ANALYTICAL

SEPARATION METHODS
LABORATORY GUIDE
3rd edition

NOR’ASHIKIN SAIM
RUZIYATI TAJUDDIN
MARDIANA SAAID
ROZITA OSMAN
Edited by:
ROZITA OSMAN
HALILA JASMANI
CONTENTS

ii
Foreword iv
Preface v
Laboratory Procedure vi
Report Writing vii

EXPERIMENT 1 1
Gas Chromatography (GC): Optimization of Flow
Rate and Column Temperature

EXPERIMENT 2 4
High Performance Liquid Chromatography (HPLC):
Method Development

EXPERIMENT 3 6
Fatty Acid Determination Using Gas Chromatography
(GC)

EXPERIMENT 4 9
Analysis of Hydrocarbons in Common Fuels by Solid-
Phase Microextraction (SPME) and Gas
Chromatography-Mass Spectrometry (GC-MS)

EXPERIMENT 5 12
Analysis of Chlorpyrifos in Water by Solid-Phase
Extraction (SPE) and Gas Chromatography-Electron
Capture Detector (GC-ECD)

iii
APPENDIX I 16
Gas Chromatography

APPENDIX II 22
High Performance Liquid Chromatography

APPENDIX III 28
Quantitation Methods in Chromatography

APPENDIX IV 32
Properties of Compounds

iv
 FOREWORD

Laboratory work is an essential part to fully understand analytical

separation techniques. Solving an analytical problem in separation
methodology involves systematic procedures and disciplines. A
detailed laboratory guide is vital in training students to acquire these
skills.

The authors should be congratulated for their attempt to compile

experiments that will provide students with helpful practical guide in
analytical separation methods. This Analytical Separation Methods
Laboratory Guide will also provide a useful basis for researchers in
related areas.

Dean
Faculty of Applied Sciences
Universiti Teknologi MARA

v
 PREFACE

Sensitivity, selectivity and speed have become vital factors in

analytical separation work. An analyst should be able to choose the
best technique for a particular problem. This Analytical Separation
Methods Laboratory Guide contains a compilation of laboratory
experiments aims at teaching students how to analyze liquid and solid
samples. Laboratory experiments emphasize on sample preparation
prior to chromatographic analysis particularly gas chromatography and
high performance liquid chromatography. The laboratory guide
attempts to provide novel approaches to analytical problem solving by
introducing the latest techniques in sample preparation such as solid-
phase extraction, solid-phase microextraction and accelerated solvent
extraction.

Compiling the experiments in this manual has been an enriching

experience for us. We are very grateful for the support we received
while writing this laboratory guide, in particular to En. Tammizi Hj.
Saaid, Puan Noor Haida Kamalul Khudzri and our students for testing
and debugging the procedures of each experiment. We would also like
to thank our reviewers, Associate Professor Zuraidah Abdullah Munir
for her comments and suggestions, most of which we have
incorporation.

Nor’ashikin Saim
Ruziyati Tajuddin
Mardiana Saaid
Rozita Osman

vi
 LABORATORY PROCEDURE

1. All students will work in groups on a given experiment. Please

come to class on time.

2. Students must be well acquainted with the instructions for the

experiment to be carried out. Make sure you have read the
procedures and the instrument operating instructions before
coming to class. Also, come to class with an outline of how you
intend to prepare your solutions and run your experiments.

3. All experimental data will be recorded and the instructor will

validate your work by placing his/her initials in your data sheets.
The original data sheets must be included in your final report.
No points will be granted for unvalidated lab sheets. You must
write everything in ink (no pencils). When correcting something
do not white-out, scratch the error with a single line and write the
correction above or below it.

4. Keep your working area always clean.

5. Always return all chemicals to their original place immediately

after use.

6. Do not leave standards solutions in volumetric flasks. Once you

are done with the preparation, transfer the solution to a labeled
vial/bottle for storage.

7. Read the operating instructions for every instrument before you

use it.

8. Always wear a lab coat. There are to be no food or drinks in the

laboratory at any time. Pay close attention to safety notes in
your lab manual or on the bulletin board concerning the
chemicals you are using.

9. Plagiarism is illegal and unethical. Plagiarizing laboratory

reports will not be tolerated and any evidence of such activities
will result in zero mark for that exercise.

10. SAFETY IS ALWAYS THE NUMBER ONE PRIORITY.

vii
 REPORT WRITING

A formal report is required for each of the experiments. A complete

report contains the following items:

1. Title Page: Title of the experiment, your name and the date.

2. Introduction: Presents a short description of the experiment,

parameters to be measured and the general approach to the
problem. This section should also state the objectives of the
experiment. (DO NOT COPY THE LAB MANUAL OR
LECTURE NOTES).

3. Experimental:

a. Analytical Procedure: This section should include a description

of how the experiment was conducted. Do not copy the
procedure section from the lab manual. Write in your own
words to describe how you performed the experiment. Be sure
to include any modifications or deviations from the suggested
protocols.
b. Raw data: Include the original data sheet with your report.

4. Results and Discussion: Present your data in tables and graphs.

Calculations and error analysis must be shown and explained. Use
SI units only. All graphs and tables must have a title/caption and
should be referenced in your discussion. Discuss your findings;
make comparisons with known values if available. Elaborate on
possible sources of errors, selectivity and sensitivity of the
technique, applicability etc. Suggest any possible improvements in
the experiment.

5. Conclusion: A summary of the results.

6. References.

7. Appendix

Since your final grade greatly depend on these reports, please

work hard on them.

viii
 EXPERIMENT 1

Gas Chromatography (GC): Optimization of Flow

Rate and Column Temperature
Introduction
Efficient separation of compounds in GC is dependent on the
compounds traveling through the column at different rates. The rate at
which a compound travels through a particular GC system depends on
several factors that include:
• Volatility of compound: Low boiling (volatile) components
will travel faster through the column than high boiling
components.
• Column temperature: Raising the column temperature
speeds up all the compounds in a mixture.
• Carrier gas flow rate: Increasing the carrier gas flow
increases the speed with which all compounds move through
the column.
• Length of the column: The longer the column, the longer it
will take for all compounds to elute. Longer columns are
employed to obtain better separation.

