MODULE 2: CHROMATOGRAPHY: GC, GLC, HPLC

Swagat K. Mohapatra
Dept. of Chemistry, University of Rajasthan, J.L.N Marg, Jaipur – 302 004

Introduction:
Chromatography is a technique mainly used for separating and/or recoginzing the components in a
chemical mixture. The origin behind this technique is that the chemical components of the mixture have
different affinities to adsorb onto a surface and/or dissolve in an organic solvent. This technique is widely
accepted in industry on a large scale to distinguish and purify the components and the respective products
in various organic syntheses.
Mikhail S. Tswett, a Russian scientist, first identified this technique as an efficient method for separation,
during the beginning of twentieth century. He used a simple form of liquid-solid chromatography to
separate a number of plant pigments, such as chlorophyll, carotenes, and xanthophylls. Since these
components are of different colors such as green, orange and yellow respectively, they gave this
technique its name as “color writing”. However, since then this technique was not popular among
scientists for a long time. In 1941, A. J. P. Martin and R. L. M. Synge at Cambridge University, UK
discovered partition chromatography for which there were awarded the Noble Prize in 1952.
Erns
Today chromatography is one of the most powerful and useful techniques available to the modern
chemist. In a single step process it can separate a mixture into its individual components and
simultaneously provide a quantitative estimate of each constituent. Samples may be gaseous, liquid or
THEORY
solid in nature and can range in complexity from a simple1 blend OF CHROMATOGRAPHY
of two isomers of the same chemical
species to a multi component mixture containing widely differing chemical species. Furthermore, the
analysis can be carried out, at one extreme, on a simple, inexpensive thin layer plate, &/or on the other
hand on a very costly and complex instrument. Separation of two sample components in chromatography
distributionthat
Thus chromatography has been defined as “a separation technique between two non-miscible
is achieved phases.
by distributing theThe one, the stati
components of a mixture between two phases, a stationary is fixed
phase andina the system.
mobile The
phase. other,
Those the mobile phase, a flui
components
held preferentially in the stationary phase are retained longer in the system than
chromatographic thoseIn
system. thatgas
arechromatography
distributed the mobile
selectively in the mobile phase. As a consequence, the solutes are eluted from the system as local
chromatography it is a liquid.
concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to
the stationary phase; thus a separation can be achieved”.[1]
Chromatography Theory:
Mobile Phase, m
Chromatography comprises a sample (or sample
extract) being dissolved in a mobile phase (which may v
be a gas, a liquid or a supercritical fluid). The mobile
phase is then forced through an immobile, immiscible A
stationary phase. The phases are chosen such that
components of the sample have differing solubilities in Stationary Phase, s
each phase. A component which is quite soluble in the
Figure  1:  Schematic  presentation  of  a  chromatographic  
stationary phase will take longer to travel through it Figurewith   1 Schematic
system   partition  of  presentation of atchromatographic
analyte  A  between   he  p hases;   system w
v  mobile  phase  velocity
between the phases; v mobile phase velocity.
1  
 
The molecules of the analytes are distributed between t
phase. When present in the stationary phase, they are retained, an
than a component which is not very soluble in the stationary phase but very soluble in the mobile phase.
As a result of these differences in mobilities, sample components will become separated from each other
as they travel through the stationary phase.

Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas
Chromatography) use columns - narrow tubes packed with stationary phase, through which the mobile
phase is forced. The sample is transported through the column by continuous addition of mobile phase.
This process is called elution. The average rate at which an analyte moves through the column is
determined by the time it spends in the mobile phase.

Distribution of analytes between phases: The distribution of analytes between phases can often be
described quite simply. The analyte A is in equilibrium between the two phases;
𝐴!"#$%&   ⇌   𝐴!"#"$%&#'(    
The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of
analyte in the stationary phase (Cs) divided by the molar concentration of the analyte A in the mobile
phase (Cm).

𝐶!
𝐾=
𝐶!
A chromatogram is a graph showing the detector response
as a function of elution time (figure 2).

