline precipitate of creatinine
zinc chloride (C 4 H 7N aOkZnCb will form at once but will increase consid~rably on standing. Decant the fluid and wash the crystals in turn with
50% and 90% alcohol.
To purify the creatinine zinc chloride, dissolve in 10 parts of boiling 4.5
I Folin's uric acid reagcnt:-Place in a flask 750 m!. watcr, 100 g. sodium tungstate
and 80 ml. 15 M orthophosphoric acid. Boil gently with a reflux condenser for 2
hours. Dilute to 1 liter.
2 Folin, 0., J. B. C., 17, 463 (1914); Benedict, S. R., J. n. ~., 18, 183 (191-1).
218
N acetic acid, and to the hot solution add one-tenth of its volume of concentrated alcoholic zinc chloride and 1.5 volumes of alcohol. The mixture
is left standing over night, filtered and the precipitate is washed with a
little alcohol. After two more such crystallizations the product is pure.
(17) Preparation of Creatinine from Creatinine Zinc Chloride. Remove the coloring matter from crude creatinine zinc chloride as follows.
Dissolve 10 g. of crude substance by heating with 100 ml. of water and 60
ml. of 1 N sulfuric acid. Decolorize the solution with about 4 g. of
animal charcoal, add 7 g. of potassium acetate and 3 ml. of a strong solution
of zinc chloride. After the fluid has cooled a little, dilute with an equal
volume of alcohol. On standing in a cool place, colorless crystals of creatinine zinc chloride are deposited. Decant and wash by decantation in
turn with a little water, 95% alcohol and absolute alcohol. If the crystals
are not perfectly white recrystallize from a small amount of hot water,
adding alcohol to complete the deposition.
Grind 10 g. of this material in a mortar. (Grind as fine as possible: a
little time spent at this point saves trouble later.) Place in a small flask
with 70 ml. of 15 N ammonia and bring completely into solution by warming and stirring. Cork the flask, allow to coolon the desk, then in ice water
for an hour (over night, if necessary). Pure, crystalline creatinine is
deposited. Decant, wash with a very small amount of concentrated ammonia and allow to dry on filter paper in the air.
(18) Weyl's Reaction. To a dilute creatinine solution add a few drops
of freshly prepared 10% sodium nitroprusside and make alkaline with
sodium hydroxide. A transient red color is produced. When the color has
faded out, acidify with acetic acid and boil. Prussian blue is formed.
(19) Picric Acid Test. Treat 5 ml. of a dilute creatinine solution with
an equal volume of saturated aqueous picric acid. Add about 10 drops of
2·N sodium hydroxide and note the rapid formation of color. On long
standing the color gradually (ades.
(20) Preparation of Creatine from Crude Creatinine Zinc Chloride.
Dissolve 10 g. of crude .creatinine zinc chloride in 70 ml. of hot water, add
15 g. of calcium" hydroxide and boil gently with occasional stirring for 20
·minutes. Filter hot, wash with hot water and precipitate the zinc with
hydrogen sulfide. Acidify the filtrate from zinc sulfide faintly with acetic
acid, evaporate rapidly but carefully to about 20 ml. and allow the concentrated liquid to stand over night in a cool place. Decant the mother
liquor and recover the unaltered creatinine which it contains by precipitation as creatinine zinc chloride. Wash the creatine crystals by decantation
in turn with 50% alcohol and absolute alcohol. Recrystallize from 7 parts
of boiling water.
220
UREA
Urea is a eolorless crystalline substance very soluble ill water but it reacts
with nitric acid to form urea nitrate which is difficultly soluble in dilute nitric acid.
An obvious method is thus furnished for isolating urea from urine.
(21) Preparation of Urea Nitrate. Evaporate 750 ml. of fresh urine
(first over a free flame but finally on a water bath) to a thin syrup. Cool
and treat with twice its volume of 8 N nitric acid that has been boiled free
from the brown oxides of nitrogen and afterwards cooled nearly to the
freezing point. The materials become almost a solid paste of urea nitrate,
and the precipitated phosphates pass into solution in the nitric acid. Filter
off the acid solution on a Buchner funnel and wash the crystals with a little
cold colorless nitric acid.
Dissolve the impure highly colored urea nitrate in a small amount of hot
0.2 N colorless nitric acid, and as the solution is gently boiled, add crystals
of potassium permanganate in small successive portiops, until the urea
nitrate solution becomes nearly or quite colorless. The permanganate
furnishes oxygen for oxidizing, the coloring matter, but does not oxidize
urea.
Treat the hot colorless urea nitrate solution with about one-fourth of its
volume of colorless 8 N nitric acid and allow to cool. Thin plates of colorless urea nitrate will be deposited. They are filtered off as before with a
pump, and roughly weighed. It is well to completely decolorize the urea
nitrate because any color that it possesses will follow the urea in the next
procedure.
(22) Preparation of Urea from Urea Nitrate. Dissolve the urea nitrate
in a small amount of hot water, add somewhat more than its equivalent of
powdered barium carbonate' and evaporate to dryness on the water bath.
Extract the powdered residue with hot alcohol and filter into an evaporating
dish. Evaporate the alcoholic solution of urea to dryness and crystallize
from a small amount of 95% alcohol. Use the material for the following
tests.
(~3) Urea Nitrate. Dissolve a fragment of urea in a drop of warm
water on a slide. Add a drop of nitric acid, cover with a cover glass and
examine under the microscope. The predominant crystal form is hexagonal.
. (24) Urea Oxalate. Make the last test, using a strong aqueous solution
of oxalic acid instead of nitric acid. The predominant form is tetragonal.
222
(25) Conversion into Biuret. Heat a portion of urea in a dry test tube.
It melts, ammonia is given off and the melted mass soon solidifies. Discontinue heating immediately. Suspend the cake in hot water and bring
into solution with a drop or two of sodium hydroxide. Acidify a part of
the solution. Cyanuric acid is precipitated.
Treat a portion of the alkaline solution with a trace of copper sulfate.
(26) Formation of Nitrogen from Urea. Warm a few drops of concentrated nitric acid with a particle of arsenic trioxide; brown oxides of nitrogen
are formed (N 203). Drop a crystal of urea into the brown solution and
notice the. evolution of a gas.
CO(NH2h
+ N 20 a =
CO2 + 2H 20
+ 2N2
(27) To a few mI. of 2 N sodium hydroxide add a drop of bromine and
warm the solution.
+
+
+H0
2NaOH
Br2 = NaBr
NaBrO
Add a crystal of urea and note the evolution of a gas.
CO(NH 2)2
+ 3NaBrO
=
CO 2
+
2H 20
2
+ N2 + 3NaBr
HIPPURIC ACID
Hippuric acid is not a metabolic constituent of the urine in the narrow sense of the
term. It is formed in the organism by the combination of benzoic acid with glycine,
the benzoic acid being present in the food eaten or formed by the oxidation of certain
substances which are due to putrefaction in the alimentary canal or are introduced
in the foo<;l. These circumstances operate to produce a greater amount of benzoic
acid in herbivora than in man, therefore their urine contains more hippuric acid than
does human urine. However, the hippuric acid in the urine of man can be increased
by the administration of benzoic acid.
(28) Take 2 g. of sodium benzoate in the evening and collect the urine
the next morning. Heat the urine to boiling and add enough milk of lime
to make the liquid markedly alkaline to litmus. Filter off the hot solution
from the precipitated phosphates and coloring matter, evaporate to 50 or
60 ml. and alter coolIng acidify carefully with hydrochloric acid. Hippuric
acid is precipitated immediately in crystalline form but highly colored.
Suspend the crystals of hippuric acid in a little hot water, bring into
solution with a drop or two of ammonia, decolorize with animal charcoal
and when cool, precipitate the hippuric acid by careful acidification with
hydrochloric acid (as before). Use the crystals for the following.
(29) Crystal Form. Examine under the microscope.
(30) Production of Benzoic Acid. Place a little hippuric acid in a dry
test tube with a drop or two of concentrated sulfuric acid and heat very
carefully over a small free flame until a white sublimate forms on the colder
224
part of the tube. Examine the sublimate under the microscope. The
hippuric acid has undergone hydrolysis forming benzoic acid and glycine.
(31) Production of Hydrocyanic Acid. Heat a very small portion of
hippuric acid in a dry test tube and note the odor of hydrocyanic acid.
INDICAN
Indican (indoxyl sulfate) may be hydrolyzed by acid to indoxyl and sulfuric acid
and the illdoxyl may be oxidized to indigo blue which is soluble in chloroform.
(32) Treat 5 ml. of urine with 2 ml. of concentrated hydrochloric acid
and a trace of ferric chloride. Shake the mixture with 3 m!. of chloroform
and allow to settle. Note the color.
226
PATHOLOGICAL URINE
All of the tests described below should be carried out with both normal
and pathological urines.
COAGULABLE PROTEINS
Abumin and globulin are not differentiated by the ordinary tests and
when excreted the condition is usually termed albuminuria. Clear urine
should be used for the following tests. If the urine is turbid, filter several
times through the same paper, asbestos or paper pulp. Among the many
tests for the detection of protein, the following are perhaps the best.
(1) Coagulation Test. Place 10 to 15 ml. of clear urine in a small test
tube. If the urine is not faintly acid to litmus, add a drop or two of 0.2 N .
acetic acid. Heat the upper portion of the column of liquid to boiling and
compare with the cooler portion. A cloud forms when protein is present.
Add a drop or two of acetic acid. If the solution is not acid enough a
precipitate of calcium phosphate may form on' heating.
(2) Heller's Test. Place 5 m!. of urine in a test tube and stratify by
introducing 16 N nitric acid carefully from a pipette which raaches to the
bottom of the tube. If protein is present, a white zone of contact appears.
In cases where the urine is concentrated or where ther!;) is an excessive
quantity of uric acid, a cloudy zone may appear on standing. This zone,
however, is always considerably higher than the zone of contact of the two
liquids, and will dissolve when the urine in which it formed is carefully
removed from the nitric acid and warmed.
Urine which contains as much as 45 g. of urea to the liter may deposit
a crystalline precipitate of urea nitrate. So concentrated a urine should
be diluted before the protein test is applied.
Certain drugs may form a white ring which is soluble in alcohol. Colored
rings may be produced when bile pigments are present.
