Separation Techniques

UNIT 3 CHROMATOGRAPHY
TECHNIQUES
Structure

3.0 Introduction

3.1 Objective

3.2 Gas liquid chromatography

3.3 High-performance liquid chromatography

3.3.1 Principle of HPLC

3.3.2 Instrumentation

3.3.3 Types of HPLC

3.3.4 Application of HPLC

3.4 Supercritical fluid chromatography

3.5 Application of chromatographic techniques in environmental monitoring

3.6 Let Us Sum Up

3.7 Terminal questions

Answers

3.0 INTRODUCTION
Analytical science in recent years has progressed enormously with the discovery
of pioneering separation methods. The subject hasfull-fledged into a separate
unit called “separation science”. Numerous journals have been dedicatedsolely
to this field. Limited instrumental methods can be directly used for the
quantitative study on account of the presence of interfering substances and
ions. These interfering elements must be separated before analysis. The
improvement of advanced method in separation rendered hasquantitative
methods of analysis more reliable. These separations are mainly based upon
the formation of a second phase with a different concentration of desired
constituent. Such separation depends upon the existence of physical or chemical
properties of the components need to be separated.

The methods usually depend on differences in properties such as mobility,

volatility, solubility, exchange equilibrium, surface activity, partition ratio,
molecular geometry and kinetic energy. Various chromatographic methods
based upon these differences are discussedin the following chapters.

Some chromatographic techniques require some modification, expertise and

are time consuming. It requires constant care while performing the experiment.
To overcome this problem, nowadays various advanced techniques such as
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GC, GLC, HPLC, UPLC and UPCC (Super Critical chromatography) are using Chromatography
Techniques
by pharmaceutical and research industries. In this unit, we shallstudy the modern
aided chromatography such as Gas-liquid chromatography or GLC, high-
performance liquid chromatography or HPLC and supercritical fluid
chromatography. And also, we shall state the application of these
chromatographic techniques in monitoringthe environment.

3.1 OBJECTIVES
After studying this unit, you should able toable to understand:

z Advanced chromatographic techniques

z Gas-liquid chromatography

z High-performance liquid chromatography

z Supercritical fluid chromatography

z Application of chromatographic techniques in environmental monitoring

3.2 GAS-LIQUID CHROMATOGRAPHY

Gas-liquid chromatography orGLC is a technique employed for the separation
of two phases in a mixture where one is a stationary phase and other is a
mobile phase. Stationary phase is a non- volatile liquid coated on a solid surface
and mobile phase is a carrier gas which is inert in nature i.e. unreactive gas
such as Neon, Helium and Argon. Gas-liquid chromatography is a type of
partition chromatography. Therefore, it is also known as gas-liquid partition
chromatography (GLPC) or vapor phase chromatography (VPC). The
instrument used for gas liquid chromatography technique is known as gas
chromatograph.

HISTORY

In most of the chromatographic techniques, the mobile phase used was liquid
but in 1950, Dr. Martin with his young colleague A.T. James demonstrated a
chromatographic technique at the October meeting of ‘Biochemical Society’
and in that chromatographic technique the stationary phase was liquid and
mobile phase was a gas. Thus, that technique was named as Gas-Liquid
chromatography or GLC.

PRINCIPLE OF GLC

GLC is based on the partition of stationary phase and mobile phase. Stationary
phase is a liquid and it is selected on the basis of polarity by the rule of “like
dissolves like”. The selected liquid solution is a polar liquid which is able to
interact with the solute particles. The sample mixture after passing through
the stationary phase and mobile phase will separate on the basis of partition
coefficient.

Partition coefficient (Kg) = Conc. of solute in liquid / Conc. of solute in gas

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Separation Techniques Partition coefficient is directly proportional to the concentration of solute in
liquid solution, if the partition coefficient will be low then the elution of
component will be fast and vice-versa. More volatile substances having low
boiling point and high vapor pressure will have more concentration in mobile
phase and therefore, will elute faster. Branched chain compounds and
compounds having lower number of carbon atoms have low boiling point and
are more volatile and therefore, will elute faster than the compounds having
higher number of carbon atoms.Similarly, less polar compounds have lower
boiling point and will elute faster than the compounds having more polarity.
In conclusion, more polar compounds are retained for a longerperiod in the
column.