The time between sample injection and an analyte peak

reaching a detector at the end of the column is termed the
retention time (tR). Each analyte in a sample will have a
different retention time. The time taken for the mobile Figure  2:  Schematic  diagram  of  a  Chromatogram
phase to pass through the column is called dead time (tM).
tR and tM are easily obtained from chromatogram.

The term Retention factor or Capacity factor (Rf) is often used to describe the migration rate of an
analyte on a column. The retention factor for analyte A is defined as;

𝑡! − 𝑡!
𝑅! =  
𝑡!

When an analytes retention factor is less than one, elution is so fast that the accurate determination of the
retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long
time. Ideally, the retention factor for an analyte is between one and five.

Selectivity factor (α) is a quantity, which describes the separation of two species (A and B) on the
column:

(𝑅!)! (𝑡! )! − 𝑡!
𝛼 =   =            (∵ 𝑠𝑖𝑛𝑐𝑒  𝑡!  𝑖𝑠  𝑠𝑎𝑚𝑒  𝑓𝑜𝑟  𝑏𝑜𝑡ℎ  𝑡ℎ𝑒  𝑠𝑝𝑒𝑐𝑖𝑒𝑠)
𝑘′! (𝑡! )! − 𝑡!

When calculating the selectivity factor α, species A elutes faster than species B. The selectivity factor

2  
 
 Branches of Chromatography
1) interaction of the solute 2) chromatographic bed shape
with the stationary phase 3) physical state of (geometry)
i) Adsorption the mobile phase
i) Planar
1) paper
ii) Partition 2) Thin layer
i) Gas ii) Liquid
iii) Ion-exchange chromatography chromatography ii) Column
1) Gas-Liquid 1) Liquid-Liquid 1) packed
iv) molecular 2) open-tubular
exlcusion chromatigraphy chromatigraphy
2) Gas-solid 2) Liquid-solid
chromatography chromatography
is always greater than one.

Classification of Chromatography methods:

Chromatography methods can be mainly categorized on the basis of followings: 1) the stationary phase
(adsorption, partition, ion-exchange, and molecular exclusion), 2) chromatographic bed shape (planar,
column), 3) the mobile phase (gas chromatography, and liquid chromatography).
1. On the basis of the stationary phase:
i) Adsorption Chromatography: It includes a mobile liquid or gas phase which is adsorbed onto the
surface of a stationary solid phase. The equilibration between the two phases (mobile and stationary)
differentiates the solutes, and thus help to separate.
ii) Partition Chromatograph: It involves the formation of a thin film on the surface of a solid supported
by the liquid stationary phase.

iii) Ion-exchange Chromatograph: In this case a resin is used as the solid stationary phase, which is
required to covalently attract ions (cations/anions) towards it. The oppositely charged ions of the solute
present in the mobile phase liquid undergo an electrostatic force of attraction and attach to the resin.
iv) Molecular exclusion / gel permeation Chromatograph:
This method does not hold the attraction between the stationary phase, and the solute. The mobile phase,
either a liquid or gaseous phase, when pass through a porous gel, are separated based on their size. As the
pore size of the gel are kept usually small, this allows the smaller molecules to go into the gel while
excluding the larger solute particles. This retards the flow rate of the smaller molecules while passing
through the column, where as the large molecule pass through the column at faster rate.
2. On the basis of chromatographic bed shape:
i) Planar chromatography: Here the stationary phase is presented as or on a plane. This can be a paper;
can be used as such or some substance can be impregnated on the surface as the stationary bed (called

3  
 
paper chromatography). It can also be a layer or solid particles uniformly spread over a glass plate, called
thin layer chromatography (TLC). The different components in the organic mixture, while passing
through the planar surface vertically, can be distinguished over time based on the distance they travel.
This mainly depends on the interaction between the solute and the stationary phase with respect to the
mobile phase. The specific retention factor of each component will also be used to characterize the
unknown substance.

ii) Column Chromatography: In this case the stationary bed is kept inside a glass tube or column. There
are to possibilities in such arrangements. In the first case the solid stationary phase or support coated with
a liquid stationary may be tightly packed the whole inside volume of the column, called as Packed column
chromatography. While in the second the solid stationary phase concentrate on or along the inside wall of
the column, leaving an open, unrestricted path for the mobile phase at the middle portion of the column,
called Open tubular column.