Resolution
Resolution (Rs) is a measure of how well species are separated. The Rs
of two species, A and B, is defined as:

2Z 2[( t R ) A − ( t R ) B ]
Rs = =
wA + wB wA + wB

Where ∆Z is the separation between peaks A and B; and Wa and Wb

are the widths at the base of peaks A and B, respectively. Acceptable
resolution is on the order of Rs = 1.0, and baseline resolution between
two peaks (as shown in the figure) requires an Rs > 1.5.

The objective of this experiment is to explore gas chromatography,

including the concepts of retention time and resolution using a mixture
of methyl esters; methyl laurate, methyl myristate, methyl palmitate,
methyl stearate and methyl linoleate. The effects of column
temperature and flow rate on the separation of these compounds will
be investigated.

1
Reagents and Solutions
a. Individual methyl esters compounds: methyl laurate, methyl
myristate, methyl palmitate, methyl stearate and methyl linoleate.

b. Standard mixture of methyl laurate (0.20 mg mL-1), methyl

myristate (0.20 mg mL-1) and methyl palmitate (1.0 mg mL-1),
methyl stearate (0.70 mg mL-1) and methyl linoleate (0.35 mg mL-
1
).

Instrument
Gas chromatograph (Agilent Technologies 6890N) equipped with
flame ionization detector (FID) and 30 m x 250 m x 0.25 m HP5-
MS capillary column

Analytical Procedure
a. Instrument set-up (may vary depending on instrument):
Injection port : Split (40:1)
Injection port temperature : 250 oC
Column temperature : 210 oC
Carrier gas flow rate : 30 cm sec-1
Detector temperature : 250 oC

b. Effect of carrier gas flow rate on isothermal GC separation of

methyl esters.
Inject 0.4 µL standard mixture isothermally at 210 oC at carrier gas
flow rate of 30 cm sec-1. Increase the flow rate to 50 cm sec-1.
Allow a few minutes for the system to equilibrate and inject the
standard mixture again. Repeat the same procedure at flow rate of
70 cm sec-1. Which flow rate is most suitable for this separation?

c. Effect of column temperature on the isothermal GC separation

of methyl esters.
Inject 0.4 µL standard mixture isothermally at 170 oC, followed by
190 oC, at the optimal carrier gas flow rate. Evaluate the effect of
column temperature on the separation, resolution, and analysis
time.

d. Separation of methyl esters using column temperature

programming.
Inject standard mixture at the optimal carrier gas flow rate using a
linear temperature ramp from 100 oC to 290 oC at optimal flow
rate. Comment on the separation of the compounds. If the
resolution is not adequate for your needs, attempt a modification to

2
 the temperature programming in order to improve the resolution of
the compounds.

e. Identification of components in methyl esters mixture.

Inject each methyl ester individually to identify the various
compounds in the standard mixture using the optimized GC
conditions.

3
 EXPERIMENT 2
High Performance Liquid Chromatography
(HPLC): Method Development
Introduction
The fundamental basis for HPLC consists of passing a sample (analyte
mixture) in a high pressure solvent (mobile phase) through a column
packed with sorbents (stationary phase). The mobile phase acts as a
carrier for the sample solution. As the analytes pass through the
column they interact between the two phases, mobile and stationary, at
different rates. The difference in rates is primarily due to different
polarities for the analytes. The analytes that have the least amount of
interaction with the stationary phase or the most amount of interaction
with the mobile phase will exit the column faster. Therefore, changes
in the polarity of the mobile phase can affect the interactions of the
sample and the stationary phase and thus changes the efficiency of
your HPLC separation. Changes in mobile phases can be done either
by isocratic elution or gradient elution.

Isocratic Elution: The mobile phase composition remains unaltered

during the separation. The mobile phase may comprise of a single
solvent or a pre-mixed mixture of solvents.

Gradient Elution: The composition of the mobile phase is changed

during the separation. Two or more solvents that differ in polarity are
employed. After sample introduction, the ratio of these solvents is
programmed to vary either continuously or in steps.

In this experiment you will be optimizing a separation of a mixture of

5 compounds (caffeine, acetone, methyl benzoate, phenatole and
phenanthrene) using HPLC by varying the mobile phase composition.

Reagents and Solutions

a. HPLC grade acetonitrile

b. Deionized water

c. Standard mixture of caffeine, acetone, methyl benzoate, phenatole

and phenanthrene (~100 ppm).

4
Instrument
Liquid chromatograph (Agilent G1314A HPLC) equipped with diode
array detector (DAD), 5 m RP C18 column and 20 L sample loop.

Analytical Procedure
a. Instrument set-up (may vary depending on instrument):
Detector wavelength : 254 nm
Flow rate : 1.5 mL min-1
Mobile phase : acetonitrile:water

b. Effect of mobile phase on HPLC separation

Set the instrument to use a mobile phase ratio of acetonitrile:water
(50:50 v:v) and inject the sample. Then, change the mobile phase
composition to 90:10. Which composition of mobile phase is
suitable for the separation of these compounds?

c. Identification of components in the mixture

Inject each compound individually to identify the components of
the mixture using the selected HPLC conditions.

d. Separation using gradient elution

Based on the separation above, perform a gradient elution
separation to improve the efficiency of the column.

5
 EXPERIMENT 3
Fatty Acid Determination using Gas
Chromatography (GC)
Introduction
Fatty acids are not sufficiently volatile for GC analysis. In addition,
acids are reactive compounds and are too polar to be well separated by
GC. Direct GC analysis of acids tends to cause peak tailing due to the
adsorption and non-specific interaction with the column.
Derivatization is the process of chemically modifying a compound to
produce a new compound which has properties that are suitable for GC
analysis. This experiment introduces a derivatization procedure
routinely used for fat analysis in which nonvolatile fatty acids are
chemically converted to the corresponding volatile methyl esters
(FAME). The resulting volatile mixture can be analyzed by GC.

Esterification
Esterification is the most popular method of derivatization technique.
In a typical reaction, esterification involves the condensation of the
carboxyl group of an acid and the hydroxyl group of an alcohol, with
the elimination of water. Results are best in the presence of a catalyst,
like BF3 or H2SO4, which is then removed with the water.

