Sample Preparation

(Analytical Methods)

Course Learning Outcomes

Students should be able to:
1. Discuss the importance of sample preparation prior
to sample analysis.
2. Explain the principles & procedures of separation
involve in the extraction method.
3. Describe the advantages & limitations of each
extraction method as compared to other extraction
method.
4. Choose the right & suitable extraction method for
sample analysis.
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STEPS IN MEASUREMENT
1. SAMPLING
9 Process of collecting a representative
sample for analysis.

2. SAMPLE PREPARATION
9 Extraction
9 Clean-up & preconcentration

3. SAMPLE ANALYSIS
9 Chromatographic / spectroscopic methods
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SAMPLE PREPARATION
Despite the great technological advances in
the analytical field, most sophisticated
instruments cannot handle complex sample
matrices directly &, as a result, a sample
preparation step is commonly involved in
an analytical procedure.

SAMPLE PREPARATION

Also known as sample treatment/sample

pretreatment.

Mean : Is a series of step required to bring the

sample into the correct size & form that is
suitable for analysis.

Often the bottleneck of analysis.

Time consuming (~ 61 %).

Laborious step.

Error prone.
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 Efficient sample preparation is therefore

important for a successful analysis.
Generally,
9 a clean sample helps to improve the
separation & detection,
9 while poorly treated sample may invalidate
the whole assay.

Objective

To isolate & concentrate the analytes of interest

from interfering sample components.

To convert the analytes to a form that is

compatible with the instrument for the final
analysis.

To provide the analytes of interest at a

concentration appropriate for detection or
measurement.
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Extraction methods

Liquid samples
1. Liquid-liquid extraction (LLE)

Liquid, gaseous samples

1. Solid phase extraction (SPE)
2. Solid phase microextraction (SPME)

Extraction methods
Solid, semi solid samples
1. Soxhlet
2. Sonication extraction
3. Microwave assisted extraction (MAE)
4. Pressurized liquid extraction (PLE)
5. Supercritical fluid extraction (SFE)
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Liquid-Liquid Extraction (LLE)

A method to separate
compounds based on the their
relative solubility in 2 different
immiscible liquids, usually
water & organic solvent.
An extraction of a substance
from one liquid phase into
another liquid phase.
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 Commonly performed after a chemical

reaction as part of the work-up.
A distribution ratio (D) is often quoted as a
measure of how well-extracted a species is.

Distribution ratio (D) =

Concentration of a solute in the organic phase
Concentration in the aqueous phase

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Disadvantages :
Operate manually.
Large volume of organic solvents.
Formation of emulsion.
Time consuming.
Different polarity of samples result in low
recoveries.

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Solid Phase Extraction (SPE)

An extraction method that uses a solid phase & a
liquid phase to isolate analyte from a solution.
Commercially available sorbents with different
phases, including non polar (C8 & C18), polar
(cyano & amino) & ion exchange sorbents.
Different interaction (Van der Waals, H bonding,
cation or anion exchange).
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 A selection of SPE cartridges, available in

many sizes, shapes & types of stationary
phase.

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Column
Disk

Cartridge

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How to use SPE ???

9 5 step processes to follow

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 An SPE disk is recommended

for large volume samples,
samples containing high
amounts of particulates, or
when a high flow rate is
required during sampling.

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 Conditioned with a watermiscible organic solvent

such as methanol, followed
by water or an aqueous
buffer.
Methanol wets the surface
of the sorbent & penetrates
bonded alkyl phases,
allowing water to wet the
silica surface efficiently.
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 Accurately transfer the

sample to the tube or
reservoir, using a
volumetric pipette or
micropipette.
The sample must be in a
form that is compatible with
SPE.

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If compounds of interest are

retained on the packing, wash off
unwanted, unretained materials
using the same solution in which
the sample was dissolved, or
another solution that will not
remove the desired compounds.

