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Unit 2
Spectrophotometry
This lab uses the following hazardous chemicals:
I. Biuret Indicator Reagent
As a result students are required to wear, at minimum, Goggles & Gloves.
Abstract:
When you look at a green leaf, it appears green to you because of the leaf pigments’
ability to absorb red and blue/violet and transmit green visible light. In this lab you will
learn about the spectrophotometer, an instrument used to detect light energy that is
transmitted by various substances. The spectrophotometer is useful because the
absorption and transmission of light energy, the basis for one of the most widely used
procedures for determining the concentration of substances in a solution. You will
learn to use the spectrophotometer, become familiar with its parts, and use this
instrument to determine the concentration of an unknown substance through the
construction of a standard curve. You will use this instrument several times during this
laboratory course, so make sure that you become comfortable in using this instrument!
You will also learn the principles of the Scientific Method, a series of steps commonly
used to make observations, formulate hypotheses, carry out experiments and interpret
the data generated from these experiments. Throughout this manual we will test your
understanding of the definition and application of various terms that apply to this
technique. At the end of this Unit you will find a list of definitions and explanations
regarding the Scientific Method. Use it to answer some of the questions throughout
experiment 2.2. In future labs, anytime you see “SM” by a question, use the Scientific
Method index to help you answer the question.
2.1 Relationship between Wavelength and Color
Introduction:
Visible light is only a small portion of a broad spectrum of available energy called
electromagnetic radiation. Electromagnetic radiation travels in the form of a wave, with
the distance between two crests termed a wavelength. Visible light consists of light
wavelengths from approximately 380-720 nanometers. Shorter wavelengths have higher
frequency and higher associated energy.
It is important for you to be familiar with the approximate wavelengths for the colors of
the visible spectrum. To do this, you will utilize the spectrophotometer which is
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comprised of: 1) a light source; 2) a focusing device that transmits an intense straight
beam of light; 3) a monochromator to separate the beam of light into its component
wavelengths; 4) a device for selecting the desired wavelength; 5) a sample holder; 6) a
photoelectric detector; and 7) a meter to display the output of the detector.
Note
Students will be using one of two types of spectrophotometers in this
lab. The two spectrophotometers are shown below. Identify the one that
you will be using, and make sure that you are following the instructions
for that particular spectrophotometer.
Spectronic 200
(Used at SCC)
Spectronic 20D
(Used at CPC and PRC)
Materials and Methods
Spectronic 20D (CPC and PRC)
1. Locate the following on the spectrophotometer. As you complete the lab, come
back to this section and record its use:
a. On/Off Switch and zero control knobb. Mode buttonc. Wavelength control knobd. Sample holdere. 100%T knob-
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2. Take a strip of provided white paper and place it into the tube. Insert the tube into
the sample holder so that the paper is perpendicular to the direction of the light.
Leave the sample holder open!
3. Set the wavelength control knob to 700nm.
4. Turn the 0 %T and the 100 %T control knob all the way to the right until they
cannot be turned anymore. (%T is percent transmittance)
5. Look into the sample holder to see the light on the paper.
6. Decrease the wavelength by slowly turning the wavelength control knob, and
record the range of wavelengths at which you see a particular color.
Spectronic 200 (SCC)
1. Locate the following on the spectrophotometer. As you complete the lab, come
back to this section and record its use:
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Display
Storage
Compartment
Sample Holder
Home
Button
Zero button
Enter
Button
Wavelength
Knob
a. On/Off Switch
b. Wavelength control knob c. Zero buttond. Home buttone. Enter buttonf. Sample Holder
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2. Turn on the spectrophotometer using the on/off switch on the back of the
machine, allow it to initialize, and select “Spec 200 Modern Interface”.
3. On the home screen use the arrow buttons, the enter button and the wavelength
knob to set the spectrophotometer to the following settings:
a. Application: Live Display
b. Measurement Mode: Abs
c. Measurement λ: 620 nm
4. Move down to “Go” (it will be highlighted green) and press the enter button
5. You should see a bar at the bottom of the display representing the visible
spectrum of light. Slowly turn the wavelength knob and you will notice that the
pointer arrow moves along the colors as you change the wavelength.