Reagents and Solutions

a. Individual FAME in the following concentrations:

FAME Concentration
1. Methyl laurate C12:0 0.10 mg mL-1
2. Methyl myristate C14:0 0.10 mg mL-1
3. Methyl palmitate C16:0 1.50 mg mL-1
4. Methyl stearate C18:0 0.70 mg mL-1
5. Methyl linoleate C18:2 0.35 mg mL-1

b. Methanolic solution (0.5 M): NaOH in methanol

c. Saturated NaCl

d. Analytical grade diethyl ether

e. Esterification reagent: Add 7.5 mL H2SO4 and 5 g of NH4Cl in a

500 ml flask and reflux for 15 minutes.

6
Sample
Oil or fat samples (margerine or butter) – 10 g.

Instrument
Gas chromatograph (Agilent Technologies 6890N) equipped with
flame ionization detector (FID) and 30 m x 250 m x 0.25 m HP5-
MS capillary column.

Analytical Procedure
a. Preparation of fatty acid methyl ester samples from fat
samples
i. Weigh out approximately 2 g of oil or fat and record the exact
weight.

ii. Transfer the sample into a 50 mL flask equipped with air

condenser.

iii. Add 5 mL of 0.5 M methanolic solution and reflux for 3-4

minutes.

iv. Add 15 mL of esterification reagent and reflux for 3 minutes.

v. Transfer the mixture into a separatory flask. Add 50 mL of

saturated NaCl and 25 mL of diethyl ether. Shake the mixture
vigorously for 2 minutes and discard the aqueous layer.

vi. Repeat step v with another 25 mL of saturated NaCl and again

discard the aqueous layer.

vii. Transfer the organic layer into a screw cap vial. Make sure
that only the organic layer is injected into the GC as water
can ruin the GC column.

NOTE: You may want to run your calibration standards while the
esterification reaction is in progress.

b. Instrument set-up (may vary depending on instrument):

Injection port : Split (40:1)
Injection port temperature : 250 oC
Column temperature : Ram:190 oC (1min) to 210 oC (5min)
at 30 oC min-1; 210 oC to 230 oC at 30
o
C min-1.
Carrier gas flow rate : 70 cm sec-1
Detector temperature : 250 oC
7
c. Quantitative analysis of FAME
i. Inject 0.4 μL of standard esters onto the column. Repeat
injection to get reproducible peak areas.

ii. Inject 0.4 μL of the derivatized sample. Repeat injection to

get reproducible peak areas.

iii. Using the data from the standard esters, calculate the amount
of each fatty acid in the sample.

Alternate Method for Derivatization of Fatty Acids

i. Combine 500 mg of fatty acid sample with 5 mL of boron

trifluoride-methanol (BF3-methanol) in a 50 ml volumetric
flask.

ii. Heat on a steam bath for 5-10 minutes.

iii. Add enough saturated NaCl solution to raise the fluid level in
the flask well into the neck.

iv. Cap the flask and mix well by repeatedly inverting the flask.

v. Allow the organic phase to collect in the neck of the flask,

and remove it from the top of the aqueous phase (bottom
layer).

vi. Dry the hexane extracts over anhydrous sodium sulfate and
transfer to a clean, dry container.

vii. Analyze the hexane layer by gas chromatography directly or,

if concentration is desirable, evaporate the hexane and
analyze the residue.

8
 EXPERIMENT 4

Analysis of Hydrocarbons in Common Fuels by

Solid-Phase Microextraction (SPME) and Gas
Chromatography - Mass Spectrometry (GC-MS)
Introduction
Sample preparation is an essential step in analysis, greatly influencing
the reliability and accuracy of analysis. Solid-phase microextraction
(SPME) is an alternative to the conventional liquid-liquid extraction
(LLE) technique. It is a rapid, inexpensive and solventless technique
for the isolation of organic compounds from gaseous and liquid
samples. It is based on the enrichment of analytes on a polymer or
adsorbent-coated fused-silica fiber by exposing the fiber either directly
to the sample or to its headspace and thermal desorption of the
analytes in the GC injector. In this experiment, the components of
hydrocarbons in common fuels are identified using SPME-GC-MS.

SPME Apparatus
The SPME apparatus consists of a fibre holder and a fibre assembly,
the latter containing a 1-2 cm long retractable SPME fibre. The SPME
fibre is a thin fused-silica optical fibre (such as polydimethylsiloxane,
PDMS).

Factors that affect SPME performance

Coating Material: The most important feature determining the
analytical performance of SPME is the type and thickness of the
coating material. The type of fibre used affects the selectivity of
extraction. In general, polar fibres are used for polar analytes and non-
polar fibres for non-polar analytes as with conventional GC stationary
phases. The thickness of the coating material affects both the
equilibrium time and sensitivity of the method. The use of a thicker
fibre requires a longer extraction time but the recoveries are generally
higher.

Extraction Procedure: Extraction can be achieved either by exposing

the fibre directly into the sample or headspace of the sample. After
equilibrium has been reached, the fibre is withdrawn and transferred to
a GC injection port. The time required to reach equilibrium and the
extraction efficiency can be influenced by several factors that include
extraction temperature, sample agitation, volatility of analytes and type
and thickness of fibre coating. For desorption, both temperature and
desorption time influence recovery.
9
Sample
Accelerants: Unleaded petrol, diesel, paint thinner and kerosene

Apparatus
a. SPME holder with 100 m polydimethylsiloxane (PDMS) fibre

b. Glass vial with septum

Instrument
Gas chromatograph (Agilent Technologies 5890 Series II) equipped
with HP 5971A mass spectrometry detector (MSD) and a 30 m x 250
m x 0.25 m HP5-MS capillary column.