To remove unwanted, weakly

retained materials, wash the
packing with solutions that are
stronger than the sample matrix,
but weaker than needed to remove
compounds of interest.
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 Rinse the packing with a small

volume (typically 200L to 2mL
depending on the tube size, or
5- 10mL depending on the disk
size) of a solution that
removes compounds of
interest, but leaves behind any
impurities not removed in the
wash step.
Collect the eluate.
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9 Relying on mainly non polar, hydrophobic

interactions, only non polar or very weakly polar
compounds will absorb to the surface.
9 When silica particles are bonded with a
hydrophobic phase, they become waterproof &
must be conditioned in order to interact with the
aqueous samples.
9 This is accomplished by passing methanol or a
similar solvent through the sorbent bed.
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9 This penetrates into the bonded layer & permits

water molecules & analytes to diffuse into the
bonded phase.
9 After conditioning, water is passed to remove the
excess solvent prior to adding the sample.
9 Following conditioning, the analyte & other
sample constituents are adsorbed on the sorbent
bed.

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9 A rinsing step removes some of the undesired

constituents, while elution removes the desired
analytes, perhaps leaving other cosituent
behind, depending on the relative strength of
interaction with the solid phase or the solubility
in the eluting solvent.

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Applications :
9 SPE is used most often to prepare liquid
samples & extract semivolatile or nonvolatile
analytes.
9 Also can be used with solids that are preextracted into solvents.

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Advantages :
9 Simplicity.
9 High analyte recovery.
9 Extraction reproducibility.
9 Lower solvent consumption.
9 Ability to automate the extraction process.

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Soxhlet Extraction
Typically, a Soxhlet
extraction is only required
where the desired
compound has a limited
solubility in a solvent, &
the impurity is insoluble in
that solvent.

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A schematic representation of a Soxhlet extractor

1: Stirrer bar
2: Round bottom flask (the flask
should not be overfilled & the
volume of solvent should be 3 to 4
times the volume of the soxhlet
chamber)
3: Distillation path
4: Thimble 5: Solid
6: Siphon top 7: Siphon exit
8: Expansion adapter
9: Condenser
10: Cooling water in 11: Cooling
water out

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Step in soxhlet extraction

The solid sample to be extracted is placed in a
thimble.
The solvent is added to the round bottom flask.
The solvent is heated to reflux.

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Step in soxhlet extraction

The solvent vapour travels up a distillation arm &
floods into the chamber where it comes into
contact with the sample.
The condenser ensures that any solvent vapour
cools & drips back down into the chamber housing
the solid material.

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 The chamber containing the solid material slowly

fills with warm solvent.
Some of the desired compound will then dissolve
in the warm solvent.
When the Soxhlet chamber is almost full, the
chamber is automatically emptied by a siphon
side arm, with the solvent running back down to
the distillation flask.
This cycle may be allowed to repeat many times,
over hours (12-18 hrs).
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 During each cycle, a portion of the non-volatile

compound dissolves in the solvent.
After many cycles the desired compound is
concentrated in the distillation flask.
The advantage of this system is that instead of
many portions of warm solvent being passed
through the sample, just one batch of solvent is
recycled.

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 After extraction the solvent is removed, typically

by means of a rotary evaporator, yielding the
extracted compound.
The non-soluble portion of the extracted solid
remains in the thimble & is usually discarded.

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Type of solvent used : less polar solvent

(e.g. hexane, acetone).

Advantage of Soxhlet extraction :

9 Cheap apparatus.

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Disadvantages of Soxhlet extraction :

1. Large solvent consumption (50-200 mL for a
10g sample).
2. Long extraction time (16-24 hours).
3. Dirty extracts require tedious clean up
steps.
4. Not suitable for thermally labile compounds.
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Sonication Extraction

Vibrational disruption of samples.