6. Record the starting and ending wavelengths that correspond to each color of the
visible spectrum of light in Figure 1.
Results:
Color
Starting Wavelength (nm)
Ending Wavelength (nm)
Red
Orange
Yellow
Green
Blue
Violet
Figure 1. Wavelength ranges corresponding to the colors of the visible spectrum. Figure 2. Wavelengths corresponding to the colors of the visible spectrum. 22
Tip
It is important that you learn the approximate wavelength range associated with
each color!
2.2 Determine the concentration of an unknown
substance by constructing a Standard Curve
Introduction:
When light of specific wavelength (color) is passed through a solution, some of the light
energy will be absorbed. The remaining light energy is detected and expressed on a meter
as either percent transmittance (%T) or absorbance (A). The spectrophotometer can be
utilized to determine the concentration of a solution using a set of standards of known
concentration. A higher concentration means a larger amount of light will be absorbed.
By graphing a standard curve using the absorbance you determine for each standard, the
unknown concentration can be extrapolated.
Materials and Methods
Follow these directions carefully. Do not rush, or you will end up having to do the entire
experiment again!
Based upon my understanding of the affects of concentration on absorbance, I
hypothesize that tube with the ______________________ concentration will have the
highest absorbance because _________________________________________________
_______________________________________________________________________.
Furthermore, I hypothesize that the tube with the ____________________ concentration
will have the lowest absorbance because _______________________________________
________________________________________________________________________
_______________________________________________________________________.
Scientific Method
The independent variable in this experiment is ____________________________.
The dependent variable in this experiment is ______________________________.
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Spectronic 20D (CPC and PRC)
Turn on the spectrophotometer.
1. If you turned the machine off in the previous experiment, turn the power
switch/zero control knob on and allow the instrument to warm up for 5 minutes.
2. Turn the 100%T knob counterclockwise until it cannot be turned anymore.
3. With the sample holder empty and the lid closed, turn the 0%T knob
counterclockwise until the %T shown on the display is 0.0.
Prepare your samples.
4. Obtain 7 test tubes.
a. Label one of them “U” for “unknown”.
b. Label one “B” for “blank”.
c. Label the remaining tubes #1-#5.
(Steps 3-9 pertain ONLY to tubes #1-#5)
5. Add 1 ml of distilled water to tubes #2-#5.
6. To tube #1, add 1 ml of standard protein solution. Record the concentration of the
standard protein solution here: ___________________________
Check your understanding
At this point, what is the difference between tube #1 and tubes #2-#5?
__________________________________________________
7. To tube #2, add 1 ml of standard protein solution. Shake gently.
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8. With a fresh pipette, remove 1 ml from tube #2 and add it to tube #3. Shake
gently.
9. With a fresh pipette, remove 1 ml from tube #3 and add it to tube #4. Shake
gently.
10. With a fresh pipette, remove 1 ml from tube #4 and add it to tube #5. Shake
gently.
11. With a fresh pipette, remove 1 ml of fluid from tube #5 and discard the fluid. At
this point, you should have approximately the same amount of fluid in each of
tubes #1-#5.
Check your understanding
What happened to the concentration of protein in each subsequent tube
as you proceeded from step 5 through step 8? (check with your instructor
if you are not sure)
___________________________________________________________________
12. Add 1 ml of unknown protein to tube “U”.
13. Add 1 ml of water to tube “B”.
Check your understanding
What is the difference between tube “B” and all of the rest of the tubes?
Why will you use this tube as your “blank”?
_________________________________________________________________
In the next step you will be adding Biuret, which is a hazardous substance. Goggles
and Gloves must be worn from this point on!
14. To ALL tubes that you have prepared, add 5 mls of Biuret reagent and allow the
color to develop for 5 minutes.
Use the spectrophotometer to determine the absorbance (A) for each sample (tube).
