Introduction
Different concerns with organic and inorganic
analysis
1. Organic analysis use extraction and elemental analysis
use digestion
2. Almost all trace organic analysis need clean-up and
enrichment. Elemental analysis in many cases can do direct
instrumental determination. Only metal, geological sample
and sea water need enrichment/clean up.
3. A clean environment is very critical for ultra trace
elemental analysis but has much less effect on organic
trace analysis
4. In case of organic trace analysis, the result need
qualitative confirmation using a different method.
Sample extraction techniques
Sample
collection
Plants, animal tissues and food
Solvent extraction with a Warning Blender
Extraction/
Digestion
Aqueous samples
Separatory funnel liquid-liquid extraction; solid-phase extraction (SPE);
solid-phase microextraction (SPME); Purge & Trap (volatile analytes
only).
Cleanup/
Enrichment
Solid samples (soil)
Soxhlet extraction; supercritical fluid extraction (SFE), solvent extraction
by tumbling or shaking, microwave assisted extraction.
Instrument
analysis
Result
Confirmation
Dr. Wan Hai Bin
CM4241- Trace Analysis
1
Dr. Wan Hai Bin
Sample preparation for organics
Blend with solvent
Suitable for plants, animal tissues, and
food. Sometime used for soil samples
Normal procedure:
1) Blend 50-100 g sample with 100-200
mL of solvent for 1-3 min;
2) Suction-filter the mixture in a
Buchner porcelain funnel ;
3) Blend the filter cake a second time
with another 100-200 mL solvent;
4) Combine the two filtrates for further
treatment.
Extraction techniques
Cleanup techniques
Enrichment techniques
Integrated techniques
Requirements:
understand the principles, advantages and disadvantages of important
techniques.
CM4241- Trace Analysis
3
Sample extraction techniques
Dr. Haibin Wan
email: [email protected] or [email protected]
Tel: 65616602 (home)
Dr. Wan Hai Bin
CM4241- Trace Analysis
2
Dr. Wan Hai Bin
CM4241- Trace Analysis
4
Sample extraction techniques
Sample extraction techniques
Extraction solvents
Use solvents that are miscible with water (acetonitrile, acetone) which
is easier to penetrate into the cell and get the analytes out. Solvents of
low polarity (e.g. hexane, toluene) will give low extraction efficiency.
In case of high fat sample, low polarity solvents can be used
Soxhlet extraction
For animal tissue, some Celite (a type of inorganic powder) or
cellulose can be added to facilitate filtration of the sticky mixtures.
Exhaustive extraction and gives highest extraction efficiency
Advantages
Often used as bench mark for evaluation of other extraction methods
This technique is suitable for solid samples only.
Suitable analytes are semi volatile organic compounds, pesticides, PCBs
Advantages
Well established technique
Fast, simple, low cost, and widely used.
Disadvantages
Disadvantages
Very time consuming (12-48 hours)
In case of some stubborn analytes, this extraction may not be complete.
Consumes more solvents than other extraction techniques.
Dr. Wan Hai Bin
CM4241- Trace Analysis
Some thermal labile analytes may be decomposed due to the long
extraction time and hot solvent
5
Sample extraction techniques
Dr. Wan Hai Bin
CM4241- Trace Analysis
7
Sample extraction techniques
Soxhlet Extraction
1) Sample is placed inside a cellulose or
ceramic thimble and placed in the
extractor.
Supercritical Fluid Extraction
Above certain temperature and
pressure, a substance will behave
neither like a gas nor a liquid. This
point on a phase diagram is called
critical point for this substance.
The temperature at the critical
point is called critical
temperature. The area above and
to the right of the critical point is
called supercritical region.
Condenser
2) Solvent in the flask is boiled and the
vapor rises to the condenser through the
bypass arm.
3) Solvent vapor condense to become
warm liquid and percolate through the
sample in the thimble and returns to the
flask.
4) The solvent re-boils, and the cycle is
repeated until the sample is completely
extracted, and the extract is in the lower
flask.
Dr. Wan Hai Bin
Supercritical
region
A liquid in this region is called
supercritical fluid.
CM4241- Trace Analysis
6
Dr. Wan Hai Bin
CM4241- Trace Analysis
8
Sample extraction techniques
Sample extraction techniques
Properties of supercritical fluid
Application
Solvent strength that can dissolve many substances
For solid matrix and analytes of low polarity.
Still not widely used.
Reason: There is no urgent need as other familiar techniques can meet
most of the needs. The instruments are still not robust enough for
routine analysis.