Retention Time: The time taken by a compound to travel through the column
to the detector is called as its retention time and it depends on the following
factors:

z Boiling point: If the boiling point of compound is low, then its retention
time will be less as compared to the compounds having higher boiling
point.

z Solubility in liquid phase: More solubility in liquid phase implies high

retention time.

Temperature of column: Higher the temperature of column, more readily all

the solute particles in a column will evaporate. The solute which elutes faster
will have lesser retention time.

Instrumentation:

Figure 1: Different components of Gas-Liquid Chromatography

1. Injection Port: In injection port, the sample is injected with the help of
micro-syringe through a silicon rubber septum and this rubber septum reseals
itself after the injection of sample into the column. The injection port is kept
into an oven so that if the sample is in liquid state then it can be converted into
the vapour phase first and then it is carried into the column with the help of an
inert gas i.e.carrier gas.

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 Chromatography
Techniques

Figure 2: Injection port of Gas Liquid Chromatography

In a packed column, 0.1 to 10 µL sample is injected whereas in capillary column,

only a small volume of sample is injected. If the sample is in solid state then
the sample is placed into a glass ampules and the sample is crushed after placing
it in the gas stream.

Injection Temperature: The injection temperature should be high so that the

sample can be easily vaporizedinto the column but it should not be too high,
otherwise it will degrade the rubber septum.

2. Columns: Two types of columns are used in GLC:

z Packed Columns: Packed columns are of various shapes such as coiled

tubes, U- shaped tubes and W- shaped tubes but the commonly used
packed columns are coiled tube. These are 1 to 10 cm long and 0.2 to
0.6 cm wide. Columns are made up of Teflon, glass and stainless steel.
For inertness, glass column is preferred but for longer columns stainless
steel is preferred so that they can be straightened for packing. GLC
column is coated with non- volatile liquid.

z Capillary Columns: Capillary columns are 25 to 100 mm long and

0.25 to 0.50 mm wide and these are made up of silica and glass.
Capillary columns are most commonly used instead of packed columns.
Capillary columns are also known as open- tubular columns.

Column temperature: Column temperature is less than injection temperature.

It is about 50oC to 250oC. If the temperature of column is too high, then the
sample isconverted firstly into gaseous phase and elution takes place which
will lower the quality of resolutionalso the temperature of column should not
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Separation Techniques be too low because at low temperature, resolution will increase but sensitivity
will decrease due to the slow elution of sample and peaks will separated by
larger gaps.

3. Detectors: They produce an electrical signal proportional to solute

concentration or mass flow rate. GLC detectors are of various types:

z Thermal conductivity detectors (TCD): TCD contains a heated

filament with an applied current. It is also known as cathetometer.
Thermal conductivity detectors are most commonly used detectors
for a wide range of solutes. The sensitivity of these detectors is fair. It
is about 5 to 20 ng. In TCD the carrier gas containing solute is passed
through the cell due to which the change in the filament current occurs.
Then the current change is compared with the current in reference
cell and the difference is measured and the signal is generated. In
TCD, Hydrogen and Heliumused as a carrier gas because they have
very high thermal conductivity as compared to the other gases. TCD
is used for the analysis of permanent gas such as CO2.

z Flame Ionization Detectors (FID): These detectors are used for all
organic substances. FID is good for the detection of all hydrocarbons
but some oxygenated products respond poorly. The sensitivity of these
detectors is very good in the range of about 10 to 100 pg. FID is based
on the principle of ionization. Mostly organic compounds after passing
through the flame produce ions which are attracted towards oppositely
charged electrodes and are measured. The number of ions collected is
proportional to the number of carbon atoms in the sample. Signal is
generated when the number of ions hitting the electrodes is measured.
In FID He and N2 gases are used as carrier gas.

z Electron Capture Detector (ECD):These detectors are used for all

those substances that have affinity to capture the electrons, they do
not give any signal for aliphatic and naphthenic hydrocarbons. The
sensitivity of ECD for halogen containing substances is excellent. The
sensitivity is about 0.05 to 1 pg. In ECD the carrier gas is passed
which results in the production of electrons and then which produces
current between the electrodes. The signal is generated by measuring
the amount of lost current. Here carrier gases used are nitrogen and
argon.