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 Advantages :
9 Cold extraction - good for thermally labile
compounds.
9 Quick (a few hours).
9 A sequence of solvents may be used.
Disadvantages :
Physically violent process -may cause
breakdown of macromolecules & clays.
Labor intensive.
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Microwave Assisted Extraction (MAE)

Microwave radiation is utilized to heat up the
extraction solvent & therefore also the sample
through ionic conduction & dipole rotation.
The solvent must be able to absorb the
microwave radiation & pass it to the sample
molecules in a form of heat.

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 Suitable solvents : polar solvent with high dipole

moment (water, MeOH, acetone).
Step in MAE :
9 The solid samples & solvent is placed in a
vessel & heated using microwave radiation in
a closed system.

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 Extraction perform in a closed vessel :

1. Temp. can be higher than the atmospheric b.p
of the solvent, thus extraction is faster due to
the increased diffusion.
2. Vessels are irradiated simultaneously (hence
extraction) up to 12 vessels.
3. Volume of solvent : 20 50 mL.
4. Extraction time : 20 40 min.
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Microwave reaction system

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 Advantages :
9 Wide applicability for fast extractions of
analytes including some thermal instable
substances.
9 Enhance solvent extraction method.
9 Simultaneous & automatic extraction (up to
12 vessels).
Disadvantage :
X Sample vessel has to been cooled before
the extract can be obtained.
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Pressurized Liquid Extraction (PLE)

PLE also known as pressurized solvent extraction

(PSE), accelerated solvent extraction (ASE),
pressurized fluid extraction (PFE).

Utilized solvent at high temperature & pressure.

Higher pressure causes the solvent to remain liquefied

above boiling point & allows solvent to penetrate the
sample matrix.
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 High temperatures can disrupt the strong solutematrix interactions & decrease the viscosity of
organic solvents, allowing improved penetration of
the matrix & thus improved extraction.
The solubility & diffusity of the analytes are
increased by the increased temperature, thus
making the extraction faster & more efficient.

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 5 steps in PLE :
1. Load sample into the extraction cell.
2. Fill the cell with solvent.
3. Increase temperature & pressure (for static
extraction, maintain temperature &
pressure for a specific time).
4. Pump solvent into the cell.
5. Purge solvent from cell using N2.
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PLE can be performed :

1. Dynamic mode
The solvent continuously flows through
the sample in the extraction vessel.
2. Static mode
The supercritical fluid circulates in the
extraction vessel for some period of time
before being released to the trapping
vessel.
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Advantages :
9 Fast analysis (approximately 15 min/sample).
The high temperature & pressure increase the
capability of solvent to penetrate the sample
matrix.
The solubility & diffusity of the analytes are
increased by the increased temperature, thus
making the extraction faster & less solvent is
required.
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9 Low solvent consumption (30-50 mL).

9 Use of less hazardous/environmental friendly
solvent such as acetone & hexane in the place
of methylene chloride.
9 Solvent systems similar to Soxhlet extraction,
so easy to optimize.
9 Automated (>20 samples can be processed as
a batch).
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 Disadvantages :
May not be appropriate for thermally labile
compounds.
Expensive.
Produce dirty extracts which may need
clean-up.

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Solid phase microextraction (SPME)

Introduced by Pawliszyns group (1990).
Was developed to address the need for a fast,
solvent-free, cost effective & field compatible
sample preparation method.
Is a microextraction technique, which means that
the amount of extraction solvent is very small
compared to the sample volume.
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 All steps of the LLE such as extraction,

concentration, (derivatization) & introduction to
the chromatograph are integrated into single
step & 1 device, considerably simplifying the
sample preparation procedure.
SPME reduces the time necessary for sample
preparation, decreases purchase & disposal
costs of solvents & can improve detection limits.

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 The SPME technique enables the simultaneous

extraction & pre-concentration of volatile & non
volatile analytes from gaseous, liquid & solid
samples & even inorganic species.
SPME not suitable for sample with organic
solvent because organic solvent adsorb onto
the fiber as well & might damage it.

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The SPME apparatus is a very simple device.

The device consisting of a fiber holder & a fiber

assembly, with built-in fiber (12 cm long) inside
the needle which looks like a modified syringe.