3. On the basis of the mobile phase:

i) Gas chromatography (GC): [2,3]
Gas Chromatography deals with separation and purification of compounds between two phases. One if
fixed, called to be stationary phase, which may be either a solid or a liquid held by a solid. The second
one is mobile, called to be mobile phase, which may be a gas, liquid or vapor. There are two method of
separation based on the state of stationary phase: 1) gas-solid chromatography (GSC), this involves the
mobile phase is a gas, and the stationary phase as an active solid like silica gel, alumina; 2) gas-liquid
chromatography (GLC), where the moving phase is a gas, and the stationary phase is a liquid distributed
on an inert solid support. GLC is mainly used for the preparation and identification of a variety of organic
compounds. GSC is used mainly for the separation of gases.

Principle: The principle of operating gas chromatograph involves the incorporation of small amount of
sample either in gaseous or liquid state, containing nanogram amounts of analytically pure gaseous or
vapourizable component, which are inserted into a column holding the stationary phase by an inert carrier
gas (N2 or He) under controlled temperature. Phase equilibria happen between the components in the
sample mixture, separate the components on the basis of their difference in adsorption, bonding nature,
and solubility. Each fraction then pass through the column at a varied flow rate, and thus begin to
separate. Further the carrier gas coming out of the column moves through a detector, thus develops a
distinct signal with respect to the quantity of each of the component present in the sample mixture. Then
the detector response is amplified on the recorder as a peak, called chromatogram which is a plot of time
versus the intensity of peaks, and represent the eluted components in the carrier stream. The retention
time, which is time needed for each of the peaks to reach the detector, and characteristics to each of the
component present under a set chromatographic conditions. Thus it characterizes each of the component
of the mixture, and further area of the peaks give a quantitative measurement.

An appropriate choice of the injection port, column, and the detector temperature, column materials, the
detector measures the efficiency of the chromatographic separations of each component present in the
sample.

Basic Design: There are many commercial models of gas chromatograph available till date, with

4  
 
124 Modern Chemical Techniques

considerable variations in design and arrangement of components. The basic design of the apparatus
THE ROYAL Unilever
includes: (a) carrier gas system, (b) sample injector, (c) column, (d) thermostat, (d) detector, and (e)
SOCIETY OF
CHEMISTRY recorder (figure 3).[3]

Dried
carrier
gas in
Detector
Replaceable
silicon
rubber
septum
Exit
Injector
port Recorder

Injector Detector
oven oven

. Column, typically 3 m
long and 2 mm inside
diameter
Column oven

Analytical columns tend to be narrow, and preparation columns tend to be wide to

allow for the greater volumes passing through them.

The sensitivity of the technique is such that very small samples can be analysed,
down to 10-7 dm3 (0.1 µl).