15. Adjust the wavelength control knob until the digital display shows 540 nm.
(Make sure the filter lever is in the correct wavelength range)
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16. Make sure that you use the zero control knob to set transmittance to “0” before
inserting your blank. Insert the “blank” into the sample holder and close the lid.
Adjust the meter reading to 100% transmittance using the transmittance control
knob. You have now “calibrated” the spectrophotometer. Note that if the
wavelength is changed, you must recalibrate by resetting 100% transmittance. If
the instrument if operated for an extended period of time, periodically check to
see that the machine is properly calibrated.
Check your understanding
Why do you set the “blank” to 100%T?
____________________________________________________________________
Tip
If you are not sure, think about transmittance vs. absorbance. If %T is 100%, how
much is A? Is there anything in your blank tube that should attribute an absorbance
value?
17. Use the mode button to switch to absorbance. Record the value in the data table
provided below.
18. Remove the “blank” tube from the sample holder and determine the absorbance of
each of your other tubes (one at a time) by:
a. removing fingerprints from the test tube with a soft wipe
b. inserting each tube into the sample holder
c. closing the lid and
d. recording the reading from the digital display in figure 2.
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Spectronic 200 (SCC)
Turn on the spectrophotometer.
1. Turn on the spectrophotometer, allow it to initialize and select “Spec 200 Modern
Interface”.
2. On the home screen use the arrow buttons, the enter button and the wavelength
knob to set the spectrophotometer to the following settings:
a. Application: Live Display
b. Measurement Mode: Abs
c. Measurement λ: 540 nm
Prepare your samples.
3. Obtain 7 test tubes.
a. Label one of them “U” for “unknown”.
b. Label one “B” for “blank”.
c. Label the remaining tubes #1-#5.
(Steps 3-9 pertain ONLY to tubes #1-#5)
4. Add 1 ml of distilled water to tubes #2-#5.
5. To tube #1, add 1 ml of standard protein solution. Record the concentration of the
standard protein solution here: ___________________________
Check your understanding
At this point, what is the difference between tube #1 and tubes #2-#5?
__________________________________________________
6. To tube #2, add 1 ml of standard protein solution. Shake gently.
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7. With a fresh pipette, remove 1 ml from tube #2 and add it to tube #3. Shake
gently.
8. With a fresh pipette, remove 1 ml from tube #3 and add it to tube #4. Shake
gently.
9. With a fresh pipette, remove 1 ml from tube #4 and add it to tube #5. Shake
gently.
10. With a fresh pipette, remove 1 ml of fluid from tube #5 and discard the fluid. At
this point, you should have approximately the same amount of fluid in each of
tubes #1-#5.
Check your understanding
What happened to the concentration of protein in each subsequent tube as
you proceeded from step 5 through step 8? (check with your instructor if you
are not sure)
___________________________________________________________________
11. Add 1 ml of unknown protein to tube “U”.
12. Add 1 ml of water to tube “B”.
Check your understanding
What is the difference between tube “B” and all of the rest of the tubes?
Why will you use this tube as your “blank”?
_________________________________________________________________
In the next step you will be adding Biuret, which is a hazardous substance. Goggles
and Gloves must be worn from this point on!
13. To ALL tubes that you have prepared, add 5 mls of Biuret reagent and allow the
color to develop for 5 minutes.
Use the spectrophotometer to determine the absorbance (A) for each sample (cuvette).
14. Place the blank sample in the diamond shaped sample holder.
15. Once the blank is oriented correctly, close the lid of the sample holder and press
the Zero button to set the blank to 0.00 Absorbance.
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Check your understanding
Why do you set the blank to 0.00 Absorbance?
___________________________________________________________________
Tip
If the blank is set to zero absorbance, what does this mean in terms of the percent
of light that will be transmitted through the cuvette (%T)? Is there anything in your
blank cuvette that should attribute an absorbance value?