Analytical Procedure
a. Instrument set-up (may vary depending on instrument):
Injector temperature : 250 oC
Detector temperature : 300 oC
Carrier gas flow rate : 30 cm sec-1
Column temperature : 60 oC to 170 oC at 10 oC min-1

b. SPME procedure
i. Condition the fibre (100 μm polydimethylsiloxane (PDMS))
in a GC injection port at 250 oC (10 minutes) to remove
contaminants.

ii. Add approximately 5 mL of sample in a glass vial with

septum and place the vial on a hot plate (Figure 1). Heat the
sample to 50 oC. Agitate the sample using a magnetic stirrer.

iii. Expose the SPME fibre to the headspace of the vial for 20
minutes.

iv. Withdraw the fibre into the needle, pull out from the vial
and immediately inject into the GC-MS with a desorption
time 80 seconds.

v. Using the mass spectra library, identify the major compounds

in each sample using the mass spectra library.

10
 Plunger

Z slot
Plunger retaining
screw

Hub viewing
window
Needle

Fibre Glass vial

with septum

Sample Magnetic
stirrer
Hot bath

Figure 1. Schematic diagram for SPME extraction procedure

11
 EXPERIMENT 5

Analysis of Chlorpyrifos in Water by Solid-Phase

Extraction (SPE) and Gas Chromatography-
Electron Capture Detector (GC-ECD)

Introduction
Solid-phase extraction (SPE) is an extraction method that uses a solid
phase and a liquid phase to isolate one, or one type, of analyte from a
solution. SPE is the most powerful technique currently available for
rapid and selective sample preparation. The versatility of SPE allows it
to be used for a number of purposes, such as purification, trace
enrichment, solvent exchange, and derivatization. It offers many
benefits and advantages over more traditional sample preparation
techniques (such as liquid-liquid extraction), including high recoveries
of analytes, concentration of analytes, ease of automation and
reduction in organic solvent consumption.

The general procedure in SPE is to load a solution onto the SPE

column, wash away undesired components, and then wash off the
desired analytes with another solvent into a collection tube. The key
success of SPE procedures is the interaction of the three components,
which are the sorbent, the analyte and the solvent. It involves several
steps as shown in Figure 2:

i. Column Conditioning: Non-polar columns should be conditioned

with polar organic solvent such as methanol, acetonitrile or
tetrahydrofuran.
ii. Sample Loading: Optimization of loading flow rates is an
important part of method development. A good starting point is 1
mL min-1 for a 1 mL cartridge, 3 mL min-1 for a 3 mL cartridge
and 7 mL min-1 for a 6 mL cartridge (wider diameter cartridges
yield lower linear velocities).
iii. Interference Elution: The purpose of interference elution is to
selectively remove undesired compounds from the sorbent without
eluting the analytes. A typical volume of interference elution
solvent is 1-2 mL/100 mg of sorbent. The flow rate should be
adjusted such that the solvent is in contact with the sorbent for 1-2
minutes.
iv. Analyte Elution: The elution solvent should be the one in which
the analytes are soluble.

12
 Column Conditioning Sample Loading Interference Elution Analyte Elution

Figure 2. Steps in SPE extraction

Chlorpyrifos
Chlorpyrifos (Figure 3), a broad-spectrum organophosphate
insecticide, is one of the top five residential pesticides in use for a
wide variety of food crops, turf and ornamental, and greenhouse.
Chlorpyrifos is sufficiently hazardous to human health. It is
moderately persistent in soils and is slightly soluble in water (2.0 mg
L-1). Chlorpyrifos has the potential to bioaccumulate.

S
Cl N OP(OCH2CH3)2

Cl Cl

Figure 3. Chlorpyrifos

In this experiment, analysis of chlorpyrifos in spikes water samples

will be conducted using C18 cartridge.

Reagents and Solvents

a. Analytical grade methanol

13
b. Analytical grade hexane

c. Standard chlorpyrifos (100 ppm)

Apparatus
a. C18 solid phase extraction cartridges (500 mg)

b. Glass fibre filter paper

Sample
Wastewater (50 mL). Adjust pH to 2 using HCl and add 10 %
methanol.

Instrument
Gas Chromatograph (Agilent Technologies 6890N) equipped with an
electron capture detector (ECD) and 30 m x 250 m x 0.25 m HP5-
MS capillary column.

Analytical Procedure
a. Filter water sample through a glass fibre filter paper.

b. Solid-phase extraction procedure

i. Condition C18 SPE cartridge by passing 10 mL of methanol.

ii. Rinse the cartridge by passing 6 mL of deionized water

without applying vacuum.

iii. Pass the filtered water sample (50 mL) through the
preconditioned column using a vacuum manifold at ~ 6 ml
per min (48 drops/min). The column should not be allowed to
dry during this sample enrichment step.

iv. Dry the column by vacuum for 15 minutes.

v. Remove the interference by eluting the column with 10 mL of

deionized water and again vacuum dry the cartridge for 30
minutes.

vi. Elute the pesticide using 5 mL of hexane. Concentrate to

1 mL by blowing down using gentle nitrogen and the sample
is ready for GC analysis.

c. Instrument set-up (may vary depending on instrument):

Injector temperature : 250 oC
14
 Detector temperature : 300 oC
Carrier gas flow rate : 50 cm sec-1 (nitrogen)
Column temperature : Initial temperature 165 oC for 3 min,
increase to 260 oC at 3 oC min-1 with a
final time of 2 min.

d. Quantitative analysis of chlorpyrifos

i. Inject 1 μL of sample onto the column. Repeat injection to get
reproducible peak areas.

ii. Inject 1 μL of standard chlorpyrifos. Repeat injection to get

reproducible peak area.

iii. Using the data from the standard solution, calculate the
concentration of chlorpyrifos in the sample.

15
 APPENDIX I

Gas Chromatography
Gas chromatography (GC) is an instrumental method for the separation
and identification of compounds. In GC (Figure 6), a liquid (or gas)
sample is introduced into the injection port through a rubber septum
into a stream of inert gas, which is called the mobile phase or carrier
gas. The gas then passes through a chromatographic column kept in
an oven that controls the column temperature. The column is lined or
packed with a stationary phase and as the analytes pass through the
column, they “partition” between the mobile phase and stationary
phase. Analytes with lower affinity for the stationary phase spend less
time in the column and pass through quickly while analytes with
higher affinity for the stationary phase spend more time bound to the
column and pass through more slowly. After passing through the
column, the solute is quantitatively measured by the detector. The
output from the detector is displayed as a chromatogram, a plot of
detector response versus time as shown in Figure 7.