Figure  3:  A  Schematic  representation  of  Gas  Chromatograph

Figure 9 The gas chromatograph
Applications:
column to be increased gradually over a required range. This is done by using
a) Incomputer control, and gives
Medical-Biochemical a better
field: Gas separation
chromatic ifhas
solute boiling
been points
proved to are
be close,
very and
useful in medical-
a faster separation if some components are relatively involatile.
biochemical field. It has been used for specific separations and measurements of substances like saturated
The solutes progress to the end of the column, to a detector. Two types of detector
and unsaturated
are commonly fattyused:
acidsthermal
in lowconductivity
concentrations, in determining
detectors and flame positional
ionisation and configurational isomers
detectors.
of unsaturated
Thermal conductivity detectors respond to changes in the thermal conductivity of thealkaloids, amino
fatty acids, straight and branched chain fatty acids, sterols and steroids,
acids,gas leaving
urinary the column.
aromatic acids,Abile
hot acids,
tungsten–rhenium
vitamins, and filament is kept in an in
toxic components oven
air,set at a and food. Many
water,
given temperature so that all solutes are in the gaseous phase
stress has been given on measuring the extent of alcohols in breath samples. (Fig. 10 ). When the
carrier gas – helium, for example, which will have been warmed to the temperature
b) In of
Toxicology:
the detectorGas chromatograph
block is also being
– leaves the column it coolsuseful in filament.
the hot the field However,
of toxicology,
if a particularly for the
solute emerges with the helium it will cool the filament less (unless the
substances like steroids, lipids, barbiturates, drugs, and blood alcohols. Usually thesolute is samples available in
hydrogen, because only hydrogen has a thermal conductivity greater than helium)
the field of toxicology are very limited, and in low concentrations, thus Gas chromatograph is a worth full
and the temperature of the filament will rise. Its resistance will then increase, and that
method for their complete analysis, and identification of the sample mixture. For example, to monitor the
can be measured.
trace constituents of atmospheric components, a small gas chromatograph-mass spectrometer was sent to
space in a simulated manned spacecraft. Gas chromatograph is also used in the aerospace and nuclear
submarine field to analyze the air quality of working and sleeping environments. This has also been
employed in the rempte sampling and analysis of tunnel Block atmosphere.

c) In Industry: Chromatographic method has also been seen to Gas be useful in the field of industrial
flowsolvents present in breath samples.
hygiene.This is used to identify and quantitatively measure the organic
out
Gas
There are toxic organic substances who has neither any identifiable biological metabolite nor whose
flow
in
5   Filament
 
Figure 10 A simple thermal conductivity detector
presence can be compared with atmospheric concentration of the existing substances. It is thus making
difficult to evaluate their effect of exposure. Thus breath analysis by Gas chromatography is found to be
useful to analyze quantitatively such organic substances.
Furthermore, this method is running by several industrial hygiene departments for product identification
programs for complete analysis of proprietary products. In industrial laboratory, chromatographic method
also plays a major role. For example: analysis of organic solvents used for various chemical syntheses,
analysis of products prepared from different batches, analysis of organic compounds of unknown
compositions and for full identification.
Many commercial products upon addition can increase to some extent the toxicity of less hazardous
materials. For example, mixing carbon tetrachloride with typewriter cleaner, which contains only
ethylene, mixing benzene to gasoline for increase the efficiency of the engine.
1. Food
Chromatography 129
d) In Air analysis:
129
Air quality analysis can also be determined using Gas chromatograph. For examples, in various industrial
plants, the air samples containing organic solvents can be separated and purified by chromatography
avoiding any tedious chemical method. Substances such as chlorinated hydrocarbons can be easily
monitored in a mixture and individual components can also be identified in chromatography, whereas
chemical methodUnilever
can only determine the total chlorinated hydrocarbon. Presence of nitrogen oxides in air THE ROYAL
SOCIETY OF
can be monitored by this method. Scientists Bethea, [4] and Lawson [5] were first proposed such CHEMISTRY

analysis.
Using a two section column, Bethea [4] was
able to determine the nitrogen dioxide with a
lower detection limit of 200 ppm. Gas
chromatography of gases emanating from the Porous flow adaptor –
fills the space above the
soli has been successful in separating the stationary phase so that
components mixture using a three-column the solutes cannot mix
with the solvent above
system equipped with a thermal detector. A
them
short silica column has been used to determine
nitrogen dioxide at 50-60 °C with hydrogen as Column – analytical columns
tend to be narrow; columns
a carrier gas. Nitrogen dioxide has been for preparation are wide
collected and concentrated on Molecular
Stationary phase
Sieves and determined by chromatography.
ii) Liquid chromatography:
Liquid chromatography method is similar to
gas chromatography but uses a liquid instead a
gas as the mobile phase. The stationary phase Glass wool plug to
is usually an inert solid such as silica gel prevent stationary
phase clogging the
(SiO2.xH2O), or alumina (Al2O3.xH2O) packed tap
in a glass column (figure 4).