Detector Temperature: Detector temperature should be high to prevent the

condensation of sample components.

4. Recorder: Recorder is a device that records the data from the detector
and draws that onto the chart paper.

Operational Procedure:

z The sample is injected into the gas stream first and then it enters into the
column.

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z The components of the mixture are then passed in a stream of gas through Chromatography
Techniques
the column.

z Then distribution of compound takes place between the phases in different

extents and elute out at different time and therefore, having different
retention time.

z The compound which dissolves in the stationary solvent is having more

retention time then others and hence, elute out slowly.

z The volatile compounds having lower boiling point emerges out rapidly.

z The compounds that are emerging out from the column are detected with
the help of detector.

z Then the recorder plots the signals detected by the detector as a

chromatogram.

z Then the peaks will be observed for each component of mixture on a

chromatogram.

Applications:

z GLC is used for qualitative and quantitative analysis of organic compounds.

z GLC technique is used in many fields such as in agriculture industry, food

industry, pharmaceutical, environmental field, forensic field, biotechnology
field, cosmetic industry and chemical industry.

z GLC is useful for the estimation of pesticide and insecticide residues in

food and other consumables.

z It is useful in the detection of pollutants in water and other food stuffs.

z In this technique very minute amount of substance can also be measured.

z As the volatile samples are needed for this technique so the samples of
human breath, blood, saliva and other secretions which contains large
amount of volatile organic substances can be easily analyzed by this this
technique.

z This technique is also useful for the analysis of effects of environment.

z GLC is also useful for determining the components of air in air samples.

z GC/MS is used in identifying the amount of chemicals in drugs in

pharmaceuticals industries.

z This technique is also useful to measure the amount of chemicals needed

for their products in cosmetic industries.

z It is highly sensitive technique.

z In this technique the analysis is highly accurate and precise.

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Separation Techniques ADVANTAGE OF GLC OVER GC CHROMATOGRAPHY

z In GLC the process of separating the compounds in a mixture is carried

out between a gas mobile phase and a liquid stationary phase whereas in
column chromatography mobile phase is liquid and stationary phase is
solid.

z In GLC temperature of gas phase can be controlled but in column

chromatography there is no way of controlling any temperature of mobile
phase.

SAQ 1

What are different mobile and stationary phase used in Gas Liquid
Chromatography.
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SAQ 2

What is different component used in Gas Liquid chromatography?

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3.3 HIGHPERFORMANCE LIQUID

CHROMATOGRAPHY
HPLC is a powerful technique used for separation and analysis of components
of mixtures. It yields high performance and high speed compared to traditional
columns because of forcibly pumped mobile phase creating pressure due to
which it is also referred as high- pressure liquid chromatography.

In twentieth century, liquid chromatography was discovered for separating

coloured compounds. In 1906 Mikhail S. Tswett a (Russian botanist) used this
technique to purify mixtures of plant pigments into the pure constituents, on
the basis of their interaction to the stationary phase.

Tswett filled an open glass column with particleswhich were chalk powder
(calcium carbonate) and alumina, using petroleum ether. The sample (solvent
extract of homogenized plant leaves) to be separated was then poured in the
column and allowed to pass through the particle bed.

3.3.1 Principle of HPLC

The principle of separation is adsorption. Separation of components takes place
due to difference in affinity of compounds towards stationary phase.
Compounds with stronger attachment to the particles (stationary phase) moved
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slower, while the compounds with strong attachment to the solvent (mobile Chromatography
Techniques
phase), moved faster. This is because the compounds in the sample get
distributed differently between the mobile phase and the stationary phase.

HPLC is a type of column chromatography that draws a sample mixture in the

solvent (mobile phase) through a column with chromatographic packing
material (stationary phase) at high pressure. The sample is being carried by a
moving gas (stream of helium or nitrogen).