Injection Port
Carrier gas
Detector

Recorder

Column

Oven

Figure 6. Schematic diagram of a gas chromatograph

The solute’s retention time is the characteristic interval required for it

to pass through the entire column under particular conditions of
temperature, gas flow rate, and stationary phase composition. Under a
given set of conditions, a single analyte will always have the same
retention time. The height or area of the peak is proportional to the
concentration of the solute.

16
 tB

tA

Detector
Response

tair
A B

Time

Figure 7. A chromatogram

Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases
include nitrogen, helium, argon and hydrogen. The choice of carrier
gas is often dependant upon the type of detector used. The carrier gas
system also contains a molecular sieve to remove water and other
impurities.

Flow rate is related to resolution. If the molecules spend too much time
in the column, they will tend to diffuse (causes peak broadening) and if
they diffuse into different sample molecules there will be a loss of
resolution. If the molecules don't spend enough time inside the
column, they won't interact with the stationary phase long enough to
achieve a separation.

Sample injection port

Sample is injected through a rubber septum on the injection port. The
injection port is maintained at a higher temperature than the boiling
point of the least volatile component in the sample mixture and the
sample is instantly vaporized. The temperature of the sample port is
usually about 50 ºC higher than the boiling point of the least volatile
component of the sample. The most common injection method is using
a microsyringe. For optimum column efficiency, the sample should not
be too large as large sample causes band broadening and loss of
resolution. Sample size ranges from tenths of a microliter up to 20
microliters for packed column and typically around 10-3 microliter for
capillary columns. For capillary GC, split/splitless injection is
commonly used. The injector can be used in one of two modes; split
or splitless. In the split injection mode, only a fraction of the vaporized

17
sample is transferred onto the head of the column. The remainder of
the vaporized sample is removed from the injection port via the split
vent line. Split injections should be used only when sample
concentration is high enough to allow a portion of the sample to be
discarded during the injection process, while still maintaining a
sufficient concentration of analyte at the detector to produce a signal.
With low concentration analyte, the injector should be operated in the
splitless injection mode. In the splitless injection mode, most of the
vaporized sample will be transferred to the head of the column.

Columns
There are two general types of column, packed and capillary. Packed
columns are typically a glass or stainless steel coil (typically 1-5 m
total length and 5 mm inner diameter) that is filled with the stationary
phase. Capillary columns are a thin fused-silica (purified silicate glass)
capillary (typically 10-100 m in length and 250 µm inner diameter)
that has the stationary phase coated on the inner surface. Capillary
columns provide much higher separation efficiency than packed
columns but are more easily overloaded by too much sample. When
selecting a capillary column for an application, four basic parameters
need to be considered; stationary phase, column internal diameter, film
thickness and column length.

The stationary phase in the column provides separation of the sample.

The stationary phase is selected based upon their polarity and ability to
interact with the analyte. Generally, polar stationary phase for polar
solutes and nonpolar stationary phase for nonpolar solutes. Analytes
of similar polarity elute in the order of boiling points, lowest boiling
point will be eluted first. The most common stationary phases in gas-
chromatography columns are polysiloxanes, which contain various
substituent groups to change the polarity of the phase. The nonpolar
end of the spectrum is polydimethyl siloxane, which can be made more
polar by increasing the percentage of phenyl groups on the polymer.
For very polar analytes, polyethylene glycol (eg. carbowax) is
commonly used as the stationary phase. After the polymer coats the
column wall or packing material, it is often cross-linked to increase the
thermal stability of the stationary phase and prevent it from gradually
bleeding out of the column. Table 1 shows a comparison of stationary
phase polarity.

18
 Table 1: Polarity of common stationary phases

Common Trade Name

Low Polarity Squalane
HP-1, DB-1, BP-1, CP-Sil 5, Rtx-1
DB-5, HP-5, BP-5, BPX-5, CP-Sil 8, Rtx-5
DB-1301, HP-1301, CP-1301,Rtx-1301
DB-35, HP-35, BPX-35, Rtx-35
DB-1701, HP-1701, BP-10, CP-19, Rtx-1701
DB-17, HP-17, BP-50, CP-24, Rtx-50
DB-225, BP-225, CP-Sil 43
DB-wax, HP-wax, BP-20, CP-wax52
DB-FFAP, BP-21, CP-wax58
DB-23, BPX-70, CP-Sil88
High Polarity OV-275

Column oven
In GC, the elution time and resolution of analytes are highly dependent
on the temperature of the column. Therefore, it is essential to control
the column temperature as accurately and precisely as possible using
an oven. The usual temperature ranges from room temperature to 400-
500 ºC. The optimum column temperature is dependant upon the
boiling point of the sample. The operating temperature is usually
slightly above the average boiling point of the sample. Minimal
temperatures give good resolution, but increase elution times. If a
sample has a wide boiling range, then temperature programming can
be useful. The column temperature is increased (either continuously or
in steps) as separation proceeds.

Detector
There are many detectors which can be used in GC. Different detectors
will give different types of selectivity. A non-selective detector
responds to all compounds except the carrier gas, a selective detector
responds to a range of compounds with a common physical or
chemical property and a specific detector responds to a single chemical
compound.

19
Common GC detectors include: flame ionization detector (FID),
thermal conductivity detector (TCD), electron capture detector
(ECD), flame photometric detector (FPD) and nitrogen
phosphorus detector (NPD). Another GC detector that is powerful
is the mass spectrometer or mass selective detector (MSD). When
coupled to a GC, it is called gas chromatography-mass spectrometry
(GC-MS). GC-MS is a very useful technique for the identification of
compounds using their mass-to-charge ratios. The applications,
detection limit and linear range of selected detectors is shown in Table
2.