Liquid chromatography is particularly useful Figure  4:  Schematic  diagram  of  a  Liquid  Chromatography  column
for the analysis of highly polar, materialsFigure
with 15 Liquid chromatography column

The adsorbing properties of silica and alumina are reduced if they absorb water,
6  
but the reduction is reversed by heating to 200–400 °C. Silica is slightly acidic, and
  readily adsorbs basic solutes. On the other hand, alumina is slightly basic and
strongly adsorbs acidic solutes. Other stationary phases that can be used include
magnesia, MgO.xH2O (good for separating unsaturated organic compounds); and
dextran (a polymer of glucose) cross-linked with propan-1,2,3-triol (glycerol,
high molecular weights having a very low volatility. This is where Gas chromatography lacks in
separating. Furthermore, this technique can run even with samples in trace amounts, such as in drugs and
drug metabolites in blood.

In principle, the sample mixture needs to pass through a short length of tube packed column with an
adsorbent, which selectively removes the material of interest. The adsorbent is then washed and the
adsorbed material extract with a small amount of suitable solvent and the solvent then inject onto the
column.

A more elaborate variation on liquid chromatography is high performance liquid chromatography

(HPLC). This technique generally uses very small packing particles with a relatively high pressure.

High Performance Liquid Chromatography (HPLC):

132 Modern Chemical Techniques
The effectiveness in separating the various components of an organic mixture increase with the decrease
in the particle size in the stationary phase, due to the rapid equilibrate of solute between the two phases.
Further, when particles sizes are smaller, the capillary action increases, as a result the draining of the
solvent through the column become difficult under gravity. Therefore, a high pressure has to be needed to
THE ROYAL Unilever
apply on the solvent SOCIETY
to runOF the column. This is where a new chromatographic method called High
CHEMISTRY
pressure Liquid Chromatography aroused. This is widely known as High Performance Liquid
Chromatography (HPLC).
Injection valve
A schematic diagram of the HPLC
system is given in Figure 5. [6]

Here the stationary phase is made up

of uniform porous silica particles of
10-6 diameter. Further the surface Column
Mobile phase reservoir High pressure pump
pores are having diameter range
between 10-6 to 10-9, which results a
solid of high surface area.
The particles bonded to the non-
volatile liquid permits to interact the Waste

solutes with different polarities. The Detector

particles of the stationary phase are
tightly packed into the HPLC column, Data collection/integrator
and kept in place using glass fibres
coated with inert alkyl silane
molecules.
Generally the length of columns are
not required to be longer because of
their effectiveness in the separating
the components of the organic
mixture. In the case of long columns, Recorder/plotter
Figure  5:  A  Schematic  diagram  of  an  HPLC  technique

Figure 17 Schematic representation of an HPLC system

7  
  long the pressure needed would be excessive.) Columns are typically 10–30 cm long,
with an internal diameter of 4 mm.
Reproducibility is essential, and this is only possible if a constant flow rate is
maintained. This means that the pump used must be capable of generating a uniform
the pressure applied needs to be increased.
A constant flow rate is always necessary to have the reproducibility, which is indispensable for such
measurements. This demands a suitable pump, which should be able to produce uniform pressure. Twin
cylinder reciprocating pumps are idea for this purpose. These kind of pumps generally contain two
chambers with piston of 180 ° out of phase, and is able to generate pressure around 10 MNM-2. Further all
the tubing in the pump, and in the instrument as well should be inert to the organic solvents, and solutes
being go through them.