(3) Trichloracetic Acid and Sulfosalicylic Acid Tests. Urine may be
, stratified on solutions of trichloracetic acid (saturated) or sulfosalicylic acid
(20%) as in Heller's test. A white ring will be seen at the zone of contact
between the two fluids, its intensity varying with the amount of protein
present. The colored rings which are formed when nitric acid is used are
rarely observed. Proteoses are also precipitated but dissolve when the
urine is warmed and reappear on cooling: Uric acid may be precipitated
with trichloracetic acid. Uric acid and the resins are not precipitated by
228
sulfosalicylic acid. This test is undoubtedly one of the most sensitive, and
by its use it is possible to demonstrate protein in urines with which the
more common tests yield negative results.
(4) Spiegler's Test. Stratify some Spiegler's reagentl with urine that
has been faintly acidified with acetic acid and note the production of a
cloudy ring in the zone of contact. This test is sufficiently sensitive to show
the presence of protein in a dilution of 1: 250,000 and will therefore often
yield a positive result with normal urine.
GLUCOSE
The following tests should be made with urines containing about 2%
and 0.2% of glucose as well as with norIl!al urine.
(5) Fehling's Test. Dilute 5 m!. of mixed Fehling's solution with an
equal volume of water and heat to boiling. See that the solution remains
absolutely clear. Add 10 to 12 drops of urine and boil for a few minlftes.
When much sugar is present a yellow or red precipitate forms at once.
This test has the disadvantage that an alkaline copper solution is reduced
by many substances which occur in normal or pathological urine, or which
may appear in the urine after the ingestion of various drugs. 'When the
test is carried out as described above, the normal constituents seldom interfere.
(6) Benedict's Test. To 5 m!. of Benedict's reagent in a test tube add
8 drops of urine and boil for 2 minutes vigorously. Allow to cool spontaneously. When glucose is present, the entire body of the solution will
be filled with a precipitate, which may be red, yellow or green depending on
the amount of sugar present. 1f no suga~ is present, the solution either
remains perfectly clear or shows a faint blue turbidity consisting of precipitated urates, which need, however, cause no confusion.
(7) Nylander's Test. To 10 volumes of urine add 1 volume of Nylander'S
reagent and boil the mixture for 5 minutes. The presence of sugar is shown
by the appearance of a black precipitate.
Nylander's solution is a preparation containing bismuth, which is extremely sensitive to the reducing action of sugar but in case the urine contains sulfide, the bismuth reactions have no value.
(8) Osazone Test. Place in a test tube 1 g. of phenylhydrazine hydrochloride and 1 g. of sodium acetate, add 10 m!. of urine and heat in a boiling
water bath for! hour, or boil gently for several minutes. Allow to cool.
In the presence of glucose yellow phenylglucosazone crystals will appear,
which may be identified by their melting point and by their appearance
1
Tartaric acid 20 g., mercuric chloride 40 g., glycerol 100 g., water 1000 g.
230
under the microscope. Glucose (or fructose) may be easily distinguished
from lactose and maltose by this test.
(9) Fermentation Test. Shake a portion of the suspected urine with
some compressed yeast and fill a fermentation tube. Allow to stand in a
warm place (preferably in a th~rmostat which is kept at 40°) and note the
accumulation of gas in the upper part of the tube. A check test should
also be made with a 1% solution of glucose which has been shaken with a
portion of the same specimen of yeast. It is also well to make a second
check test with normal urine that has been shaken with the yeast. The
fermentation in each case should proceed overnight.
(10) Optical Test. Before applying this test it is necessary to remove
all proteins from the urine and to free it from a greater part of the coloring
matter by agitating with a little solid lead acetate. Filter the solution,
place it in a polarimeter tube and determine the rotation.
The sensitiveness of the test depends entirely upon the construction of
the instrument and as most instruments are not capable of showing a trace
of sugar it is generally useless to apply this test when no positive reaction
has been obtained by reduction tests. A rough quantitative estimation
can be made by means of this method.
ACETONE
(11) Rothera's Test. Add a little ammonium sulfate (0.5 g.) and 2 to
3 drops of freshly prepared sodium nitroprusside solution to 5 ml. of urine
and then stratify with 2 ml. of 15 N ammonia. A slow development of a
permanganate color indicates the presence of acetone. This test is also
positive with acetoacetic acid.
(12) Gunning's Iodoform Test. To 5 ml. of urine or distillate add a
few drops of iodine-potassium iodide and enough ammonia to form a black
precipitate of nitrogen iodide. Allow the tube to stand. Iodoform is
precipitated and can be recognized by its odor and the hexagonal arrangement which it exhibits under the microscope.
Acetone may be'detected more accurately by testing the distillate from
50' to 100 ml. of urine. The acetone may be formed from acetoacetic acid
by heating with acid.
. (13) Scott-Wilson Test. This is best for distinguishing between acetone and acetoacetic acid. Place 5 to 10 ml. urine in a large test tube fitted
with an aerating tube. Connect with another tube containing 5 ml. of the
Scott-Wilson reagentl and an absorbing tube as described in the quantita1 Dissolve 1 g. mercuric cyanide and 18 g. potassium hydroxide in 120 m!. of water.
Add with violent stirring, 40 m!. of a 0.726% solution of silver nitrate. The solution
should be clear. If turbid, allow to stand and syphon off the clear liquid. The
reagent will keep for 2 to 3 months but will gradually deposit a sediment.
232
tive ammonia determination. Place in a beaker of water at 40°. Aerate
for 5 to 10 minutes. An opalescence or white precipitate will appear if
acetone was present in the urine.
Acetone in the breath may be detected by allowing the patient to breathe
through the reagent. A safety bottle should be interposed between the
mouth piece and the aerating tube containing the reagent in order to prevent the poisonous solution being drawn back into the patient's mouth.
ACETOACETIC ACID
(14) Gerhardt's Test. Add ferric chloride, drop by drop, to 5 m!. of
urine until no more precipitate forms. A Bordeaux-red color develops in
the presence of acetoacetic acid 1: 7000. If the color is masked by the
precipitate, allow to stand or filter. After the ingestion of certain drugs
this test is also positive. If the color is due to acetoacetic acid, it will disappear after boiling for 2 to 3 minutes and will not reappear on cooling.,
(16) Harding and Ruttan Modification of Le Nobel's Test. Acidify
the urine with acetic acid, add 0.5 m!. of 0.1 M sodium nitroprusside (1 m!.
= 0.0298 g. Na 2Fe(CN)6NO· 2H 20) then stratify with concentrated
aqueous ammonia (hood). A purple ring develops rapidly. Acetone does
not give the test unless present in considerable concentration. The test
is positive when acetoacetic acid is present 1: 30,000. This is much more
delicate than Gerhardt's ferric chloride test.
BILE
Urine containing much bile froths greatly on shaking and the greenish
colored foam persists for some time.
(16) Gmelin's Test; Stratify 5 m!. of urine with concentrated nitric
acid. Allow to stand for a few minutes, then observe the colored rings near
the zone of contact.
(17) Huppert's Test.' Treat the urine with barium hydroxide until
the precipitation is c~mplete. The precipitate is yellow and contains the
bile pigments. Filter and boil out the precipitate with alcohol which is
acidified with sulfuric-acid. The precipitate is decolorized and the alcohol
becomes green or blue according to the amount of bile present.
BLOOD
(18) Examine the urine spectroscopically.
(19) Guaiac Test. To 5 m!. of urine add 6 drops of tincture of guaiac
and 6 drops of hydrogen peroxide. The blue color may be concentrated by
extracting with 1-2 m!. of chloroform. With chloroform the test can be
used to detect 1 part of laked blood in 5,000,000 parts of urine.
234
METABOLISlVI
A quantitative study of the urine offers a favorable method for observing
certain phases of metabolism. The Qomposition of the urine depends upon
a number of variables such as the quantity and composition of the food
ingested, bodily acitivities, pathological conditions, etc., so that the influence of any variable can be observed best when the others are kept constant.
In metabolism studies therefore one should know and control the composition of the diet and should observe bodily habits that are as nearly
constant as possible.
It is of the greatest importance to note that the excretion of materials by
the kidneys varies continually throughout the day, but that this variation
repeats itself from day to day. Therefore, for most studies one should use
for chemical examination the mixed urine voided throughout an entire day.
To obtain the normal urine picture, collect a 24 hour specimen of urine
while the regular diet is being ingested. A 2 liter bottle may be used containing a suitable preservative (thymol, toluene or chloroform) to prevent
decomposition. Start the period of collection in the morning, before breakfast. The urine is voided (and discarded), the time noted, and a complete
collection made thereafter including the voiding 24 hours later. This
period should end on the morning when the quantitative studies are to be
made, as some of the urinary constituents are slowly decomposed on standing. Keep a list of foods eaten during the period of collection with the
approximate amounts of the same. From this list calculate as accurately as
possible the quantities of proteins, fats, carbohydrates, purines and total
calories ingested.l
For the experimental period (lasting three days) make a list of foods
satisfactory for the chosen ,dietary and the principal foods that are unsatisfactory. Collect a 24 hour specimen of urine on the third day during which
the' prescribed diet is being ingested. This period should end on the morning when the quantitative studies are to be made.
, Examine the specimen of urine and notice especially any deposit which
may be present. The amor.rhous precipitate of phosphates or urates
usually remains suspended for some time after shaking while crystals of
1 For analysis of foods see: Locke, Food Values; Lusk, Science of Nutrition;
Hammarsten, Physiological Chemistry; Wiley, Foods and their Adulterations; Hawk
and Bergeim, Practical Physiological Chemistry; Sherman, Chemistry of Food and
Nutrition.
236
uric acid, triple phosphate (ammonium magnesium phosphate) calcium
oxalate, etc., settle rapidly. After measuring the volume of the urine,
it is well to allow a portion of it to sediment in a conical glass or to centrifuge a portion of the urine taken from the bottom of the bottle by means
of a pipette. Examine a little of the sediment u~der the microscope.
Refer to charts and descriptions of urinary sediments.
Amorphous precipitates usually remain suspended long enough after
shaking so that uniform samples of urine may be obtained for analysis.
If crystals are present of any compound containing any element which is
to be determined, they must be dissolved even though by so doing, other
determinations are rendered impossible. A precipitate of phosphate will
dissolve after adding a few drops of acetic acid; urates dissolve on warming;
uric acid crystals may be dissolved by adding sodium hydroxide to the
urine.