High diffusivity to allow fast mixing
Low viscosity to allow fast penetration of solid matrix
Application to sample extraction
Many substance can become supercritical fluids at their supercritical
points (ethylene, 9.3 degrees/50 bar; water 374 degrees/220 bar).
Advantages of SFE
No waste disposal problem and environment friendly
Fast extraction (<30 minutes)
The extraction power can be adjusted by changing the pressure or
adding modifiers (such as methanol).
CO2 becomes the best choice due to its low cost, safety, and
convenience (31 degrees and 73 atm is easy to achieve).
Disadvantages
The instruments available are still not robust enough
Extraction is mainly for analytes of low polarity
Dr. Wan Hai Bin
CM4241- Trace Analysis
9
Dr. Wan Hai Bin
11
CM4241- Trace Analysis
Sample extraction techniques
Sample extraction techniques
Solid-phase extraction (liquid-solid extraction)
A technique for extracting organics from water
Normal Steps
1. Adjust pH of the sample if necessary
2. Conditioning with a solvent compatible
with the sorbent (e.g. methanol)
3. Add water sample to the cartridge
4. After all the sample has gone through
the cartridge, blow away extra water
5. Rinse with a solvent to bring
the analytes down
6. Concentrate the eluate if necessary
Supercritical Fluid Extraction Instrumentation
Dr. Wan Hai Bin
CM4241- Trace Analysis
10
Dr. Wan Hai Bin
Sorbent
Vacuum
CM4241- Trace Analysis
12
Solid phase extraction
Sample extraction techniques
Why adjust pH?
Most common sorbent is silica
bonded with C18. This sorbent has
stronger adsorption to nonpolar
compounds (it is like a reverse
phase C18 column)
Some analytes have different retention at different pH.
The pH can affect stability of some analytes
The extraction material (normally bonded with C18 or other groups) may
break down if the pH is not appropriate.
Graphtized carbon black is used for
trapping polar compounds
Why conditioning?
The extraction material is normally highly hydrophobic (does not like
water). You need a solvent to bring the two together (better reaction). It
also help to remove contamination from the system.
There are also some cartridges
packed with silica gel, florisil, or
alumina. They are normally used
for cleanup purpose
Dr. Wan Hai Bin
CM4241- Trace Analysis
13
Dr. Wan Hai Bin
Sample extraction techniques
CM4241- Trace Analysis
15
Sample extraction techniques
Solid phase extraction using disk extraction media
Advantages of solid phase extraction
1. Assemble the apparatus. Add filter aid (small fibrous
particle) to prevent clogging
Consume very little organic solvent
2. Adjust pH of sample if necessary
There is no emulsification problem (often occurred in liquid/liquid
extraction)
3. Condition the extraction disk with an organic solvent
(methylene chloride followed by a water miscible solvent
such as methanol and acetonitrile)
Easy to be automated
4. Add sample continuously.
The resulting sample is of very small volume, so concentration is fast or
unnecessary
5. After all the sample has passed the disk, blow dry the disk
Disadvantages
6.Rinse the analytes down with organic solvents. Use a water
miscible solvent first then a less polar solvent.
In case of dirty water, the sorbent can be easily saturated or blocked.
Some components in natural water may reduce the recovery
7. Concentrate the eluate if necessary
Dr. Wan Hai Bin
CM4241- Trace Analysis
14
Dr. Wan Hai Bin
CM4241- Trace Analysis
16
Sample extraction techniques
Sample extraction techniques
Microwave assisted extraction
Microwave assisted extraction normal steps
Microwave is a radiation with wavelength between infrared and radio
wave.
Dry the sample by air dry or mixing with anhydrous sodium sulphate
It can heat up materials by acting on the dipoles of molecules and
movement of ions
Allow the sample to cool down
Extraction for ca 10 min at preset temperature and pressure
Advantages
Materials of high polarity is most effective absorber of microwave energy
(e.g. water, acetone, methanol)
Fast extraction, recovery comparable to Soxhlet extraction, much less solvent
consumption (10% of amount used in Soxhlet or shaking extraction).
In microwave assisted extraction, the extraction is accelerated by fast and
homogenous heating.
Disadvantages
Pressurized container allows higher extraction temperature.
Sample drying process can be long (several hours).