16. Remove the “blank” test tube from the sample holder and determine the
absorbance of each of your other test tube (one at a time) by:
a. removing fingerprints from the cuvette with a soft wipe
b. inserting each test tube into the sample holder
c. closing the lid and
d. recording the reading from the digital display in Figure 2.
Results:
Serial Dilution
Tube #
1
mg/ml protein
Absorbance
0
0.00
2
3
4
5
B (blank)
“U” (Unknown)
Figure 3. Comparative concentrations and absorbances in known and unknown samples. 29
Tip
It is important to note that you must construct a “fresh” standard curve
every time you are determining the concentration of unknowns. You should
prepare the standards and your unknowns together. You cannot use a
friend’s standard curve and expect accurate results for your unknown!
To construct your standard curve, you will plot the absorbance of each dilution on
the Y-axis against the concentration of each dilution on the X-axis, as shown in
figure 4. You will draw a line of best fit using the data points you plot. By drawing
a straight line from (a) to the curve at (b) and dropping from the curve to (C), the
concentration of an unknown solution can be determined.
Figure 4. Using a standard curve to determine the concentration of an unknown protein sample. 30
Use the blank graph provided in Figure 5 to create your standard curve by plotting each
of the absorbance values that you determined from tubes #1-#5. Make sure that you label
both the X axis and the Y
Figure 5. Standard curve correlating sample concentration to absorbance. Conclusions
What is the concentration of your unknown (include units!)? ___________________
Points for Discussion:
1. Why are you not able to use a standard curve generated in one lab to assess the
concentration of an unknown in another lab? List at least 2 reasons.
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2. How does the way you connect the data points affect your conclusions about the
concentration of your unknown? If all of the data points lie in a straight line
except for one, should you ignore the one point or shift the line to connect the
points?
3. Would a hand generated line of best fit or a computer generated line yield the
most accurate results? Why?
4. Using the standard curve generator provided by your instructor, create a computer
generated graph with your data. Extrapolate the concentration of your unknown
from the computer generated graph. How does this concentration compare to your
answer from your hand drawn graph?
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Index
Color
Wavelength
Violet
380-450 nm
Blue
450-500 nm
Green
500-570 nm
Yellow
570-590 nm
Orange
590-620 nm
Supplemental Figure 2. Abridged Red
620-750 nm
Overview of the Scientific Method Supplemental Figure 1. Colors of the visible spectrum and their correlative wavelength ranges The Scientific Method is a series of steps that enable researchers to ask a question based
off of observations they have made, hypothesize potential answers to that question, and
then perform and interpret experiments to determine if their hypotheses were supported
or nullified.
Important Terms
Independent Variable: The variable in an experiment that is being manipulated by the
researcher.
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Example: A tube containing a certain chemical reaction is placed at a variety of different
temperatures ranging from 0-100 C to see what effect the change in temperature may
have on the products produced by the reaction. Independent Variable: Temperature
Tip
An easy way to think about the Independent variable is that it begins with
I, and I, the researcher, am the one who changes this variable at the offset
of the experiment.
Dependent Variable: The variable in an experiment that has the potential to change in
response to the experimental conditions altered by the researcher. The dependent variable
often times is defined as the variable that changes in response to the independent
variable.
Example: A tube containing a certain chemical reaction is placed at a variety of different
temperatures ranging from 0-100 C to see what effect the change in temperature may
have on the products produced by the reaction. Dependent Variable: Products
produced by the reaction.
Hypothesis: A hypothesis is not an educated guess, but instead is a statement based off
of previously supported data. A hypothesis must be falsifiable, and therefore must
provide a means to test and either support or nullify the hypothesis. Note that one does
not “prove” a hypothesis as being true, but instead determines whether or not the
hypothesis is supported by the data obtained from experiments.
Example: Based on the previously observed data found in the lab manual, I hypothesize
that if deionized water reaches a temperature above 100 C, it will begin to boil. Notice
that the hypothesis is based on previous results, and that it provides the researcher
with a means of proving or nullifying it. If water is placed at or above 100 C and it
boils, it supports the hypothesis, but if it does not boil, then it will nullify this
hypothesis.
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