All determinations should be made in duplicate, and repeated if the
duplicates do not agree suitably. Tho compounds which are subject to
decomposition on standing should be determined as soon as possible.
237
URINE: QUANTITATIVE NIETHODS
(1) Volume. Before any urine is taken for analysis, the volume of the
complete 24 hour specimen should be measured in a large graduated cylinder.
This volume is used as the basis for ~alculating the quantities of all of the
constituents studied.
(2) Color, Odor, Transparency. Note each.
(3) Specific Gravity. This may be determined by means of a urinometer. Some clear urine (filter if necessary) is placed in a wide test tube
or cylinder, being allowed to run down the side to avoid foaming. Any
foam may be removed with a filter paper. The urinometer is introduced,
allowed to float free in the liquid and the specific gravity reading taken
where the bottom of the meniscus cuts the stem. For accurate readings
the urine should be at the temperature for which the instrument is calibrated (usually 15°C.). If it is not, correct the observed reading by adding
0.001 for every 3° above the standard temperature or by subtracting the
same for every 3° below.
Check the accuracy of the urinometer by determining the specific gravity
of distilled water. Any variation from 0.999 at 15° should be corrected
for in all readings.
(4) Total Solids. Total solids may be calculated approximately by
multiplying the second and third decimal figures of the specific gravity by
2.6. The product represents the number of grams of solids in 1 liter of
urine. For example: If the specific gravity of a urine is l.020 then 20 X 2.6
= 52.0 g. total solids per liter.
From this, the output during 24 hours may
be calculated.
(6) Reaction to Litmus. For a rough indication of the reaction of the
urine, testing with sensitive litmus paper is sufficiently accurate. A much
more accurate metho'd of studying the reaction is described in the next
section.
(6)
HYDROGEN ION CONCENTRATION
A suitable indicator is added to diluted urine and the resulting color compared
with standard tubes of varying hydrogen ion concentration containing the same
concentration of indicator. Thc yellow color of the urine when mixed with the
color of the indicator produces a result which makes a comparison with standards difficult. This is overcomc by using a comparator ill which a tube of the diluted urinc
is placed behind the standard tube. By observing light which passes through the
two tubes, the two colors are blended so that comparison with the unknown (with a
238
tube of water similarly placed) is much more satisfactory. As the pH of urme may
vary from 4.8 to 8.0, a series of standard buffer tubes should be available covering
that range at suitable intervals. For class work intervals of 0.4 pH are satisfactory.
A particular indicator is used only within the range of its color change. See pp. 28-34.
The Determination. Place 2 m!. of urine in a clean test tube and add 8
mI. of water. (Use distilled water which has been recently boiled and
cooled and tested for acidity.) Add 5 drops of brom thymol blue and
compare with the standard containing this indicator. If the color is within
the range of the standards make an accurate comparison by placing the
tube in a comparator and matching against a standard tube behind which
is placed a tube of urine similarly diluted. If the urine is outside the range
of brom thymol blue, discard the portion, add 5 drops of another indicator
to another portion of diluted urine and compare with standards. In this
determination, a measuring cylinder is sufficiently accurate for the measurements because they involve only the dilution of the colors of the indicator
and the urine.
(7) TITRATABLE ACIDITY
(A Modification of L. J. Henderson's Method)l
In order to remove the acids formed in the body without removing too much
base, urine which is much more acid than the blood is usually excreted by the kidney.
The excretion of acids may therefore be determined by estimating the amount of
material excreted which is more acid than the blood. The pH of the blood under
normal conditions is about 7.4. By titrating the urine to this reaction th!l physiological excretion of acids may be determined. Conversely L. J. Henderson has pointed
out that this determination shows the amount of base retained by the kidney in
forming urine more acid than blood.
Phosphate Buffer (PH 7.4). See p. 32.
Standard Sodium Hydroxide 0.05 N. Transfer with a pipette 25 m!. of
0.1 N sodium hydroxide to a clean dry flask. Rinse the pipette and add
25 m!. of water to the sodium hydroxide and mix. Further standardization
is not necessary.
The Determination. Transfer 5 m!. of urine to a large test tube, and add
25 ml. of water and 15 drops of phenol red. Measure 5 m!. of phosphate
buffer (pH 7.4) into another tube of similar diameter, and add 25 m!. of
w~ter and 15 drops of phenol red. Place the tubes in a comparator and
place a tube of urine similarly diluted behind the buffer tube. Titrate the
urine containing the indicator with 0.05 N sodium hydroxide until the color
matches the standard when viewed so that light passes through both the
standard and the control tube of diluted urine.
1
Henderson, L. J. and Palmer, W. W., J. B. C., 17, 305 (1914) •.
240
Calculate the milliliters of 0.1 N acid and the milliequivalents of acid
excreted in 24 hours.
(8)
TITRATABLE ACIDITY
(Folin's :Method for Total Acidity)
See under Ammonia, Rough Method, page 248.
(9)
TOTAL NITROGEN
(Kjeldahl l\Iethod)
Practically all of the nitrogen in physiological materials can be converted into
ammonium sulfate by oxidation and hydrolysis with sulfuric acid and a catalyzer.
The ammonia thus formed may be set free by the addition of sodium hydroxide,
and distilled from the solution into a known quantity of standard acid. The amount
of ammonia can be calculated from the acid neutralized.
Place 5 ml. of urine in a 500 ml. Kjeldahl flask, add 15 ml. of concentrated
sulfuric acid, a spoonful of potassium sulfate (3 to 4 g.) and 10 drops of
copper sulfate. Wash the materials from the neck into the flask with a
few spurts of water. Boil' on the digestion rack until colorless (actually
light green) and rotate the hot fluid in the flask to make sure that undecomposed material is washed down into the acid. Boil 30 to 40 minut·es
longer. (Foods and feces require much longer digestion than urine.)
While the digestion proceeds, introduce a knmm quantity of 0.1 N acid
(50 ml. is usually sufficient) from a burette into a 500 ml. flask and add 2
drops of sodium alizarine sulfonate. Carefully rinse a delivery tube, place
it in the flask and attach to a condenser. See that the delivery tube extends slightly below the surface of the liquid.
As soon as the digestion is finished, allow the flask to cool, and dilute
with about 200 rn!. of tap water. Add one small piece of granulated zinc,
about] ml. of phenolphthalein and more than enough concentrated sodium
hydl"Oxide to neutralize the acid (50 m1. of 45 per cent sodium hydroxide is
sufficient). Incline" the flask and pour in the alkali steadily but not too
rapidly so that it flows under the liquid in the flask forming a layer at the
bottom. Why? Do not wet the neck of the flask within an inch of the
top. Why? Without delay connect the flask by means of the safety tube
to the condenser carrying the receiving tube and flask. To make the connection tight, hold the rubber stopper firmly and twist the flask instead of
attempting to twist the stopper. Light the burner, shake the flask vigorously and heat with a full flame until boiling just begins when the flame
should be turned down and then increased cautiously as the tendency to
froth ceases. Distil until 150 to 200 ml. of distillate have been collected.
212
Should all of the acid in receiving flask be neutralized (as shown by change
in color of the indicator) while ammonia is still coming over, the determination may be rendered inaccurate through loss of ammonia by volatilization.
The determination may be saved by adding, without delay, a known
amount of acid from a pipette. When the distillation is finished, disconnect
. the delivery tube from the condenser then turn out the flame and loosen the
stopper in the distilling flask.
Remove the delivery tube from the receiving flask, rinsing carefully with
distilled water and titrate the excess of acid in the flask with 0.1 N sodium
hydroxide. The difference between the amount of alkali used and the acid
originally introduced is the amount of acid neutralized by the ammonia
during the distillation. A small portion of the ammonia arises from impurities in the reagents. A blank determination should therefore be made
and the amount found subtracted from the determinations before calculation. Calculate the total nitrogen in the 24 hour specimen.
(10)
TOTAL NITROGEN
(Method of Folin and Denis modified by Wongl )
By digestion with sulfuric acid and potassium persulfate, all of the nitrogen of
the urine is changed into ammonia which reacts with Nessler's solution yielding a
dark brown color. The depth of color produced is compared with the color of a
standard ammonia solution similarly treated.
Reagents. Fifty per cent sulfuric acid, saturated potassium persulfate,
Nessler's solution, Nessler's solution with extra alkali, standard ammonium
sulfate solution, Pyrex test tubes. See pp. 140.
The Determination. The determination is carried out as described on
p. 142. Dilute urine so that 1 ml. contains 0.2-0.3 mg. of nitrogen.
(Urines having specific gravities between 1.018 and 1.030 should be diluted
one in twenty to one in fifty. 'When the specific gravity is outside these
limits the dilution sho.uld be varied accOI:dingly.) Measure 1 m!. of the
diluted urine into a large dry Pyrex test tube and add 1 ml. of 50% sulfuric
. acid and 2 glass beads. Proceed with the determination as described on
p. 142. Calculate the-total nitrogen in the 24 hour specimen.
(11)
UREA2
The ammonia formed from urea by urease is aerated into an excess of standard
acid and the excess determined by titration.
For references, see p. 140_
Marshall, E. K., J. B. C., 15,487,495 (1913).
J. B. C., 19, 211 (1914).
1
2
244
Van Slyke, D. D. and Cullen, G_ E.,
Urease Solution p. 144.
Aerating Apparatus.
Air is to be drawn through the apparatus by suction. The air first
passes through a layer of greatly diluted sulfuric acid to remove any trace
of ammonia. It then passes through the sample of urine into the standard
acid. Duplicates are run at the same time. The small perforated bulbs
are placed in the standard acid to increase the absorption.
The Determination. Carefully introduce 1 ml. of urine into the bottom
of a tube of the aerating apparatus, dilute with 3 m!. of water and add 1 m!.
of urease solution. Put the stopper in place, mix the contents of the tube
and allow it to stand for 15-30 minutes in a water bath warmed to 40°.
Insert into the receiving cylinder 15 m!. of 0.1 N hydrochloric acid, 10
m!. of water, and a drop of sodium alizarine sulfonate.