Cooling down also need half an hour or longer
Some thermal labile analytes can be decomposed
Only suitable for dry solid samples
Dr. Wan Hai Bin
CM4241- Trace Analysis
17
Dr. Wan Hai Bin
Sample extraction techniques
CM4241- Trace Analysis
19
Sample cleanup techniques
Purpose of cleanup
Microwave assisted extraction
Instrumentation requirement
To remove substances that will adversely affect analysis (it is not for
getting pure analytes).
Can control temperature and pressure of the extraction
These substance can either affect performance of the instruments
Stir during the extraction
or interfere with the separation/detection of analytes
Safety feature like solvent leak sensor and over pressure vent
Considerations in selection of cleanup techniques
Properties of sample matrix (type of interference)
properties of analytes
Concentration level to be analyzed
final instrumental analysis
Dr. Wan Hai Bin
CM4241- Trace Analysis
18
Dr. Wan Hai Bin
CM4241- Trace Analysis
20
Sample cleanup techniques
Sample cleanup techniques
Selection of the two phases
Available cleanup techniques
The extracts are normally polar,
water miscible but can also
dissolve in non polar solvents
liquid/liquid partition
Column chromatography using various adsorbents
The other solvent for partitioning
has to be less polar and not
miscible with water
Gel permeation chromatography
Co-distillation
To allow phase separation, the
extracts need to be diluted with
water. NaCl may be added to salt
out the analytes and facilitate the
phase separation.
Ideal
situation
Weak
solvent
Strong
solvent
The extraction strength need to be
considered also.
Dr. Wan Hai Bin CM4241- Trace Analysis
21
Sample cleanup techniques
Dr. Wan Hai Bin
CM4241- Trace Analysis
23
Sample cleanup techniques
The extraction strength of solvents
Liquid/liquid partition
ethyl acetate>CHCl3>CH2Cl2>toluene>hexane>petroleum ether
Separation is based on the difference in solubility between the analytes
and the interference.
Commonly used solvents for partitioning
Advantages
CH2Cl2
easily concentrated due to low boiling point (+). Easy to
remove as it stays at the bottom of the funnel (+), appropriate strength for most
analytes (+). Much higher density than water make the phase separation fast and
clear (+). It may affect instrument analysis as some GC detectors does not like it
(-) .
Simple technique, low cost, widely used.
Disadvantages
Cannot remove interfering substances of similar solubility as analytes.
Hexane easily concentrated due to low boiling points (+), appropriate strength
for non polar analytes (+), low toxicity (+), not good for extraction of polar
analytes.
May become difficult to separate due to emulsification
The shaking duration and extent affect the recovery.
Petroleum ether (saturated hydrocarbon with boiling points 30-60 degrees or
60-90 degrees) Low cost (+), mainly for non polar analytes, extraction rate
not as good as hexane.
Dr. Wan Hai Bin CM4241- Trace Analysis
22
Dr. Wan Hai Bin
CM4241- Trace Analysis
24
Sample cleanup techniques
Sample cleanup techniques
Commonly used adsorbents
Cleanup by gel permeation chromatography
Alumina (Basic) Good for basic and neutral analytes(+), can cause
polymerization and hydrolysis to some compounds, cannot use acetone and
ethyl acetate as eluate.
The separation is based on size of molecules.
Large molecules come out first.
Alumina (neutral) Good for neutral and reactive compounds (aldehydes,
ketones, esters; less active than basic and acidic Alumina.
A very effective method for removing lipids,
fats, proteins, polymers, resins and other large
molecules
Alumina (acidic) Good for acidic analytes (they may be strongly adsorbed
by neutral or basic alumina)
These material can block HPLC or GC
columns and contaminate the GC inlet system.
Florisil A magnesium silicate type adsorbent with acidic properties
(Florisil is the trade name from Floridin Co.); widely used for pesticides,
chlorinated hydrocarbons, phthalate esters, nitrosamines, notroaromatics.
(start with Florisil if not sure what adsorbent to use).
Can be used with almost any type of organic
analytes (no matter polar or non polar)
The sample after this step is normally ready for
instrumental analysis
Silica gel A slightly acidic adsorbent, good for phenolic compounds and
PCBs.
Dr. Wan Hai Bin
CM4241- Trace Analysis
flow
26
Dr. Wan Hai Bin
28
CM4241- Trace Analysis
Sample cleanup techniques
Sample cleanup techniques
Packings for the GPC column
Alumina and Florisil: heat at 450-500 degrees overnight and then deactivate
to required activity by adding certain amount of water (2-6%)
Porous particles.
Sephadex LH-20
Silica gel: Heat at 130-140 degrees overnight followed by deactivation with
water (up to 10%).