The SPME fiber itself is a thin fused-silica optical

fiber, coated with a thin polymer film (such as
PDMS).

This film acts like a sponge, concentrating the

analytes on its surface during adsorption from
the sample matrix.
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Design of the commercial SPME device

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SPME Principle
When the coated fiber is placed into an aqueous
matrix for a pre-determined time, the analyte is
transferred from the matrix into the coating.
The extraction is considered to be complete when
the analyte has reached an equilibrium distribution
between the matrix & fiber coating.

58

 SPME relies upon the extraction of solutes from a

sample into the SPME absorptive layer.
After a sampling period - during which extraction
has ideally reached equilibrium - the absorbed
solutes are transferred into an inlet system that
desorbs the solutes into a gas (for GC) or liquid
(for LC) mobile phase.
During desorption of the analyte, the polymeric
phase is cleaned & therefore ready for reuse.

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Two Basic Types of Extractions

1. Direct Immersion (DI-SPME)
In DI-SPME, the fiber is directly
immersed/inserted in liquid samples.
DI-SPME works best for low concentration
water based sample matrices.
Position the fiber just below the sample
surface & maintain this position consistently
for all extractions.
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2. Head Space (HS-SPME)

In HS-SPME, the fiber is exposed in the vapor
phase above a gaseous, liquid or solid sample.
HS-SPME is suitable for the analysis of VOCs.
It is extremely important to keep the headspace
volume constant & keep the fiber position at the
same depth every time.

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62

SPME procedure (extraction)

1. Pierce septum on sample
container;
2. Expose SPME
fiber/extract analytes;
3. Retract fiber/withdraw
needle.

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SPME procedure (desorption)

4. Pierce septum in GC inlet
(or introduce needle into
SPME/HPLC interface);
5. Expose fiber/desorb
analytes;
6. Retract fiber/withdraw
needle.
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 Parameters affect the extraction efficiency :

9 Types of fiber coating
9 Thickness of the fiber coating
9 Extraction time
9 Extraction temperature
9 Sample agitation
9 Sample pH
9 Salt addition
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Optimizing extraction condition

1. Type of fiber coating
The fiber is coated with a thin polymeric film,
which concentrates the analytes.
The type of fiber used affects the selectivity of
extraction (in general, polar fibers are used for
polar analytes & non-polar fibers for non-polar
analytes).
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2. Thickness of the fiber coating

The thickness affects both the equilibrium
time & sensitivity of the method.
The use of a thicker fiber requires a longer
extraction time but the recoveries are
generally higher.

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 The thinnest film is employed to reduce

extraction times (faster the partition equilibrium
can be reached).
Volatile compounds require a thick film & a thin
film is effective for semivolatile compounds.

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3. Extraction time
The longer the extraction time, the more
solutes adsorbed onto the fiber until it reach
optimum extraction time whereby the amount
of the adsorbed solutes is max.
Increasing the extraction time then will not
increase the amount anymore.
If the extraction is lower than the optimum
time, possibility only a fraction of solutes will
be adsorbed.
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The extraction time depends on the size of the

compounds, fiber coating, type of extraction used &
sample concentration.

Extraction times can be shorter when :

9 Analyzing small compounds (< 150 MW)
9 Using thinner fiber coatings.
9 Using the headspace technique.
9 Working with high concentration samples (high
ppb or ppm range).

Extractions typically take 15-20 minutes.