When the digestion is complete, connect the digestion tube with the receiving tube and aerate for half a minute to draw over any ammonia in the
gas space above the liquid. Then open the digestion tube and add 5 drops
of caprylic alcohol and 5 mI. of saturated potassium carbonate. Stopper
again and aerate slowly 2-3 minutes then rapidly for 1 hour. The air
should first pass through water containing a few drops of dilute sulfuric
acid to remove traces of ammonia. Titrate the excess of acid in the receiving tube with 0.1 N sodium hydroxide. A,s the determination includes
the preformed ammonia of the urine as well as the ammonia formed from
the urea, the ammonia nitrogen must be subtracted in calculating the urea
nitrogen. Calculate the urea nitrogen in the 24 hr. specimen.
(12)
UREA
(Nesslerization method l )
The urea is converted into ammonium carbonate by urease and the resulting
ammonia is adsorbed by permutit and determined as described below. (Method of
Folin and Bell.)
Urease Solution. S~e p. 144.
Nes,sler's Solution. See p. 140.
Standard Ammonium Sulfati} Solution.
See p. 140 .
. The Determination. Rinse a 100 ml. volumetric flask with dilute nitric
acid and then with several portions of water. Introduce enough urine to
contain 0.5 mg. of urea nitrogen. (Dilute urine 10 times and take 0.5 m!.)
Add 5 ml. of water and 1 ml. of urease solution and allow to stand 20
~
Folin, O. and Youngburg, G. E., ,J. B. C., 38,111 (1919).
Wakeman, A. M. and Morell, C. A., Arch. Int. Med., 46, 290 (1930).
246
minutes at room temperature. Add about 1.5 g. washed, dust-free permutit, swirl the flask gently for 5 minutes, decant the liquid (without losing
any of the solid) and wash the solid by decantation with about 100 ml.
of distilled water. Add 10 ml. of water and 1 m!. of 2 N sodium hydroxide
and agitate the flask gently for about 5 minutes. Dilute to. 75-80 m!. with
water, add 10 ml. of Nessler's solution and make up to volume with water.
To another 100 m!. volumetric flask, add 5 m!. of the diluted standard
ammonium sulfate (0.5 mg. of nitrogen) and 1 m!. of 2 N sodium hydroxide
and dilute with water to 75-80 ml. Add 10 ml. of Nessler's solution, make
up to volume and make the color comparison with the unknown. The
ammonia nitrogen is included with the urea nitrogen in this determination.
(13)
AMMONIA
(Rough Method)
Formaldehyde combines with ammonium salts forming hexamethylenetetramine,
a neutral substance, and liberates an equivalent amount of acid. The liberated
acid may be titrated with standard alkali. Amino acids react in a similar manner
and are included in the determination.
A saturated solution of potassium oxalate and a 40% form~ldehyde solution are made neutral to phenolphthalein with dilute sodium hydroxide.
The formaldehyde should be tested by diluting a portion with two or three
volumes of water which contain a little indicator.
The Determination. To each of two large test tubes add 25 m!. of urine,
5 ml. of neutralized oxalate solution and two drops of phenolphthalein.
Titrate the contents of one tube with 0.1 N sodium hydroxide until the
color of the solution is a shade darker than the urine in the other tube.
This constitutes Folin's titration for total acidity. Titrate the urine in the
second tube until it matches the first. Calculate the milliliters of 0.1 N
acid and the milli-equivalents of acid excreted in 24 hours.
For the determination of ammonia now add 5 m!. of neutralized formalin
to one tube and titrate again to the same end point using the second tube
for comparison. Th~n repeat the procedure with the second tube. The
alkaii used in the last titration is equivalent to the amount of ammonia in
the urine used. Calculate the grams of ammonia nitrogen and the per'centage of ammonia nitrogen to total nitrogen in the 24 hr. specimen.
(14)
AMMONIA
(Aeration Method)
To 10-20 m!. of urine accurately measured into an aerating tube of an
aerating apparatus, add 2 drops of caprylic alcohol and connect with a
248
receiving tube containing 15 mI. of 0.1 N hydrochloric acid, 10 mi. of water
and one drop of sodium alizarine sulfonate. Add 5-10 g. dry potassium
carbonate and aerate the liberated ammonia into the standard acid. Allow
the air current to pass slowly for a couple of minutes and then rapidly for
1 hour. Titrate the excess of standard acid with 0.1 N sodium hydroxide
and calculate the amount of ammonia nitrogen in the 24 hour specimen.
(15)
AMMONIA
(Method of Folin and Bell)l
In this method the ammonia is absorbed from the urine by a synthetic zeolyte
powder called permutit and the other compounds are removed by the decantation
of the solution. The ammonia is then liberated from the pcrmutit with alkali and
Nesslerizcd. Permutit is an insoluble sodium silicate in which the sodium may
be easily replaced by ammonia in neutral or faintly acid solutions. The reaction
is reversible, the ammonia being forced out of combination by certain concentrations
of sodium chloride and sodium hydroxide.
Wash by decantation about 2 g. of permutit2 in a 100 ml. volumetric
flask, once with 0.2 N acetic acid and twice with water. Add 5 m!. of
water and 1 or 2 m!. of urine. Rinse down the urine with 2 or 3 m!. of water
and rotate gently but continuously for 5 minutes. Rinse the powder to the
bottom of the flask with 25 to 30 m!. of water and decant. Wash once more
by decantation and add a little water to the powder. Introduce 5 m!.
(accurately measured) of 2 N sodium hydroxide, shake for a few moments
and allow to stand while preparing the standard solution.
To 5 ml. of standard ammonium sulfate (0.5 mg~ of nitrogen) in a 100 m!.
volumetric flask, add 5 m!. of 2 N sodium hydroxide (to balance the alkali
in the unknown). Dilute the standard and unknown to about 75 ml.
Rotate each flask vigorously and add to each 10 m!. of Nessler's solution.
Dilute to the mark and compare the colors.
'(16)
URIC ACID
(Method of Benedict and Franke)3
Stock Standard Uric Acid Solution. See p. 150.
Standard Uric Acid Solution. Transfer 50 ml. of the stock solution to a
500 m!. volumetric flask, add 300 ml. of water, 16 ml. of 2 N hydrochloric
Folin, O. and Bell, R. D., J. B. C., 29, 329 (1917).
Permutit should be used which has passed through a 60 mCdh sieve and will
not pass through an 80 mesh sieve,
'3 Benedict, S. R. and Franke, E., J, B. C., 52, 387 (1922) ..
1
2
250
acid and dilute to the mark. 20 ml. contains 0.4 mg. of uric acid. The
solution will keep for 2 weeks.
Arsenotungstic Acid Color Reagent. See p. 150.
Sodium Cyanide Solution. See p. 150.
The Determination. Transfer 5 mi. of urine to a 100 ml. volumetric flask,
dilute to the mark with distilled water and mix. (If 20 ml. of this diluted
urine should contain less than 0.30 mg. or more than 0.60 mg. of uric acid,
make a suitable dilutioq and repeat the determination.) 20 ml. of the
diluted urine are measured into a 100 ml. volumetric flask, 10 ml. of sodium
cyanide solution are added from a burette, followed by 2 ml. of the color
reagent. The contents of the flask are mixed by gentle shaking, at the
end of 5 minutes diluted to the mark with distilled water and again mixed.
This blue solution is then compared in a comparator with a simultaneously
prepared solution obtained by treating 20 ml. of the standard uric acid
solution (0.4 mg. of uric acid) in a 100 ml. flask with 10 ml. of the sodium
cyanide solution and 2 ml. of the reagent and diluting to the mark at the
end of 5 minutes.
Caution, Poison. When the determination is finished pour the liquids
directly into the waste pipe as the volatile hydrocyanic acid may easily
be formed from the cyanide present.
Calculate the grams of uric acid and uric acid nitrogen excreted in 24
hours.
The photoelectric photometer may be used. The solution employed for
obtaining the zero setting contains the reagents, viz., distilled water,
sodium cyanide and the arsenic phosphotungstic acid reagent in concentrations the same as those occurring in the unknown.
(17)
CREATININE
(Folin's Method)!
A mixture of creatinine and picric acid in alkaline solution quickly develops a
deep red color which ,may be' compared with a standard solution.
Standard Creatinine Solution. Dissolve 1 g. of creatinine or 1.61 g. of
creatinine zinc chloride in 1 liter of 0.1 N hydrochloric acid.
, The Determination. Place 1-2 ml. of urine in a 100 ml. volumetric flask
and 1 ml. of standard creatinine solution in another. Add to each flask
20 ml. of saturated picric acid and 1.5 ml. of 2.5 N sodium hydroxide and
mix. Allow to stand 10 minutes, dilute to the mark and compare the
colors (within the next 10 minutes), setting the standard at 20 mm.
I
Folin, 0., J. B. C., 17,469 (1917).
252
If the readings of the standard and unknown differ by more than 50 per
cent of the lower value, repeat the determination using a suitable amount
of urine.
The photoelectric photometer may be used. The solution employed for
obtaining the zero setting contains the reagents (except creatinine) in
concentrations the same as those occurring in the unknown.
(18)
CREATINE
(Folin's Method)l
Creatine is changed into creatinine by beating with acid.
may then be determined colorimetrically.
The total creatinine
Heat 1 m!. of urine with 5 m!. of 0.5 N hydrochloric acid on a boiling
water bath for 3 hours. Cool, introduce a small square of litmus paper
and neutralize the contents of the flask with 2.5 N sodium hydroxide.
Add 20 m!. of saturated picric acid and 1.5 m!. of 2.5 N sodium hydroxide,
shake and allow to stand 10 minutes. Rinse the solution into a 100 m!.
volumetric flask and make up to the mark. Compare with a standard
creatinine solution as described under creatinine.
The ereatine (in terms of ereatinine) may be ealeulated by subtraeting
the preformed creatinine from the "total creatinine" determined above.
(19)
CHLORIDE
(Modified Volhard-Harvey .i'lIethod)2
A known quantity of silvcr nitrate is added to the urine. The chloride is thus
prccipitated and the excess of silver is determined by titration with thiocyanate
uSlDg a ferric salt as the indicator. When the titration is carried out in thc prescnce
of the silver chloridc, the end point though not sharp, is sufficiently so for most
urine analyses.