SX-3 Biobeads (a cross-linked divinylbenzens-styrene copolymer)
They are all polar material. The separation of the analytes from the
interfering substances is based on the difference in polarity.
The columns can be used many times (allowing automation)
The eluant must be organic solvent and dissolve the sample extracts well.
As they are all very polar and have strong adsorption to polar
compounds, the recovery of polar analytes could be low.
Procedures Swell the packing and then pack the column (dimension 0.6m
x 25mm id); calibrate the column; add sample extract (c.a. 1 mL); elute the
column; collect the fraction containing the analytes; concentrate if necessary.
Limits
GPC cleanup cannot remove interfering substances of similar molecular size
as the analytes. The interference may be removed by using suitable column
or selective detectors.
Dr. Wan Hai Bin
CM4241- Trace Analysis
27
Dr. Wan Hai Bin
29
CM4241- Trace Analysis
Integrated techniques
Sample enrichment techniques
Application of SPME
Aqueous samples or headspace samples. The extracted
analytes can be introduced into a GC or a HPLC system.
Concentrate using Rotary evaporator
Fast (+), Low temperature (+), easy to dry out
(-), may lose sample if too fast (-)
Advantages
Snyder
column
Almost solvent free, easy to be automated, easy operation,
normally no further cleanup is needed, low cost.
Concentrate using Kuderna-Danish
concentrator
High recovery (+), can go down to very
small volume (0.5 mL), slower than rotary
evaporator (-)
Concentration
flask
Concentration by blowing nitrogen
can be used to reduce volume further (0.2
mL). Tedious (-)
Receiver
Water
bath
SPE also includes enrichment
Dr. Wan Hai Bin
Headspace
Disadvantages
CM4241- Trace Analysis
The sorbent only absorb a fraction of the analytes from
the sample. The quantitation is based on the distribution
equilibrium of analytes between the aqueous phase and
the sorbent.
Not very good for quantitation (more suitable for pre
screening), the results can be affected by the sample
matrix, carryover from previous run if not treated
properly.
30
Dr. Wan Hai Bin
Integrated techniques
SPME Procedures:
SPME using a stirring bar
1. Immerse the sorbent segment in
the water sample and stir the
sample for 20-40 min.
2. Pull back the sorbent segment
into the needle and insert the needle
into a hot GC inlet.
The stir bar is coated with adsorbent.
During fast stirring process, the
analytes are quickly caught by the
sorbent. The extraction is near
exhaustive as the surface of the
sorbent is much larger than
conventional SPME
3. Analytes desorbed from the
sorbent and are focused at the head
of the column.
Advantages
Fast, more
quantitative than conventional SPME,
better sensitivity.
4. Remove the needle from GC
inlet and start GC run.
Disadvantages Need to use thermal
desorption unit, thermal labile analytes
may decompose
5. Heat or rinse the sorbent to
remove any organics left from
previous run
31
CM4241- Trace Analysis
32
CM4241- Trace Analysis
Integrated techniques
Dr. Wan Hai Bin
Liquid
sample
Sorbent
Polymer
Magnet
bar
Thermal
desorption
unit
Carrier
gas
GC
Dr. Wan Hai Bin
33
CM4241- Trace Analysis
Integrated techniques
Integrated techniques
Headspace sampling
Headspace sampling --- How to increase the detection limit?
The technique is based on the following equation:
Add salt to aqueous sample -- Example:
P i = Ki Xi ;
P i = k Ai ;
Xi=(k/K)Ai
septum
Xi concentration of compound i in liquid phase.
headspace
Ai Peak area given by the headspace sample.
Pi Vapor pressure of compound i in headspace.
Liquid
sample
K Henry constant
To establish the equilibrium between the liquid and the
gas phase, a closed container must be used.
Add water to organic solution to reduce solubility of analytes
Add water to DMF (N,N-dimethyl formamide) containing 120 ppm
styrene can increase the response of styrene: 0% water, 4; 10% water, 9;
50%, 144; 90%, 504.
Increase the temperature
A compound with higher volatility and lower solubility
in the liquid phase will have higher concentration in the
headspace.
The response may double if temperature is increased by 10 degrees.
If the temperature is too high, decomposition may occur. Maximum is
150-180 o C
Increase the temperature also increase the amount of
analytes in the headspace
Dr. Wan Hai Bin
Addition of NaCl to Aqueous sample containing 2% ethanol increases the
response by 3 times. Replace the NaCl with K2CO3, the increase become
5 times. This approach is not very effective to non polar analytes or
analytes of low polarity (e.g. toluene, hexane).