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4. Extraction temperature
Optimization of extraction temperature is
generally more important in the headspace mode.
The use of heat during headspace SPME will help
release the analyte from the sample, improve
sensitivity & shorten the extraction time.
A constant temperature is advisable for all
extractions to obtain good precision.
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5. Sample agitation
Agitation accelerates the transfer of analytes
from the sample matrix to the fiber & reduce
the equilibrium time.
Agitation methods include magnetic stirring,
sonication & vibration.
Vigorous or harsh agitation modes such as
sonication may affect the coating, thus they
should be used with caution.
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6. Sample pH
Adjustment of sample pH may improve the
extraction yield for compound that can be
protonated.
pH is adjusted in order to obtain the analyte in
its neutral form to enhance the extraction
yield.
E.g., a sample may be acidified for extracting
acidic compounds or made alkaline for
extracting basic compounds.
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6. Salt addition
The addition of salt into the sample solution often
improved the extraction efficiency due to the
salting-out effect.
The addition of salt decreases the solubility of
analytes in the sample solution & enhances their
partitioning into the fiber.
E.g. of salt; NaCl, NaHCO3, K2SO4 & (NH4)2SO4.
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Advantages of SPME
It is a rapid (analysis time very fast).
The configurations & operation of the SPME
devices are very simple.
Reducing cost.
Solvent free & sensitive method for the
extraction of analytes.
Clean extracts & do not need any clean-up
or concentration.
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 It gives highly consistent, quantifiable results

from very low concentration of analytes.
SPME is becoming widely used as an
extraction & concentration step prior to MS
analysis.

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 It has small size, which is convenient for

designing portable devices for field or on-site
sampling.
On-site sampling can be done even by
nonscientists without the need to have
equipment at each location.
When properly stored, samples can be
analyzed days later in the laboratory without
significant loss of volatiles.
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Limitations of SPME
The volume of the polymer extraction phase is
very small & requires extreme precision during
manufacturing of the coating.
The quality of the fibers depends on the
manufacturer & sometimes the performance is
different from batch to batch.

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 Some level of degradation of the fiber occurs

during repeated usage.
The carry-over of the fiber is also a problem
that in some cases is difficult to eliminate.
Fibers are fragile & can easily be broken.

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Application of SPME in various fields

of analytical chemistry
Environmental (water analysis).
Food samples (analysis of wines & other
alcoholic beverages).
Biological fluids (blood, urine).
Forensic, clinical & pharmaceutical (analysis
of drugs).
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Sample problem
Briefly discuss how the used of fiber give advantages to
SPME method when compared to SPE method.

82

How if we use LLE ???

(in term of extract quality, may or may
not require clean-up & concentration
step)

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Separation methods
Method

Basic of method

Extraction

Different in solubility in two immiscible

liquids

Ion exchange

Difference in interaction of reactants

with ion exchange resin

Chromatography

Difference in rate of movement of a

solute through a stationary phase

Electrophoresis

Difference in migration rate of

charged species in an electric field
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Derivatization
A process of chemically modifying a compound
to produce a new compound which has
properties that are suitable for analysis.
Organic compounds with readily exchangeable
H (acids, alcohols) generally exhibit poor
chromatographic behavior on a polar GC
stationary phases.

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Advantages of derivatization :

Improved chromatography (better sensitivity

& quantification).

Prevents GC column degradation.

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Common derivatization methods:

1. Fatty Acids
Methylation (BF3/MeOH or MeOH/HCl) to yield
Fatty Acid Methyl Esters (FAMES).
Silylation (BSTFA) to yield trimethylsilyl (TMS)
esters.
2. Alcohols (n-alkanols, sterols)
Acylation (pyridine, acetic anhydride) to yield
acetates.
Silylation (BSTFA) to yield trimethylsilyl (TMS)
ethers.
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Pyrolysis
Pyrolysis is a form of incineration that
chemically decomposes organic materials by
heat in the absence of oxygen.
Pyrolysis typically occurs under pressure & at
operating temperatures above 430 C (800 F).
Organic materials are transformed into gases,
small quantities of liquid & a solid residue
containing carbon & ash.
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Application
Pyrolysis treats & destroys :
Semi-volatile organic compounds (SVOCs),
fuels & pesticides in soil.
The process is applicable for the treatment of
organics from refinery wastes, coal tar wastes,
contaminated soils, hydrocarbons & volatile
organic compounds (VOCs).

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