HPLC has the ability to separate and identify the compounds, present in the
sample which are in very trace amounts (as low as parts per million) and can
be dissolved in the liquid. It is applicable for both volatile and non-volatile
compounds. It is used in qualitative as well as quantitative analysis, due to its
high resolution. Sample retention time will vary depending on the interaction
of sample with stationary phase, the solvent used and the molecule being
analysed. As the sample passes through the column it interacts between two
phases at different rate due to different polarities of the analytes. Because of
this versatility, this technique is being used in many industries such as
pharmaceutical, environmental, forensics and chemicals.

COMPONENTS OF HPLC

z Pump to force mobile phase through a system; suitable pressure gauges

i.e., flow meters control such pressure (fig3).

z Sample injection port or valves to inject sample in the system of the mobile
phase as head of separation column.

z Separation column in which sample components are separated into separate

peaks before elution and finally.

z The detectors and read out device, including computer accessories. First
we would consider mobile phase, stationary support and stationary phase,
kind of the columns and finally various kinds of detectors employed in
analysis.

3.3.2 Instrumentation

Figure 3: Different components of HPLC

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Separation Techniques FUNCTIONING OF HPLC:

z The liquid sample is injected insmall volume into a tube packed with small
particles (3P to 5P in diameter) called stationary phase.
z This is where the individual components of the sample are moved down
to the column with a liquid (mobile phase) forced through the column by
high pressure delivery by a pump.
z The components of the mixture are separated by the column packing, due
to various chemical or physical interactions between their molecules and
the packing particles.
z The separated components are detected at the exit of the column by a
flow-through device (detector) that measures their amount. The response
obtained from the detector is called a liquid chromatogram.
z In terms of principle, LC and HPLC workthe same way except the speed,
efficiency, sensitivity and ease of operation of HPLC is much more superior.

Chromatogram: It is a graphical presentation of detector response which is

used as a measure of the concentration of the analytes in the effluent versus
effluent volume or time.

Baseline: Baseline is a portion of chromatogram recording which is attained

when mobile phase arises from the column.

Column heater is used to maintain consistent temperature in the column in

order to get accurate results.

3.3.3 Types of HPLC

I BASED ON THE MODE OF SEPARATION

1. Normal Phase HPLC

In this method analytes are separated on the basis of polarity. Normal

Phase HPLC uses polar stationary phase (hydrophilic) and a non-polar
mobile phase (hydrophobic). Therefore, stationary phase is mostly silica
and mobile phasemay be hexane, chloroform, methylene chloride, diethyl
ether or mixtures of these.Polar sample interacts better with the polar
surface of column and persists for longer timethan less polar material, as
they elute out fast. Alumina, a porous particulate form of aluminum oxide
is used as a stationary phase. It has a highly active basic surface and is
more hygroscopic than silica. Its activity is measured according to
Brockmann scale for water content.

2. Reverse phase HPLC

The stationary phase is non-polar (hydrophobic) in nature, while the mobile

phase is a polar liquid (hydrophilic), such as mixtures of water and methanol
or acetonitrile. Reverse phase HPLC functions on the principle of
hydrophobic interactions hence the non-polar material in the sample will
retain for longer in the column than the polar.
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Non polar-non polar bonds and polar-polar bonds have more affinity than non Chromatography
Techniques
polar-polar bonds. Reverse phase chromatography is more commonly used
for drugs,as they are usually hydrophilic.

Table 1: Difference between Normal Phase and Reverse Phase HPLC

Normal phase Reversed phase

Stationary phase Polar (silica gel) Non-polar (C18)
Mobile phase Non-polar(organic Polar(aqueous/organic)
solvent)
Sample movement Non-polar fastest Polar fastest
Separation based on Different polarities Different hydrocarbon
(functionality) Content

II BASED ON PRINCIPLE OF SEPARATION

1. Adsorption Chromatography

In this chromatography the molecules of solute bond directly to the surface

of the stationary phase. The efficiency of separation depends on:

z Solubility of molecule in mobile phase.

z Binding strength to the stationary phase.