34
Dr. Wan Hai Bin
CM4241- Trace Analysis
36
CM4241- Trace Analysis
Integrated techniques
Integrated techniques
Headspace sampling
Headspace sampling --- How to calibrate?
Application : volatile analytes in liquid or solid samples. (volatile
organic compounds in water, residue solvent in pharmaceutical
products, alcohol in blood, flavor components in beverages, trace
organic material in solid material).
External standard calibration:
1) Make a set of solution of similar composition as the sample matrix; 2) Add
different amount of analytes to these solutions; 3) Carry out analysis under
the same condition as those for sample analysis; 4) draw calibration curve
using the response and the concentration data.
Manual injection: low cost (+) but the temperature of the syringe must
be maintained during the injection.
To use this calibration method, you must know the composition of your
sample matrix (e.g. QC samples, drinking water).
Automatic headspace sampler: more reproducible, can transfer the
headspace sample directly to a GC inlet
Method of standard addition
1) Put sample in 4-5 sampling vials; 2) add different amount of analyte to
these vials (volume added should be the same); 3) carry out Headspace-GC
analysis; draw calibration curve based on the response and the added
concentration.
Injection
Fill loop
Dr. Wan Hai Bin
35
CM4241- Trace Analysis
Dr. Wan Hai Bin
37
CM4241- Trace Analysis
Integrated techniques
Integrated techniques
Headspace sampling --- How to calibrate?
Instrumentation for purge and trap
Method of standard addition
This approach is suitable to samples whose composition is unknown. (waste
water, wine sample given by a customer).
Must remember the equation Xi=(k/K)Ai is correct only when the
concentration of analytes is low. If the concentration is too high, you may not
get a linear relationship. In such case, you can dilute the sample and repeat the
analysis.
Peak area
25
20
15
10
5
60
40
20
0 20
40
60
80 100
Concentration (ug/mL)
38
Dr. Wan Hai Bin
CM4241- Trace Analysis
Integrated techniques
Integrated techniques
Purge and Trap sampling
Construction of the trapping tube
Applicable to analytes of boiling points below 200 degrees and insoluble or
slightly soluble in water. For compounds of high solubility in water, the
recovery may be low. Sample matrix can be aqueous or soil (dispersed with
water). This is a very sensitive method ( can go done to 1 ppb level). To
prevent cross contamination, sample should be pre- screened using HeadspaceGC approach.
Activated charcol: for Freon or
compounds of similar volatility
Trapping
tube
High
purity He
or N2
40
CM4241- Trace Analysis
Glass wool
Silica gel: for compounds with boiling
points below 35 degrees and higher than
Freon
Tenax (diphenylene oxide polymer): for
compounds of boiling points above 35
degrees.
OV-1 (methyl silicone) coated on inert
powder: for heavy hydrocarbons. It can
extend the lifetime of the trap.
Purging
tube
The trap can be reused after
conditioning at 180 degrees for ca. 10
min.
Activated
charcol
Silica gel
Desorb
direction
Adsorption activity
Dr. Wan Hai Bin
Glass wool
Tenax
Tenax
3%
OV-1
3%
OV-1
Analyte inlet
Dr. Wan Hai Bin
39
CM4241- Trace Analysis
Dr. Wan Hai Bin
41
CM4241- Trace Analysis
Sample cleanup techniques
Cleanup by column chromatography
Integrated techniques
Comparison of Headspace sampling and Purge and Trap technique
Headspace
Design of the column:
Purge&Trap
must be easy to clean
Reservoir
---------------------------------------------------------------------------------------Sample Matrix
aqueous/solid
aqueous/solid
Analytes
volatile
volatile
Normal procedures:
Na2SO4
Change the solvent to a non polar type
Principle
equilibrium in G and L
total extraction
Detection limit
above 1 ppm
above 1 ppb
Cost
less
more
Add the concentrated extract to the column
Sample handling
easier
more complicated
Continue the elution with the eluting solvent
Analysis time
shorter
longer
Collect the fraction that contain the analytes
Requirement to GC
simpler
more
Concentrate the eluate if necessary
Dr. Wan Hai Bin
Sample
Concentrate the extract from the extraction
Slide 42
Pre-elute the column with the eluting solvent
adsorbent
Cotton
wool
42
CM4241- Trace Analysis
CM4241- Trace Analysis