HPLC columns normally have slow flow rates, between the range 0.5 – 5 cm3.min-1. Further these
columns are very narrow. Thus injection of the sample should be accurate, and this should not disturb the
solvent flow in the column. In case of Gas Chromatograph, the flow rate of solutes passing through the
column can be increased by raising the temperature of the column. While in case of HPLC, this is
achieved by modifying the composition of the mobile phase. For example, in the concentration gradient
of methanol and water, the ratio of methnol can be increased linearly from 10 – 60 % of the total volume,
during the separation.

As the concentration of the solute in their respective solution passing through the column are very low.
Instead of extracting them from the solution, they are analyzed directly for identification once they come
8
through the pump. In often cases the different components of the organic mixture are separated by HPLC
absorb ultraviolet light. The eluate are generally made to
glass tube. passsample
The through was
a small cell so that
reduced in can come across
volume by evaporation in
the ultraviolet radiation. stream of nitrogen and finally made up to a volume of 250µl wi
Application:
methanol.

Gas Chromatograph analyzes compounds which are Urine Extract Calibration

stable at high temperatures, while High Performance (50ng/ml of drug) Standard
Liquid Chromatography (HPLC) technique is ideal for
those sample which are likely to decompose at high
temperature or low volatile organic solids. It is one of
the widely adopted analytical procedure; its application
involve in a range of areas such quality control, process
control, forensic analysis, environmental monitoring and
clinical testing. Furthermore HPLC technique remain as
one of the most sensitive analytical method and is an
exclusive method which is suitable for multi-component
Tetrahydrocannabinol
mixtures.

1. An example of the use of this technique is in the

identification of tetrahydrocannabinol carboxylic acid in
urine, which is generally present in the urine of those
persons who have smoked marihuana. The
tetrahydrocannabinol carboxylic acid can be extracted
from the urine by means of a solid state extraction
Minutes Minutes
cartridge packed with a C18 reverse phase (a strongly
dispersive packing containing bonded Figure  6:  Chromatogram  
Courtesy of of  Supelco
urine  sample  
Inc.of  a  person  
smoking  marihuana.

8 Figure
  42. Chromatograms of Tetrahydrocannabinol Car-boxy
  Acid from Urine

The chromatograms obtained from a urine sample and a referen

octyldecyldimethyl chains). The urine sample can be used direct, without pretreatment and the materials
of interest are irreversibly adsorbed on the reverse phase solely by dispersive interactions. [2] The
chromatograms obtained from a urine sample and a reference standard by this procedure is shown in
figure 6. This is a characteristic application for liquid chromatography using solid phase extraction
cartridges. It is seen that the tetrahydrocannabinol carboxylic acid is clearly and unambiguously separated
from the contaminating materials with an extraction efficiency of over 90%. [2]

A similar example of using this technique is analysis of tricyclic antidepressant drugs in blood serum.

2. Pharmaceuticals: HPLC provides a reliable quantitative analysis of pharmaceutical products with high
accuracy and precision in a single run. An appropriate method for preparing sample for solid dosage is
dispersion in water or a mixture of water and organic solvents such as acetonitrile/methanol. HPLC
provides various possibilities for separating chiral organic molecules into their respective enantiomers,
which involves precolumn derivatization to form diastereomers. Furthermore, typical columns may be
prepared with cyclodextrins for special chiral moieties as stationary phases. The common use in analyzing
the pharmaceutical products are: Assay, Related Substances, Analytical Method Validation, Stability
Studies, Compound Identification, Working Standards.

2. Foods: HPLC technique has been proved to be suitable in analyzing food components. Food
components are usually in complex forms and extraction of these components is not easy. Again both the
desirable and undesirable substances are often seen to be present in trace amounts, and thus their
extraction, and identification become further complicated and thus do not provide the necessary amount
of accuracy and precision. HPLC provides a doable solutions because of a variety of choices available in
selecting the stationary and mobiles phases. The Common components in food those can be analyzed in
food are: fat and water soluble vitamins, Residual pesticides, Antioxidants, Sugars, Cholesterol and
sterols, Dyes and synthetic colors, Mycotoxins, Amino acids, Residual antibiotics, Steroids and
flavonoids, Aspartame and other artificial sweeteners, and some active ingredients of farm products.