Table 2. Common GC Detectors

Dynamic
Detector Applications Detectability
range
Flame
ionization Most organic compounds 100 pg 107
(FID)
Thermal
conductivity Universal 1 ng 107
(TCD)
Halides, nitrates, nitriles,
Electron
peroxides, anhydrides, 50 pg 105
capture (ECD)
organometallics
Nitrogen-
phosphorus Nitrogen, phosphorus 10 pg 106
(NPD)
Sulphur, phosphorus, tin,
Flame
boron, arsenic,
photometric 100 pg 103
germanium, selenium,
(FPD)
chromium

Procedures for running the gas chromatograph

a. Turn on main cylinder valves of gases for the detector and carrier
gas.
b. Turn on flow valve on GC and set the pressure.
c. Set the temperature for injection port, column and detector.
d. Press igniter button (for FID).
e. After completing work turn off main valves for all gasses.

20
Care of syringes
1. Slowly fill clean, dry syringe with sample by pulling back on the
plunger (empty and repeat 4-5 times to rinse).
2. Hold syringe vertical and slowly empty the syringe until you’ve
obtained the exact amount required.
3. Pull back the plunger to fill a small amount of air in the syringe.
4. Insert straight down into the injection port.
5. Remove by pulling straight up out of the injection port.
6. Rinse the syringe by filling of acetone several times and dry by
repeatedly filling with air and emptying.

Injection reproducibility
1. Inject at least three successive samples, including approximately
5 µL of air into the column at one minute intervals.
2. You will need to adjust both the attenuation and the range factors
on the instrument to get the peaks on scale. Setting the recorder’s
sensitivity to a higher setting will cause the peaks to become
smaller; increasing the attenuation to a larger number will cause
the peaks to become dramatically smaller.
3. In your calculations, use the average peak areas and determine
the relative standard deviation for your injections.

21
 APPENDIX II

High Performance Liquid Chromatography

Introduction
HPLC instrumentation includes mobile phase reservoirs, pump,
injector, column, detector and recorder or data system, connected as
shown in Figure 8. The heart of the system is the column where the
separation occurs. The column is packed with stationary phase,
composed of micrometer size porous particles. Thus, a high pressured
pump is required to move the mobile phase (solvent) through the
column.

The chromatographic process begins by injecting a sample solution

into the mobile phase through the injector port. As a sample solution
in the mobile phase flows through a column, the
components/compounds of that solution migrate according to the
interactions of the components with the stationary phase. The chemical
interactions of the mobile phase and sample solution, with the
stationary phase, determine the degree of migration and separation of
components contained in the sample. For example, components which
have stronger interactions with the mobile phase than with the
stationary phase will elute from the column faster and thus have a
shorter retention time compared to those components which have
stronger interactions with the stationary phase. The type and
composition of the mobile phase is one of the variables influencing the
separation. Therefore, the mobile phase can be altered in order to
manipulate the interactions of the sample components and the
stationary phase. In isocratic elution, components are eluted using
constant mobile phase composition. In gradient elution, different
components are eluted by changing the mobile phase composition as
the separation proceeds. By increasing the strength of the mobile phase
will subsequently results in elution of retained components.

Each component elutes from the column as a narrow band (or peak),
detected and displayed as a chromatogram on a computer screen.
Detection of the eluting components can be either selective or
universal, depending upon the detector used. To collect, store and
analyze the chromatographic data, computers, integrators, and other
data processing equipment are frequently used.

22
 Figure 8: Schematic diagram of an HPLC instrument.

Instrumental components
The basic instrument consists of the following components:

Mobile phase reservoir

The most common type of mobile phase reservoir is a glass bottle with
the special cap, teflon tubing and filter to connect to the pump inlet
and to the purge gas (helium) used to remove dissolved air. Common
properties of solvents used in HPLC as mobile phase are as follows:
• Purity
• Detector compatibility
• Solubility of the sample
• Low viscosity
• Chemical inertness

Pump
Pump provides a continuous constant flow of the eluent through the
HPLC injector, column and detector. The pumping system must be
capable of delivering the mobile phase reproducibly through the
system. A constant flow rate between 0-10 mL/min is required in
HPLC, creating backpressures up to 6000 psi. Higher flow rates would
require too high pressure.

The two basic classifications of pump are the constant-pressure pump

which is mainly used for column packing and the constant-flow pump
which is the most widely used in all common HPLC applications.
Frequent types of pump available for use with HPLC analysis are
reciprocating piston and syringe type pumps.

Injector
Samples are injected into the HPLC via an injection port which
consists of an injection valve and a sample loop. Loop volumes can
range between 10 µL to over 500 µL. The sample, typically dissolved

23
in the mobile phase prior to assay, is drawn into a syringe and injected
into the sample loop via the injection valve. A rotation of the valve
rotor closes the valve and opens the loop in order to introduce the
sample into the stream of the mobile phase. In most modern HPLC
systems, the sample injection is typically automated.

Figure 9a shows a schematic drawing of a six-port Rheodyne valve in

which the sample fills an external loop at load position. A clockwise
rotation of the valve rotor places the sample-filled loop into the
mobile-phase stream, shown in Figure 9b (inject position), with
subsequent injection of the sample onto the top of the column through
a low-volume, cleanly swept channel.

to column to column

from pump from pump

loop
loop

sample in waste sample in waste

(a) (b)

Figure 9. Rheodyne valve at (a) load position and (b) inject position

Separation Column
The separation/analytical column is typically a stainless steel tube with
10, 15 or 25 cm in length. The stationary phase is micro-particulate
packing with commonly uniform porous silica particles several μm in
diameter. The internal diameter of the column is typically 4.6 mm;
this is considered the best compromise among sample capacity, mobile
phase consumption, speed and resolution.

Guard column
Guard column is placed prior to the separation column to serve as a
protective factor that prolongs the life of the separation column. They
are dependable columns designed to filter or remove: 1) particles that
clog the separation column; 2) compounds and ions that could
ultimately cause "baseline drift", decreased resolution, decreased
sensitivity, and create false peaks; 3) compounds that may cause
precipitation upon contact with the stationary or mobile phase; and 4)
compounds that might co-elute and cause extraneous peaks and

24
interfere with detection and/or quantification. These columns must be
changed on a regular basis in order to optimize their protective
function. Size of the packing varies with the type of protection needed.

Detector
HPLC detector is positioned immediately posterior to the stationary
phase in order to detect the compounds as they elute from the column.
It emits a response due to the eluting sample compound and
subsequently signals a peak on the chromatogram.