Greater the binding strength slower will be the movement of the molecules.
The matrix of stationary phase can be made of alumina or silica. Silica
(SiO2) is acidic in nature whereas alumina can be acidic, basic or neutral.
To increase the resolution/ separation, the surface should be of uniform in
size and the surface area should be increased.

2. Affinity Chromatography

This is most selective chromatographic techniqueis based on the

specificreversible interaction between the one kind of solute molecule need
to be purified and the affinity ligand immobilized to a solid support.

Example: the molecule that is immobilized on a stationary phase could be

an antibody to some specific protein. When solute containing mixture of
proteins is passed through this immobilized molecule, only a specific
protein reacts with this antibody, thereby binding it to the stationary phase.
By changing the ionic strength or pH, this protein can be later extracted.

3. Ion- Exchange HPLC

Ion exchange chromatographyallows the separation of ions and polar

molecules based on the charge they carry.Here like charges repel each
other and unlike attracts. This technique is used for ionic or ionisable
samples like- proteins, small nucleotides and amino acids. The stationary
phase is based on the nature and strength of the acidic and basic functions
and the ions types they attract and retain. The positively charged ions are
separated by cation exchange method on a negative surface. The negatively
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Separation Techniques charged ions are separated by the anion exchange method on a positive
surface. Strong ion exchangers (bear functional groupslike (quaternary
amines or sulfonic acids) are alwaysionised,and separate weak ions. Weak
ion exchangers (secondary amine or carboxylic acid functional group)
may get neutralized at certain pH and lose their ability to retain ions by
charge, but when charged they separate strong ions.

Retention is based on the attraction between solute ions and charged

species. The stronger the charge on the sample, the stronger will be the
attachment to the ionic surface and thus, the longer it will take to elute
out. The mobile phase actslikean aqueous buffer, where both,ionic strength
and pH control the elution time. Resin (stationary solid phase) is used to
attach anions or cations covalently onto it. Oppositely charged solute ions
in the mobile phase are attached to the resin by electrostatic forces of
attraction.

4. Ion-pair chromatography

In this chromatographic technique, ions in the solution can be neutralized

or paired and then separated as an ion-pair on a reverse-phase column.
Ion-pairing agents are nothing but the ionic compounds comprising of
hydrocarbon chain which imparts hydrophobicity to retain the ion pair on
a reversed-phase column.

5. Gel permeation chromatography

The interaction between the stationary phase and solute does not take place.
The materials used to fill the column have precisely controlled pore sizes.
The gaseous or liquid phase passes through a porous gel separating the
molecules based on their size. The pore size is normally small and excludes
the larger solute molecules, but allows smaller molecules to enter the gel,
due to which they flow through a larger volume. This result in larger
molecules passing through the column at a faster rate compared to the
smaller ones. Larger molecules get washed first from the column, as smaller
molecules penetrate inside the porous of the packing material and get
separated later.

6. Chiral chromatography

This technique helps in separation of stereoisomers. In enantiomers, there

are no physical or chemical differences apart from being three-dimensional
mirror images. To enable this technique, either mobile phase or stationary
phase should themselvesbe chiral to providedifferent affinities between
the analytes.

BASED ON ELUTION TECHNIQUE

1. Isocratic elution

In thisseparation technique, the mobile phase composition remains constant

throughout the procedure.

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2. Gradient elution Chromatography
Techniques
It is the process of separation in which the mobile phase composition is
changed.In this technique, the retention of the later eluting components is
decreased, so that they elute faster.

BASED ON TYPE OF ANALYSIS

1. Qualitative analysis

It is the analysis of the substance in order to know the nature of its chemical
constituents. We can separate the individual components but cannot find
the quantity.

2. Quantitative analysis

It is used to determine the amount and proportion of the chemical

constituents. Quantity of the impurity and individual components can be
found out.

3.3.4 Applications of HPLC Techniques

Pharmaceutical Application

z Tablet dissolution study of pharmaceutical dosages form.

z Pharmaceutical quality control.