3. Air analysis: HPLC technique is also being introduced in detecting the level of several pollutants
present in air. For example, recently the extent of particulate matter with aerodynamic diameter < 2.5
(PM2.5) bound polycyclic aromatic hydrocarbons (PAHs) present in air is measured by HPLC method
with fluorescence-ultraviolet detector (HPLC-FLD). Incomplete combustion and pyrolysis of fossil fuels
and other organic materials are generally the source for PAHs in the atmosphere. Their presence in the
atmosphere are serious threats to the human life due to their toxic, carcinogenic, and mutagenic effects. In
particular, epidemiological evidence confirms the incidences of respiratory diseases, including lung
cancer in persons from the exposure of PM2.5 bound PAHs.

References:

1. Scott Raymond P. W., Principles and Practice of Chromatography, Chrom-Ed Book Series, 01-02,
www.library4science.com.
2. http://www.cdc.gov/niosh/pdfs/74-177-k.pdf
3. http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatogra
4. R. M. Bethea, M. C. Meador Journal of Chromatographic Science, 7, 655 (1969)
5. A. Lawson, H. G. McAdie, Journal of Chromatographic Science, 8, 723 (1970)

9  
 
6. http://faculty.ksu.edu.sa/Dr.almajed/Books/practical%20HPLC.pdf

Suggestions for further Readings:

1. Textbooks:
i) Chromatography, By B. K. Sharma, Goel Publishing House, Krishna Prakashan Media (P) Ltd, fift
edition 2007.
ii) Analytical Chromatography, By Gurdeep R. Chatwal, Himalaya Publishing House 2006.
iii) Chromatography, By David Robert Browning, McGraw-Hill, 2008.
2. Websites:
i) http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatogra
ii) http://faculty.ksu.edu.sa/Dr.almajed/Books/practical%20HPLC.pdf
iii) http://shodhganga.inflibnet.ac.in/bitstream/10603/3866/7/07_chapter%201.pdf
iv) http://lincolnparkhs.org/ourpages/auto/2010/9/26/37121222/Chapter%2018%20-
%20Chromatography.pdf
Multiple Choice Questions:

1. Who did first recognize the technique Chromatography?

(A) A. J. P. Martin
(B) R. L. M. Synge
(C) M. S. Tswett
(D) None of the above
2. In which year A. J. P. Martin and R. L. M. synge got the nobel prize in chemistry for their
invention of partition chromatography.
(A) 1952
(B) 1955
(C) 1925
(D) 1975
3. The chromatography method is useful for the process of ___.
(A) separation
(B) identification
(C) purification
(D) all of the above
4. The number phases invloves in the Chromatography technique is:
(A) 2
(B) 4
(C) 3
(D) 1
5. In Gas-liquid Chromatography, the mobile phase or carrier is a__
(A) solid
(B) liquid

10  
 
 (C) gas
(D) None of the above
6. In HPLC, the mobile phase is a__
(A) solid
(B) liquid
(C) gas
(D) none of the above
7. What are the commonly used carrier gases in GC.
(A) N2, He
(B) O2, water vapor
(C) Ne, Ar
(D) None of the above
8. For the accurate determination, the retention factor Rf for an analyte should be in the range?
(A) Rf < 1
(B) Rf > 20
(C) 1< Rf < 5
(D) None of the above
9. Highly polar and large molecular materials those have very low volatility are ideal for use in
which chromatography technique:
(A) GLC
(B) LC
(C) GSC
(D) All of the above
10. The substance which appear in the urine of those who some marihuana.
(A) Catachin
(B) Pantothenic acid
(C) Tetrahydrocannabinol acid
(D) Folic Acid

Answers: 1. (C), 2. (A), 3.(D), 4.(A), 5.(C),6. (B), 7. (A), 8.(C), 9.(B), 10. (C)

11