Standard ,Silver Nitrate Solution. Dissolve 29.06 g. of silver nitrate in
about 100 m!. of distilled water in a liter volumetric flask. Add 250 m!.
of concentrated nitric acid and 250 m!. of a saturated aqueous solution
of fen'ic ammonium sulfate. Dilute to 1 liter. 1 m!. of this solution is
equivalent to 10 mg. of sodium chloride or 6 mg. of chlorine.
Standard Ammonimn Thiocyanate Solution. Dissolve about 7 g. of ammonium thiocyanate in about 800 m!. of water and standardize the solution
as follo,,"s: Place 10 m!. of the standard silver nitrate in an Erlenmeyer flask,
1
2
Folin, 0., J. B. C., 17, 469 (1917).
.
See Peters and Van Slykc, Quantitative Clinical Chemistry, vol. ii, 1932.
254
and titrate by adding the thiocyanate solution from a burette until the
first tinge of brown appears in the solution. Dilute the thiocyanate solution so that 2 m!. are equivalent to 1 m!. of the standard silver nitrate.
The Determination. To 5 ml. of urine in a 200 ml. Erlenmeyer flask add
100 ml. of water and 10 ml. of the standard silver nitrate solution. If a
pink color develops, it may be dispelled with a few drops of permanganate
solution. Titrate with the standard thiocyanate solution until a brownish
tint forms and persists for several seconds after mixing. If the urine contains an unusual concentration of chloride this color may appear upon the
addition of the first drop of thiocyanate. II). this case add another 10 m!.
of silver nitrate solution at once and resume the titration. Do not attempt
to obtain a permanent brown color for an end -point.
In calculating the amount of chloride remember that the thiocyanate
solution is half as strong as the silver solution. Calculate the grams of
sodium chloride and of chloride excreted in 24 hours.
(20)
INORGANIC PHOSPHATE
The phosphate may be determined by direct titration with uranium acetate which
forms insoluble uranyl phosphate.
H.PO;-
+ UO:+ + Ac- -> UO.HPO. + HAc
When all of the phosphate has been precipitated, an excess of uranium is shown
by the formation of a brown precipitate with potassium ferrocyanide.
Fe(CN)~-
+ UO";+ + 2K+ -> K 2UO.Fe(CN)e
Standard Phosphate Solution. Dissolve 4.39 g. of pure dried monopotassium phosphate in water and dilute to 1 liter~ 1 ml. contains 1 mg.
of phosphorus.
Special Acetate Solution. A solution containing 10 g. sodium. acetate
and 3 g. acetic acid per 100 m!.
Standard Uranium Acetate Solution. Dissolve 34 g. of uranium acetate
and 100 ml. of 50% a,cetic acid in water and make up to 1 liter. If solution
is nQt complete, allow to stand and syphon off the clear liquid. Standa:z:dize
the solution as follows:
Place 50 ml. of the standard phosphate solution in a beaker with 5 m!.
of the special acetate solution and heat to boiling. Add 15 to 20 ml. of
the uranium acetate from a burette and boil. Remove a drop with a stirring rod and test by mixing it with a drop of potassium ferrocyanide on a
test tablet. If a brown color does not form immediately, add about 0.5
m!. of uranium solution, boil and test again. A brown color indicates an
excess of uranium. When the approximate amount of uranium solution
necessary to completely precipitate the phosphate has· been determined,
256
make another estimation adding nearly all of the uranium solution at once.
Complete the titration keeping the solution at the boiling point and testing
after the addition of a few drops of uranium solution, until the first immediate brown coloration appears when a drop of the hot solution is mixed
with a drop of ferrocyanide solution. From the burette reading calculate
the amount of phosphorus to which 1 ml. of the uranium solution is equivalent.
The Determination. Place 50 m!. of urine in a beaker with 5 m!. of special
acetate solution, heat to boiling and titrate as directed above, first carrying
out a preliminary determination. Repeat, adding nearly all of the required
uranium solution at once. Boil the solution and titrate exactly to the end
point. Calculate the grams of phosphorus excreted in 24 hours.
(21)
INORGANIC PHOSPHATE
(Method of Fiske and Subbarow, Modified)
Special Reagents. See p. 164.
The Determination. Measure into a 100 ml. volumetric flask enough
urine to contain between 0.2 and 0.8 mg. of inorganic phosphate phosphorus
(usually 1 or 2 ml.). Dilute to about 60 mI., add 20 rnl. of rnolybdatesulfuric reagent and 10 rnl. of reducing reagent.
At the same time transfer to a similar flask 5 m!. of the standard phosphate solution (containing 0.5 mg. of phosphorus), about 60 mI. of water,
20 m!. of molybdate-sulfuric reagent and 10 m!. of reducing reagent.
Dilute the contents of each flask to the mark, mix, and compare the colors
after 15 minutes. Set the standard in a Duboscq comparator at 20 mm.
Calculate the grams of phosphorus in the 24 hr. sample.
The photoelectric photometer may be used. The solution employed for
obtaining the zero setting contains the reagents (except the phosphate)
in concentrations the same as those occurring in the unknown.
(22)
INORGANIC SULFl\.TE
The following methods involving benzidine are modifications of methods of Rosenheim and Drummond.' Accuracy has been sacrified somewhat to make them more
available for class work. _ See also Fiske, C. H., J. B. C., 47, 59 (1921).
The sulfate is precipitated as benzidine sulfate_ Because of the slight dissociation
of benzidine, the salt may be titrated with phenolphthalein as if it were free sulfuric
acid. Note that both hydrogens of the sulfuric acid are titrated_
Benzidine Solution. Grind 4 g. of benzidine to a fine paste with 10 m!.
of water, wash into a flask with 500 m!. of water, add 5 mI. of concentrated
1
Rosenhcim, O. and Drummond, J. C., Biochem. J., 8, 143 (1914).
258
hydrochloric acid and make up to 2 liters. 150 ml. of this solution is sufficient to precipitate 0.1 g. of sulfuric acid.
The Determination. Introduce 25 rul. of urine and a small piece of
Congo red paper 4-5 mm. square into a 250 rul. Erlenmeyer flask, and treat
with 2 N hydrochloric acid, a drop at a time, until the reaction is distinctly
acid to the Congo red paper. 1 to 2 ml. of dilute acid is usually sufficient.
Add 100 ru!. of benzidine solution (0.2%) and allow the precipitate to settle
for lO minutes. Filter on a small paper and wash with water saturated
with benzidine sulfate by pouring small amounts of the wash solution
around the top edge of the filter. Continue to wash until the top edge
of the filter and the drops of filtrate are neutral to litmus. . Transfer the
filter paper and precipitate to the original flask with about 50 m!. of water
and titrate at the boiling point with 0.1 N sodium hydroxide after the
addition of a few drops of phenolphthalein. Calculate the grams of sulfate
sulfur in the 24 hr. sample.
(23)
TOTAL SULFATE
The esters of sulfuric acid (commonly called ethereal sulfates) are hydrolyzed
by boiling with acid. The "total sulfate" is then determined as described in the
previous method.
Place 25 ml. of urine in a 250 rul. flask and acidify to Congo red paper
with dilute hydrochloric acid. Cover with a watch glass and boil gently
for 30 minutes adding water if necessary. Allow to cool and proceed as
directed under inorganic sulfates. The difference between the two determinations represents ethereal or conjugate sulfate.
(24)
TOTAL SULFUR
(Benedict's Method 1 modified)
All of the sulfur of the urine is oxidized to sulfate by heating with copper nitrate
and potassium chlorate and is then precipitated with benzidine as described in
the previous methods.
Benedict's Sulfur. Reagent. Crystalline copper nitrate, 200 g.; sodium
or potassium chlorate, 50 g.; distilled water to make 1 liter. A determination of the sulfur in the re~gent should be made.
The Determination. To 10 to 20 ml. of urine in a small porcelain evaporating dish, add 5 to 10 m!. of Benedict's sulfur reagent and evaporate on
the water bath or over a free flame just below the boiling point. When the
mixture is dry, increase the heat gradually until it blackens, fuses, and is
finally heated to dull redness. Continue heating for 10 minutes. Allow to
J
Benedict, S. R., J. B. C., 6, 363 (1009).
260
cool and dissolve in 10 to 20 m!. of 2 N hydrochloric acid using a little heat
if necessary. Evaporate the solution to dryness, dissolve the residue in
water and proceed as directed under inorganic sulfate.
The difference between the quantities of total sulfur and the sulfur estimated as total sulfate represents "neutral sulfur."
(25)
SUGAR
(Benedict's Method1)
Urine containing sugar is added to a boiling solution, which contains a known
quantity of copper, until all of the copper has been reduced as shown by the disappearance of the blue color. The formation of a white precipitate of cuprous thiocyanate (Cu SCN) aids in detecting the end point. Knowing the amount of sugar
necessary to reduce the copper under the conditions given, the quantity of sugar in
the urine may 1;Ie calculated.
Benedict's Quantitative Sugar Reagent. Dissolve 200 g. of sodium (or
potassium) citrate, 100 g. of anhydrous sodium carbonate and 125 g: of
potassium thiocyanate in about 800 m!. of water and filter if necessary.
Dissolve exactly 18.0 g. of crystalline copper sulfate in 100 m!. of water
and add it very slowly, with stirring, to the alkaline solution. Add 5 m!.
of 0.1 M potassium ferro cyanide and dilute to 1 liter in a volumetric flask.
10 ml. of the standard solution will be completely reduced by 20 mg. of
glucose.
The Determination. Dilute 10 m!. of urine to 100 m!. (unless the amount
of sugar present is small), and fill a burette with the diluted urine. Pipette
exactly 10 ml. of Benedict's reagent into a 100 ml. Erlenmeyer flask or an
evaporating dish, add about 5 gm. of anhydrous sodium carbonate (one
spoonful) and heat to boiling. When most of the carbonate has dissolved,
run the diluted urine from the burette into the boiling copper solution
rapidly at first, then more slowly and finally a few drops at a time until the
solution becomes colorless.
The solution should be practically saturated with sodium carbonate.
When a flask is used, more than 5 g. of sodium carbonate are required. Too
little sodium carbonate may result in the formation of a red precipitate.
When a dish is used, -water may need to be added to keep the suspension
from becoming too concentrated. The end point should be determined
without too much delay and while the solution is still hot, as reoxidation by
oxygen of the air may occur slowly. When urine is used, the end point is
not colorless but a slightly yellowish green.