The detector passes a beam of light through the flowing column

effluent as it passes through a low volume (~10 mL) flowcell. The
variations in light intensity caused by UV absorption, or fluorescence
emission, or change in refractive index (depending on the type of
detector used) from the sample components passing through the cell,
are monitored as changes in the output voltage. These voltage changes
are recorded and fed into an integrator or computer to provide
retention time and peak area data.

The most commonly used detector in HPLC is the ultraviolet (UV)

absorption detector. Fixed wavelength UV detector measures at one
wavelength, usually 254 nm; variable wavelength UV detector
measures at one selected wavelength at a time, but can detect over a
wide range of wavelength from 190 to 460-600 nm; and photo diode
array UV detector measures a spectrum of wavelengths
simultaneously. Other common detectors include: refractive index
(RI), fluorescence (FL), electrochemical (EC) and mass-spectrometric
(MS).

Data system
Since the detector signal is electronic, use of modern data acquisition
techniques can aid in the signal analysis. The main goal in using
electronic data system is to increase analysis accuracy and precision,
while reducing operator attention.

Types of HPLC
Based on the mechanism of the separation, four modes of HPLC can
be specified: partition, adsorption, ion-exchange, and size exclusion.

In partition chromatography, two modes are defined depending on the

relative polarity of the two phases: normal and reversed-phase
chromatography.

25
In normal-phase chromatography, the stationary phase is strongly
polar in nature, and the mobile phase is non-polar (n-hexane) or less
polar solvents such as dichloromethane and carbon tetrachloride. Polar
compounds are retained longer on the polar surface of the stationary
phase, thus eluted later than those which are less polar.

Reversed-phase chromatography is the inverse of normal-phase. The

stationary phase is non-polar (hydrophobic) in nature, while the mobile
phase is a polar solvent, such as mixtures of water and methanol or
acetonitrile. Hence, the more non-polar the compound is, the longer it
will be retained by the stationary phase.

In adsorption chromatography, the stationary phase is a solid

adsorbent (e.g. silica gel or any other silica based packings) and the
separation is based on repeated adsorption-desorption steps.

In ion-exchange chromatography, the stationary phase has an ionically

charged surface of opposite charge to the sample ions. The stronger the
charge in the sample, the stronger it will be attracted to the ionic
surface and thus, the longer it will take to elute. The mobile phase is an
aqueous buffer, where both pH and ionic strength are used to control
the elution time. Ion-exchange chromatography is only applicable with
ionic or ionizable samples.

In size exclusion chromatography, the column is filled with precisely

controlled pore sizes packing, and the sample components are
separated according to its solvated molecular size. Large molecules
which could not enter the pores are rapidly eluted through the column
followed by moderate size molecules; small molecules which could
penetrate inside the pores of the packing particles are thus eluted later.
Two types of size exclusion chromatography are gel filtration and gel
permeation chromatography.

Basic procedures for running HPLC

1. Switch on the instrument.
2. Enter values for mobile phase composition and flow rate.
3. Start the pump and wait until the pump stabilizes.
4. Turn on the UV detector and set at desired wavelength.

Helpful hints
1. Liquid samples may be injected directly. Solid samples need only
be dissolved in an appropriate solvent, preferable the mobile phase

26
 in order to avoid detector interference, column interference or loss
in efficiency.
2. It is advisable to remove particles from the sample by filtering or
centrifuging since continuous injections of particulate material will
eventually cause blockage of injection devices or columns.
3. Always make sure that the injection valve is at LOAD position
before injecting any sample using the syringe.

27
 APPENDIX III
Quantitation Methods in Chromatography
Using individual standards and reproducible conditions, individual
components are identified by the retention time, tR. Under a given set
of chromatographic conditions, a specific analyte will always have the
same retention time.

Quantitative analysis in chromatography depends upon the direct

relationship between the area under a peak or peak height in the
chromatogram and the amount of the compound corresponding to that
peak in the analyzed sample. Each peak should be totally resolved
from any neighboring peaks. A co-elution or other anomalies such as
tailing or fronting will distort or obscure the beginning and ending
points of the peak, making it difficult to accurately determine the size
of the peak. There are several types of quantitation methods commonly
used. The five most common are area percent, single point external
standard, multiple point external standard, single point internal
standard, and multiple point internal standard.

Area Percent Method

Area percent is the simplest quantitation method. This method assumes
that the detector responds identically to all compounds. This
assumption, however, is not valid. This method provides a rough
estimate of the amounts of compounds present. To calculate area
percent take the area of a compound and divide it by the sum of areas
for all peaks. This value represents the percentage of a compound in
the sample.

Single Point External Standard

Chromatographic detectors have different responses to each
compound. In order to determine quantitative amounts of various
compounds in a separation mixture, the detector response must be
calibrated using standards. Inject a standard solution of the compound
and record the peak area. Then calculate the response factor for the
compound using Equation 1.

Equation 1

sample amount
Re sponse Factor (RF) =
peak area

28
Inject the sample containing the unknown compound and record the
peak area. The amount of unknown compound in the sample can be
determined using Equation 2.

Equation 2

Amount of unknown compound = peak area of unknown compound x RF

Example 1
The following gas chromatographic data were obtained in the analysis
of two phthalate esters, dioctyl and dibutyl phthalate in a sample.

Area
Dioctyl phthalate 230714
Dibutyl phthalate 218977

An injection containing dioctyl phthalate and dibutyl phthalate at a

concentration of 5 ppm resulted in a peak area of 150305 and 130000
respectively. Calculate the amount of each phthalate present in the
sample.

5
RF for dioctyl phthalate = = 3.3 x 10 −5
150305
5
RF for dibutyl phthalate = = 3.8 x 10 −5
130000

Using the response factor, the unknown concentration can now be

calculated:

Amount of dioctyl phthalate in mixture = 230718 x 3.3 x 10 −5 = 7.6 ppm

Amount of dibutyl phthalate in mixture = 218977 x 3.8 x 10 −5 = 8.3 ppm

29
Multiple Points External Standard
A series of different concentrations of standard solution (at least five
levels) should be prepared and the response was measured. The
relationship between standard solution concentration and detector
response ie. peak area versus concentration, can be plotted to give a
calibration curve and generating regression linear equation, y = mx + c
where m is the slope and c is the intercept. The concentration of
unknown in samples can be calculated using the linear equation.