1
Benedict, S. R., J. B. C., 9, 57 (1911).
Quick, A. J., J. Ind. Eng. Chern., 17, 729 (1925).
262
The amount of urine just necefisary to completely reduce the copper in
Calculate the 24 hour
output and the percentage of sugar in the urine.
10 m!. of the reagent contains 20 mg. of glucose.
(26)
ASCORBIC ACID
In acid (pH approximately 3.0), the dye, 2,6-dichlorophenol indophenol, may exis t
in two forms, as a red, oxidized form, or as a leuco (colorless) reduced form.
Cl
O=C)=N-L)-OH
+ 2 H+
+
2e
[oxidant), at pH 3.0
I
If
I
J
H*
*HO
(red)
Cl
Cl
I
,f'-~ ~ ,f'-~ OH
~~
(colorless)
[reductant] at pH 3.0
Cl
At pH 3.0 this system has a high potential, E~ = 0.488 volt.
At the same pH of 3.0, ascorbic acid may exist in two forms
OH H
OH"
I I
I
f:-~-CLC
o
OHH
OH*
C0
I
-2 H+ - 2 e
0
I I " "
r:-~-Cl-C-C
o
-0
H
0
0
---0
H
Ascorbic Acid
(Reductant)
Dehydroascorbic Acid
(Oxidant)
At pH 3.0 this system has a potential significantly lower than that of the dichlorophenol indophenol dye, namely E~ = 0.210 volt. Thus, the dye may oxidize the
ascorbic acid to dehydroascorbic acid, and at the same time the dichlorophenol
indophenol is reduced to the colorless form. In the process two hydrogen ions and
two electrons (i.e. two hydrogen atoms) are transferred from the ascorbic acid to
the dye. This is an excellent example of the operation of balanced oxidation-reduction reactions.
Advantage is taken of the above phenomena in the determination of ascorbic
acid in biological fluids. The solution containing the ascorbic aeid is titrated with
the dye until the production of the first "permanent" pink color (lasting about t
minute). The greater the content of ascorbic acid the gre~ter will be the quantity
264
of dyestuff reduced to the colorless form before a sufficient excess is added to yield
the pink color. Substances (dyes) such as dichlorophenol indophenol, whose oxidation-reduction potentials are well established, are oft!)n referred to as oxidationreduction indicators, since the change in color can be utilized in the estimation of
the potential of the system titrated by the dye.
Standard Dye Solution: A stock solution is made up to contain 100 mg.
of dichlorophenol indophenol per 50 ml. The dye is dissolved in recently
boiled, hot distilled water. The solution is kept in a refrigerator. The
working solution is made from the above by careful dilution of 10 ml. to
100 mL with recently boiled, cold distilled water. This solution contains
0.2 mg. of the dye per mL
Standard Ascorbic acid: A stock solution is made up to contain 50 mg.
per 100 ml. of 5 per cent acetic acid, and is kept in a refrigerator. The
working standard is made from the above by dilution of 2 ml. to 50 ml. with
5 per cent acetic acid. The working standard, which contains 0.02 mg.
.
of ascorbic acid per mI., is also kept in a refrigerator.
Standardization: 2 mL of the working standard of ascorbic acid (containing 0.04 mg. of ascorbic acid) are titrated with the working dye solution.
This titration may be designated ml s • (This titration may be of the order
of 0.40 mL In this case 0.04 mg. of ascorbic acid would be equivalent to
0.40 mL - 0.05 mL (blank) = 0.35 mL of the dye.)
Blank: Titrate a 1.3 ml. sample of the 5 per cent acetic acid with the dye.
This value must be substracted from the unknown. This titration may be
designated mlb. (The blank is usually of the order of 0.05 mL)
The Determination. Urine is collected over acetic acid (10 mL of glacial
acetic acid per liter of urine). This is done to prevent oxidation of the
ascorbic acid present by atmospheric oxygen, which would take place at
more alkaline pH.
Pipette 2 mL of urine into a small test tube. Add 4 mL of 5 per cent
acetic acid. Filter, if solution becomes turbid. Measure out 2 ml. of
this solution (or filtrate) into a second test tube, and. titrate with the dye
until the formatIon of the first "permanent" pink color. Be sure to mix
'very thoroughly during titration. The titration represents the "ascorbic
acid" content of i mL of urine. This titration may be designated mlu.
Calculation: The amount of ascorbic acid in mg. in the total volume of
urine, V in mL, may be calculated from the equation
"aCl
d = (I
mg. 0 f ascorbIC
mu
-
266
) ( 0.04
3 Vml
rnh·
I mg.I ) .-.
rn.-rnb 2
DIETARY DEFICIENCIES
The following experiments are for the purpose of demonstrating the
various dietary deficiencies in laboratory animals. Each student will be
required to prepare a ration for at least one group of animals. For the
records of the group he alone will be directly responsible over the period for
which he has prepared the ration. All data recorded on the bulletin board
must be transcribed in notebooks weekly. Daily pare of the animals will
be divided among members of the class.
(1) ·Rat Experiments. For these feeding trials casein, dextrin, agar,
yeast, cod liver oil, gelatin, wheat gluten, grains and salt mixtures will be
furnished ready for incorporation in the rations.
The casein is a commercial product obtained by acid from skimmed milk
and is extracted in the laboratory with successive portions of alcohol, then
dried in an oven. The dextrin is prepared by heating starch with waier
for two to three hours at 151bs. pressure and then dried. The agar, yeast,
alfalfa meal, gelatin and wheat gluten are commercial products. The
grains are merely finely ground. The salt mixtures are made from laboratory reagents mixed, dried and ground.
Salt ltfixture 35 consists of:
Salt 111ixturc 12 consists o/:'
grams
grams
NaC!. ............... '"
KH.PO •................
MgSO •.................
CaC0 3 • • • • • • • • • . • . . . • • .
FeSO.·7H.O ........... .
KI .................... .
MnSO.·2H.O .......... .
ZnCh ................. .
CuSO.·5H 20 ............ .
CoCh·6H.O ........... .
CaC0 3 • • • • • . • . . • • • • • • • • • • • • • •
NaC!.. ............... , . . .. ...
292.5
816.6
120.3
800.8
56.6
1.66
9.35
0.5452
0.9988
0.0476
1.5
1.0
Q
Ration I
Low Vitamin A
. Casein (alcohol extracted) . . . .
Agar.. . ......................
Yeast (irradiated). . .. .. .. . . ..
Salt Mixture 12. . . . . . . . . . . . . . .
Dextrin. .....................
pe, cent
18
2
5
4
il
Ration III
Poor Protein
Ration II
Low Vitamin Bl
p"",ent
percent
Casein (alcohol extracted) . . . . 18
Agar.........................
2
Cod Liver Oil. . . ... . .... .. .. . 4
Salt Mixture 12.. . . . . . . . . . . . ..
4
Dextrin containing liver extrnctt. . . . . . . . . . . . . . . . . . . . .. 20
Dextrin. ..................... 52
Gelatin... .. . . .. . . .. . . . . .. . . . . . 18
Agar.. ........................
2
Cod Liver Oil. . ... .. .... . .....
4
Salt Mixture 12.. . . . . . . . . . . . . . . 4
Yeast ............. " .. .... .. ..
5
Dextrin. ...................... 67
1 Liver extract, autoclaved and evaporated on dextrin furnishes B 2 • The liver
extract makes up about 2% of the diet.
268.
Ration IV
Low Vitamin D
(Steen bock Diet)
pe'UlIl
Yellow Corn.....................
Wheat gluten. . . . . . . . . . . . . . . . . . ..
CaCO a••••.•.••.•••..•••.••••.• ,
NaCl............................
76
20
3
1
(2) Guinea Pig Experiments. The guinea pig is one of the few known
animals satisfactory for the demonstration of the lack of the antiscorbutic
vitamin. Use Ration VI with and without 3 cc. of orange juice as supplement.
'
Ration VI
per
,en'
Rolled Oats. . . . . . . . . . . . . . . . . . . . . 80
Alfalfa Meal. . . . . . . . . . . . . . . . . . . .. 10
Casein .... :.....................
8
Salt Mixture 35.. .. . . . . . . . . . . . . . .
2
(3) Pigeon Experiments. The pigeon is satisfactorily used for the
demonstration of the lack of the anti polyneuritic vitamin. Feed polished
rice to contrast with whole .grains.