Internal Standard
Unlike external standard methods, the internal standard method
accounts for any variances in gas chromatograph performance. An
internal standard is a known amount of a compound, different from
analyte that is added to the unknown. Signal from analyte is compared
with signal from the internal standard to find out how much analyte is
present. The analyte chosen for the internal standard has a predictable
retention time and area, allowing it to be used to determine if
abnormalities have occurred.

Internal standard

A B

Internal standards are especially useful for analyses in which the

quantity of sample analyzed or the instrument response varies slightly
from run to run for reasons that are difficult to control. For example,
gas or liquid flow rates that vary by a few percent in a chromatography
experiment could change the detector response. A calibration curve is
only accurate for the one set of conditions under which it is obtained.
However, the relative response of the detector to the analyte and
standard is usually constant over a wide range of conditions. As long
as the concentration of standard is known, the correct concentration of
analyte can be derived. Internal standards are widely used in
chromatography because the small quantity of sample solution injected
into the chromatograph is not very reproducible in some experiments.
Internal standards are also desirable when sample loss can occur

30
during sample preparation steps prior to analysis. If a known quantity
of standard is added to the unknown prior to any manipulations, the
ratio of standard to analyte remains constant because the same fraction
of each is lost in any operation.

The Single Point Internal Standard method requires at least two

analyses. The first analysis contains a known amount of internal
standard and the analyte of interest and calculate the relative response
factor (Equation 3). Then add a known amount of the internal standard
to the sample containing the analyte of unknown concentrations and
calculate the amount of the analyte in the sample using equation 4.

Equation 3

amount AS x area IS
Re lative Re sponse Factor (RRF ) =
area AS x amount IS

Equation 4

area AS x amount IS x RRF

Amount of analyte in the sample =
area IS

IS = internal standard
AS = analyte in sample

Multiple Point Internal Standard uses several analyses. Each analysis

contains the internal standard whose concentration is kept constant and
the analyte of interest whose concentration covers the range of
concentrations expected. Plot the results with the ratio of the area of
the analytes to the area of the internal standard on the y-axis and the
ratio of the concentration of the analytes to the concentration of
internal standard on the x-axis. Fit this data to a curve using the
method previously described. Add known amount of internal
standard to the samples and analyze the samples with the added
internal standard. Determine the ratio of the analyte area to internal
standard area from the data. The corresponding ratio of analyte
concentration to internal standard concentration is determined from the
graph. Multiply the concentration of internal standard in the sample by
this ratio.

31
 APPENDIX IV
Properties of Compounds
A. Physical Properties of Common Solvents

Hildebrand Snyder
o solubility polarity
Solvent Bp ( C)
parameter index
δ
Acetic acid 117.9 12.4 6.2
Acetone 56.3 9.6 5.4
Acetonitrile 81.6 11.7 6.2
Benzene 80.1 9.2 3.0
Carbon tetrachloride 76.5 8.6 1.7
Chloroform 61.2 9.2 3.4-4.4
Cyclohexane 80.7 8.2 0
n-decane 174.1 7.8 0.3
Dichloromethane 40.0 9.6 3.4
Ethanol 78.3 12.0 5.2
Ethyl acetate 77.1 9.1 4.3
n-hexane 68.9 7.3 0
Methanol 64.7 13.7 6.6
Methyl acetate 56.3 9.2 4.4
i-octane 99.2 7.0 0.4
i-propyl ether 68.3 7.3 2.2
Pyridine 115.3 10.7 5.3
Toluene 101.6 8.9 2.3
Tetrahydrofuran 66.0 9.1 4.2
Water 100 21.0 9.0

32
 B. Polarity Ranking of Organic Classes

Non polar Alkanes

Alkenes
Ethers
Halogenated hydrocarbons
Aromatic hydrocarbons
Aldehydes and Ketones
Esters
Alcohols
Amines
Carboxylic acids
Polar Amides

References:

Licaberth Ismail (2014). Pharmaceuticals as potential chemical

markers in wastewater, MSc Thesis Universiti Teknologi MARA.

Cruickshank, J.M. (2010). Beta blockers in

hypertension. Lancet 376, 415.

Baranowska, I., Magiera, S. and Baranowski, J. (2011). UHPLC

Method for the Simultaneous Determination of β-Blockers,
Isoflavones and Flavonoids in Human Urine, Journal of
Chromatographic Science, 49, 764-773.

Rozita Osman, Norashikin Saim, Mardiana Saaid, Nor Nasriah Zaini.

(2016). An Experimental Design Method for the Extraction of

33
 Eurycomanone from Tongkat Ali (Eurycoma longifolia) Roots using
Pressurised Liquid Extraction (PLE). Malaysian Journal of Analytical
Sciences, Vol 20 No 2, 342 – 350.

Licaberth Ismail, Rozita Osman and Norashikin Saim. (2013). Tandem

Solid Phase Extraction for the Determination of Pharmaceuticals in
Wastewater. The Malaysian Journal of Analytical Sciences 17(2)2): 262
– 271.

R. Osman, N. Saim and M. P. Abdullah. (2009). Simultaneous

Extraction of Organic Compounds with a Wide Polarity Range in Water
Using Solid Phase Extraction Technique. Research Journal of
Chemistry and Environment 13(3):7-18. {Impact Factor:
0.292/SCOPUS/ISI}

SUMMARY

This Analytical Separation Methods Laboratory Guide contains a

compilation of ten laboratory experiments aims at teaching students
how to analyze liquid and solid samples. The first two laboratory
experiments emphasize on method development of gas
chromatography (GC) and high performance liquid chromatography
(HPLC). The other eight laboratory experiments emphasize on sample
preparation such as solid-phase extraction, solid-phase microextraction
and accelerated solvent extraction prior to chromatographic analysis.
This laboratory guide is designed for students to plan, conduct and
justify scientific analysis in separation chemistry. Theoretical notes on
GC and HPLC are provided as appendices at the end of this laboratory
guide.

34
35