269
LOGARITHMS OF NUMBERS
Proportional parts
Natural
0
numbers
--- -
2
1
-
-
3
-
4
-
-
7
6
5
-
-
9
8
-
-
10
11
12
13
14
0000 0043 0086 0128 0170 0212 0253 0294 0334 0374
0414 0453 0492 0531 0569 0607 0645 0682 0719 0755
0792 0828 0864 0899 0934 0969 1004 1038 1072 1106
1139 1173 1206 1239 1271 1303 1335 1367 1399 1430
1461 1492 1523 1553 1584 1614 1644 1673 1703 1732
15
16
17
18'
19
1
2
3
4
12
11
10
10
9
17
15
14
13
12
--- -
-
5
7
25
23
21
19
18
29
26
24
23
21
g
9
4
4
3
3
3
8
8
7
6
6
1761 1790 1818 1847 1875 1903 1931 1959 1987 2014
2041 2068 2095 2122 2148 2175 2201 2227 2253 2279
2304 2330 2355 2380 2405 2430 2455 2480 2504 2529
2553 2577 2601 2625 2648 2672 2695 2718 2742 2765
2788 2810 2833 2856 2878 2900 2923 2945 2967 2989
3
3
2
2
2
1\
5
5
5
4
8 11 14 17 20 2225
8 11 13 16 18 2124
7 10 12 15 17 2022
7 9 12 14 16 1921
7 9 11 13 16 1820
20
21
22
23
24
3010 3032 3054 3075 3096 3118 3139 3160 3181 3201
3222 3243 3263 3284 3304 3324 3345 3365 3385 3404
3424 3444 3464 3483 3502 3522 3541 3560 3579 3598
~617 3636 3655 3674 3692 3711 3729 3747 3766 3784
3802 3820 3838 3856 3874 3892 3909 3927 3945 3962
2
2
2
2
2
4
4
4
4
4
6
6
6
6
5
8 11 13 15 1719
8 10 12 14 1618
8 10 12 14 1517
7 9 11 13 1517
7 9 11 12 1416
25
26
27
28
29
3979 3997 4014 4031 4048 4065 4082 4099 4IH~ 4133
4150 4166 4183 4200 4216 4232 4249 4265 4281 4298
4314 4330 4346 4362 4378 4393 4409 4425 4440 4456
4472 4487 4502 4518 4533 4548 4564 4579 4594 4609
4624 4639 4654 4669 4683 4698 4713 4728 4742 4757
2
2
2
2
1
3
3
3
3
3
5
5
5
5
4
7
7
6
6
6
9 10 12 1415
8 10 11 1315
8 9 11 1314
8 9 11 1214
7 9 10 1213
30
31
32
33
34
4771 4786 4800 4814 4829 4843 4857 4871 4886 4900
4914 4928 4942 4955 4969 4983 4997 5011 5024 5038
5052 5065 5079 5092 5105 5119 5132 5145 5159 5172
5185 5198 5211 5224 5237 5250 5263 5276 5289 5302
5315 5328 5340 5353 5366 5378 5391 5403 5416 5428
1 3
1
1
1
1
3
3
3
3
4
4
4
4
4
6
6
5
5
5
7
7
7
6
6
9 10 1113
8 10 11 12
8 9 1112
8 9 10 12
8 9 1011
35
36
37
38
39
5441 5453 5465 5478 5490 5502 5514 5527 5539 5551
5563 5575 5587 5599 5611 5623 5635 5647 5658 5670
5682 5694 5705 5717 5729 5740 5752 5763 5775 5786
5798 5809 5821 5832 5843 5855 5866 5877 5888 5899
5911 5922 5933 5944 5955 5966 5977 5988 5999 6010
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
5
5
5
5
4
6
6
6
6
5
7
7
7
7
7
9 10 11
8 10 11
8 910
8 910
8 910
40
41
42
43
44
6021 6031 6042 6053 6064 6075 6085 6096 6107 6117
6128 6138 6149 6160 6170 6180 6191 6201 6212 6222
6232 6243 6253 6263 6274 6284 6294 6304 6314 6325
6335 6345 6355 6365 6375 6385 6395 6405 6415 6425
6435 6444 6454 6464 6474 6484 6493 6503 6513 6522
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
8
7
7
7
7
910
8 9
8 9
8 9
8 9
45
46
47
48
49
6532 6542 6551 6561 6571 6580 6590 6599 6609 6618
6628 6637 6646 6656 6665 6675 6684 6693 6702 6712
6721 6730 6739 6749 6758 6767 6776 6785 6794 6803
6812 6821 6830 6839 6848 6857 6866 6875 6884 6893
6902 6911 6920 6928 6937 6946 6955 6964 6972 6981
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
4
4
6
6
5
5
5
7
7
6
6
6
8
7
7
7
7
8
50
51
52
53
54
6990 6998 7007 7016 7024 7033 7042 7050 7059 7067
7076 7084 7093 7101 7110 7118 7126 7135 7143 7152
7160 7168 7177 7185 7193 7202 7210 7218 7226 7235
7243 7251 7259 7267 7275 7284 7292 7300 7308 7316
7324 7332 7340 7348 7356 7364 7372 7380 7388 7396
1
1
1
1
1
2
2
2
2
2
3
3
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
7
6
6
8
8
7
7
7
270
21
19
17
16
15
6
- - - -
33 37
30 34
28 31
26 29
2427
9
8
8
S
LOGARITHMS OF
NUMBERs-Concluded
Pr,oportional parts
Natural
0
numbers
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
- - - - - - - - -- - - - - - - 7404 7412 7419 7427 7435 7443 7451 7459 7466 7474 1 2 2 3 4 5 5 6 7
--- -
-
55
56
57
58
59
7482 7490 7497 7505 7513 7520 7528 7536 7543 7551
7559 7566 7574 7582 7589 7597 7604 7612 7619 7627
7634 7642 7649 7657 7664 7672 7679 7686 7694 7701
7709 7716 7723 7731 7738 7745 7752 7760 7767 7774
1
1
1
1
2
2
1
1
2
2
2
2
3
3
3
3
4
4
4
4
5
5
4
4
5
5
5
5
6
6
6
6
7
7
7
7
60
61
62
63
64
7782 7789 7796 7803 7810 7818 7825 7832 7839 7846 1
7853 7860 7868 7875 7882 7889 7896 7903 7910 7917 1
7924 7931 7938 7945 7952 7959 7966 7973 7980 7987 1
7993 8000 8007 8014 8021 8028 8035 8041 8048 8055 '1
8062 8069 8075 8082 8089 8096 8102 8109 8116 8122 1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
5
5
6
6
6
6
6
65
66
67
68
8129 8136 8142 8149 8156 8162 8169 8176 8182 8189
8195 8202 8209 8215 8222 8228 8235 8241 8248 8254
8261 8267 8274 8280 8287 8293 8299 8306 8312 8319
8325 8331 8338 8344 8351 8357 8363 8370 8376 8382
8388 8395 8401 8407 8414 8420 8426 8432 8439 8445
1
1
1
1
1
1
2
1
1
2
1
1
2
2
3
3
3
3
2
3
3
3
3
3
4
4
4
4
4
5
5
5
4
4
5
5
5
5
5
6
6
6
6
6
72
73
74
8451 8457 8463 8470 8476 8482 8488 8494 8500 8506
8513 8519 8525 8531 8537 8543 8549 8555 8561 8567
8573 8579 8585 8591 8597 8603 8609 8615 8621 8627
8633 8639 8645 8651 8657 8663 8669 8675 8681 8686
8692 8698 8704 8710 8716 8722 8727 8733 8739 8745
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
6
5
5
5
5
75
76
77
78
79
8751 8756 8762 8768 8774 8779 8785 8791 8797 8802
8808 8814 8820 8825 8831 8837 8842 8848 8854 8859
8865 8871 8876 8882 8887 8893 8899 8904 8910 8915
8921 8927 8932 8938 8943 8949 8954 8960 8965 8971
8976 8982 8987 8993 8998 9004 9009 9015 9020 9025
1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
1
2
2
2
2
2
5
5
4
4
4
5
5
5
5
5
80
81
82
83
84
9031 9036 9042 9047 9053 9058 9063 9069 9074 9079
9085 9090 9096 9101 9106 9112 9117 9122 9128 9133
9138 9143 9149 9154 9159 9165 9170 9175 9180 9186
9191 9196 9201 9206 9212 9217 9222 9227 9232 9238
9243 9248 9253 9258 9263 9269 9274 9279 9284 9289
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
4
4
4
1
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
85
86
87
88
89
9294 9299 9304 9309 9315 9320 9325 9330 9335 9340
9345 9350 9355 9360 9365 9370 9375 9380 9385 9390
9395 9400 9405 9410 9415 9420 9425 9430 9435 9440
9445 9450 9455 9460 9465 9469 9474 9479 948-! 9489
9-!94 9499 9504 9509 9513 9518 9523 9528 9533 9538
1
1
0
0
0
1
1
1
1
2
2
1
2
2
2
3
3
3
1
2
1
1
2
3
3
2
2
2
3
4
4
3
3
3
4
4
4
4
4
5
5
4
4
4
90
91
92
93
94
9542 9547 9552 9557 9562 9566 9571 9576 9581 9586
9590 9595 9600 9605 9609 9614 9619 9624 9628 9633
9638 9643 9647 9652 9657 9661 9666 9671 9675 9680
9685 9689 9694 9699 9703 9708 9713 9717 9722 9727
9731 9736 9741 9745 9750 9754 9759 9763 9768 9773
0
0
0
0
0
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
1
1
1
1
1
1
95
96
97
98
99
9777 9782 9786 9791 9795 9800 9805 9809 9814 9818
9823 9827 9832 9836 98-!1 9845 9850 9854 9859 9863
9868 9872 9877 9881 9886 9890 9894 9899 9903 9908
9912 9917 9921 9926 9930 9934 9939 9943 9948 9952
9956 9961 9965 9969 9974 9978 9983 99S7 9991 9996
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
6~
70
71
271
1
1
1
l
2
3
4
4
4
4
4
4
4
4
4
4
4
3 4
ABRIDGED TABLE OF ATOMIC WEIGHTS,
Aluminum .. " ............
Antimony. . . . . . . . . . . . . . . ..
Arsenic ...................
Barium ..................
Bismuth ..................
Boron .................... .
Brominc ................. .
Cadmium ................ .
Calcium ...................
Carbon. . . . . . . . . . . . . . . . . ..
Chlorine ................. ,
Chromium ................
Cobalt..............
Copper...... .
Fluorine............
Gold ......................
Helium ...................
Hydrogen. . . . . . . . . . . . . . . ..
Iodine ....................
Iron .....................
Lead. . . . . . . . . . . . . . . . . . . . ..
0=16
Al 26.97
Sb 121.76
As 74.91
Ba 137.36
Bi 209.00
B
10.82
Br 79.916
Cd 112.41
Ca 40.08
C 12.010
CI 35.457
Cr 52.01
Co 58.94
Cu 63.57
F
19.00
Au 197.2
He 4.003
H I . 008
I 126.92
Fe 55.85
Pb 207.21
1945
Lithium ....................
Magncsium .................
Manganese .................
Mercury ...................
Molybdenum ...............
Nickel. ....................
Nitrogen ...................
Oxygen ....................
Phosphorus .................
Platinum. .. ....... . ..
Potassium .................
Silicon .....................
Silver ......................
Sodium ....................
Strontium ..................
Sulfur ......................
Tin. . . .. ..................
Tungsten ...................
lJrsnium ...................
Zinc .... , ..................
272
o = 16
Li
6.940
Mg 24.32
Mn 54.93
Hg 200.61
Mo 95.95
Ni 58.69
N 14.008
0
16.000
P
30.98
Pt 195.23
K 39.096
Si 28.06
Ag 107.88
Na 22.997
Sr 87.63
S
32.06
Sn 118.70
W 183.92
1] 238.07
Zn 65.38
INDEX
Acetoacetic acid in urine 234
Acetone in urine, 232
'
Acid hematin, 126
Acid, standard solution 18
Acidi.ty, titratable, of u'rine, determinabon of, 240, 242
- of gastric juice, determination of
100
'
Acrolein, 78
Adenine, 204
Adsorption, 46, 64
Albumins, 66
Alcohol from maltose, 92
Alcohols, 48
Aldehyde reaction for tryptophane 62
Aldehydes, 48
'
Alkali, standard solution 18
Alkali reserve of blood,' determination
of, 172
Alkaloidal reagents 64
Ammonia in urin~, determination of,
248,250
- test, 14
Amylase, pancreatic, 102
Arsenious sulfide, 44
Atomic weights, 272
Barfoed's test, 52
Benedict's test for sugar 50 230
Benzidine test, 118
"
Bile, 210, 212
Rile in urine, 234
Bilirubin, 212
- in serum, 178, 180
Biuret, 60
Blood, 116-184
- detection of, in urine 234
- determination of:
'
- - - carbon dioxide content 172 174
- - - chloride, 162,164
"
- - - cholesterol, 176, 178
- - - creatinine, 148, 150
- - - creatine, 148,150
- - - hemoglobin, 184
- - - inorganic phosphatc 164 166
- - - non-protein nitrogen', 140,142
- - - sugar, 152-162
- - - "true" sugar, 158
- - -.urea, 142-148
- - - urie acid, 150,152
- hemolysis, 116
- in urine, 234
- preparation of protein-frce filtrate 138
- plasm~, C92 combining capacity,' determmatlOn of, 168-172
- quantitative metlwds, 130-184
- serum, 126, 128
273
- - bilirubin, determination in 178 180
- - calcium, determination in,' 166; 168
- - phosphatase, determination in 180184
'
~one, 186-190
Buffer solutions, 28
Butter fat, 112
Calcium test, 10
Calcium phosphate from milk 110
Calcium in serum, determination of 166
168
'
,
Cane sugar, see sucrose
Carbohydrates, 50-58
Carbon dioxide, acidity of 36
- - combining capacity ~f plasma determination of, 168-172
'
- - content of blood, determination of
172,174
'
Carboxyhemoglobin, 122
Casein, 110, 112
- isoelectric point, determination of,
74
Cell nucleus, 202-208
Cellulose, 58
Chloride, test, 14, 214
- ~n bl?od, determination of, 162,164
- m urme, determination of 254
Cholesterol, 210
'
- in blood, 176, 178
Coagulation of proteins 62
- test in urine, 228
'
Colloids, 4G-46
- dialysis of, 40
- electrical charge, 42
- sol and gel forms, 42
Color comparator Duboscq 130
Creatine, preparation from'muscle 192
- conversion into creatinine 194 '
- in blood, determination or' 148 150
-:. in urine, determination of' 254'
Creatinine, preparation from' urine 218
- preparation of creatine from 220
- in blood, determination of 148 150
- in urine, determination of' 252'
Cystine, preparation of, 68 '
Dextrose, see glucose
Dialysis, 40, 66
Diazo reaction, 62
Dietary deficiencies, 268, 269
Diffusion thru gelatin, 40
Digestion, gastric, 98,100
- pancreatic, 102-106
- Bali vary, 90
Duboscq comparator, 130
Electric charge on colloidal particles, 42
Electrolytic dissociation, 22-38
Emulsification of fats, 78
Emulsoids,42
Enzymes, gastric, 98,100
Enzymes, pancreatic, 102, 104
- salivary, 90
Esters, 48
Fats, 78-86
- crystallization of, 78
- emulsification of, 78
- iodine number, determination of, 82
- saponification of, 80
- saponification number, determination
of,82
- solubility of, 78
Fehling's test, 50, 230
Fatty acids, preparation of, 80
Fermentation test, 52, 232
Ferric hydroxide, colloidal, 44
Ferricyanide test, 52
Folin's test for uric acid, 218
Formaldehyde titration, 72, 104
Free hydrochloric acid in gastric contents, determination of, 100
Fructose test, 54
Gastric analysis for free Hel and total
acidity, 100
- contents, lactic acid in, 96
- - hydrochloric acid in, 96
- digestion, 98
- juice, 96-104
Gelatin, 186
- diffusion through, 40
Gerhardt's test, 234
Glass electrode, 38
Globulin, cucurbit seed, 66
Globulins, 66
Glucose tests in urine, 230
Glycogen, experiments on, 58
- preparation of, 58
Gmelin's test, 210, 212, 234
Guaiac test, 122, 234
Guanine, 202
Gum arabic, 56
Gunning's iodoform test·, 232
Gunzperg's test, 96
Hydrochloric acid in gastric contents,
determination of, 100
- - standard solution, 18
Hydrogen electrode, 38
Hydrogen ion concentration, colorimetric determination, 28
- - - - sources of error, 32
- - - in saliva, determination of, 94
- - - in urine, determination of, 238
Hypoxanthine, isolation of, 194
Iodine number of fats, 82
- test, 56, 58
Indican, 226
Indicators, 26
- choice of, 26
Inorganic constitucnts, 8-16
- - in urine and bone, 16
Isoelectric point of casein, 74
Kjeldahl's method for total nitrogen, 242
Lactic acid, fermentation, isolation of,
112
.
- - identification of, 96
- - isolation from muscle, 198
- - separation, 98
Lactose, 110
Lassaigne test, 14
Le Nobel's test for acetoacetic acid, 234
Lipase, pancreatic, 102
Logarithm table, 270, 271
Magnesium test, 10
Mercuric nitrite test, 60
Metabolism, 2~6, 237
Methemoglobin, 122
Milk, 108-114
- clotting by rennin, 114
- proteins, 110, 114
- separation of constituents, 108
- sugar, see lactose
Millon's reaction, 60
Mineral constituents of bone, 186, 188
Molisch test, 50
.Mucic acid test, 54
lHucin in saliva, 88
Murexide test, 216
Muscle, 192-200
Mutarotation, 52
Neutral salts, reactions of solutions of, 28
Nitrogen tests, 14, 16
- in urine, determination of, 242, 244
- non-protein, of blood, determination
of, 140
Nucleic acid, 192
Nylander's test, 50, 230
Heller's test, 228
:Hematin, acid, 126
Hematoporphyrin, 126
Hemin, 118
Hemochromogens, 126
Hemoglobin, 116, 118, 122
- carboxy, 122
- oxy, 118, 122, 184
Hemolysis, 116
Hes,8e's Reaction, 210
Hippuric acid, isolation of, 224
Hopkins-Cole reaction, 62
Hopkin's Thiophene test, 96
Huppert's test, 234
Optical test (carbohydrate), 52, 232
Orcin test, 54
ORazone test, 52, 230
Oflsein, 186
.
Oxalic acid, standard solution, 18
Oxyhemoglobin, 118, 122
274
Pancreatic extract, 102
- juice, 102-106
Pauly's diazo reaction, 62
Pentose, tests, for, 54
Pepsin and pepsinogen, 98, 100
Peptic digestion, 98
Pettenkofer's test, 210
pH, see hydrogen ion concentration
Phloroglucin test, 54
Phosphatase, in blood serum, 180-184
Phosphate, tests, 8, 214
- in blood> determination of, 164, 166
- in urine, determination of, 256
Phosphoric acid in nucleic acid, 204
Phosphorus, partition in yeast nucleic
acid,206
- organic, test, 10
Photometer, photoelectric, 131-138
Pirias test, 104
Protein, preparation of a crystalline, 66
Proteins, 60-76
- color reactions, 60
- coagulation by heat, 62
- elementary composition, 60
- precipitation with various reagents, 64
- salting out, 64
- tests in urine, 228
Protein-free blood filtrate, 138
Polysaccharides, 56
Pyrrol test, 14
Sulfate, ethereal, in urine, determination of, 260
- inorganic, in urine, determination of,
258
- test, 12, 214
Sulfide test, 12
Sulfhemoglobin, 124
Sulfosalicylic acid test in urine, 228
Sulfur, organic, test, 12
- total, in urine, determination of, 260
- unoxidized, test, 14
Surface tension, 44
Suspensoids, 42
Thiophene test" 9 6 "
"
Titratable acidity of urme, determmation of, 240, 242
Titration curveS, determination of, 34
Toepfer's reagent, 96
Total acidity of gastric contents, determination of, 100
Total nitrogen in urine, determination
of, 242, 244
Total solids in urine, 238
Trichloracetic acid test in urine, 228
Tropaeolin 00, 96
Trypsin, 102
Tyrosine, preparation of, 68, 104
Uffelmann's reagent, 96
Urea in blood, determination of, 142-148
- in urine, determination of, 244, 246
isolation of, 222
Reaction of urine, 238,239
Uric acid in blood, determination of, 150,
Rennin, 114
152
Rothera's test, 232
- - in urine, determination of, 250
- - isolation of, 216
Saliva, 88-94
Urine, normal, qualitative, 214-226
- action 'on starch, 90
- pathological, qualitative, 228-234
- determination of pH, 94
- quantitative methods, 238-266
- inorganic tests on, 88
- determination of:
Salivary digestion, 90
- - - Ammonia, 248, 250
Saponification, 80
- - - Ascorbic acid, 264
Saponification number, determination - - - Chloride, 254
of,82
- - - Creatine, 254
Sarcolactic acid, 198
- - - Creatinine, 252
Scott-Wilson test, 232
- - - Hydrogen ion concentration,238
Schweitzer's reagent, 58
- - - Inorganic phosphate, 256,258
Seliwanoff's reaction, 54
- - - Inorganic sulfate, 258
Silver test for reducing sugar, 52
---pH,238
Soaps, 80
-" - - Reaction to litmus, 238
Sodium hydroxide standard solution, 18 - - - Specific gravity, 238
Sorensen's formaldehyde titration, 72 - - - Sugar, 262
Specific gravity of urine, 238
- - - Titratable acidity, 240, 242
Spectroscopic examinations, 118
- - - Total nitrogen, 242, 244
Spiegler's test, 230
- - - Total solids, 238
Standard acid and alkali, 18, 20
- - - Total sulfate, 260
Starch grains, 56
- - - Total sulfur, 260
- hydrolysis, 56
- - - Urea, 244, 246
- iodine test, 56
- - - Uric acid, 250
- paste, 56
- - - Volume, 238
Sucrose, inversion of, 56
Sugar in blood, determination of ,1 52- Weyl's reaction, 220
162
Xanthine, 198
- in urine, determination of, 262
Xanthoproteic reaction, 60
275