Environmental Application

z Detection of phenolic compounds in drinking water.

z Bio-monitoring of pollutants.

Applications in Forensics

z Quantification of drugs in biological samples.

z Identification of steroids in blood, urine etc.

z Forensic analysis of textile dyes.

z Identification of cocaine and other abusive drugs in blood and urine

Food and Flavour

z Determination of quality of water and soft drinks

z Sugar analysis in fruit juices.

z Analysis of polycyclic compounds in vegetables.

z Preservative analysis

Applications in Clinical Tests

z Urine analysis, antibiotics analysis in blood.

z Analysis of bilirubin, biliverdin in hepatic disorders.

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Separation Techniques z Recognition of endogenous neuropeptides in extracellular fluid of brain,
etc.

Why HPLC is preferred over other chromatography technique?

z HPLC works under pressure of around (50-350 bar), unlike liquid

chromatography which relies on the force of gravity to pass through the
mobile phase. Due to this small sample amount can be separated.
z Column dimensions are 2.1-4.6 mm diameter and 30-250 mm length, and
are made with smaller adsorbent particles (2µm-50µm), this gives it a
superior resolving power (ability to distinguish compounds).
z This technique is fast because they used pump rather than gravity to flow
a solvent.
z Highly sensitive because whole process of completion takes only 10-30
minutes only.
z Accurate because whole system is automated.

DISADVANTAGE OF HPLC

z It is costly and complex because it requires large quantities of expensive

solvents
z Lack of ideal universal detector.
z Less separation efficiency than capillary gas chromatography.
z Relatively difficult for novices.

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY

It is an advanced type of HPLC, which has better instrumentation and column

technology which leads to increase in resolution, speed and sensitivity in liquid
chromatography. Columns are made with much smaller particles (1.7 micron)
and instrumentation with specialized capabilities designed to deliver mobile
phase at 15,000 psi (1,000 bar) which gives new level of performance.

SAQ 3

What is reverse and normal phase HPLC?

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SAQ 4

What are the different components of HPLC?

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 Chromatography
3.4 SUPER CRITICAL FLUID Techniques
CHROMATOGRAPHY
Supercritical fluid chromatography was first proposed in 1958 by Jim Lovelock.
He suggested that polar components could be separated by using inorganic
gases, above their critical points as a chromatographic mobile phase, where
mobile phase was considered to be inert carrier.

Ernst Klesper was first to actually separate thermally labile porphyrins using
a fluid above its critical point. He used di-chlorodifluoromethane and mono-
chlorodifluoromethane as mobile phase.

After Klesper’s paper, scientists aimed to find the perfect mobile phase by
examining purine, nucleotides, steroids, sugar, amino acids, and many more
substances using gases such as He, N2, CO2and NH3.

Currently SFCs involve a silica (or silica + modifier) packed column with
CO2 (or CO2 + modifier) mobile phase.

Supercritical fluid chromatography

This techniqueuses highly compressed gases above its critical temperature

and pressure as mobile phaseinstead of an organic solvent. Its principle is
similar to those of high -performance liquid chromatography (HPLC), but
Supercritical fluid chromatography (SFC) typically uses carbon dioxide as
mobile phase.

Supercritical fluid is any substance which exists above its critical point of
pressure and temperature, where distinct liquid and gas phases do not exist.

Critical point is the point at which two phases of a substance become

indistinguishable from one another. It is the end point defined by critical
pressure and critical temperature.

The fluid can solubilize the material of intersect with manipulation of pressure
and temperature. The sample is placed in an extraction vessel and to dissolve
the sample it is pressurized with CO2. Sample is transferred to a fraction
collector and CO2 loses its solvating power after depressurizing it causing the
precipitate of desired material. Condensed CO2 can be recycled.

Why CO2 is used?

Supercritical fluid chromatography uses CO2 modified with the organic solvents
and sometimes a highly polar additive. CO2 is preferred because:

z Easily available

z Has an accessible critical point

z Relatively safe

z Inexpensive

z Miscible with wide range of highly polar modifiers

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Separation Techniques z Non-toxic in nature

z Non-flammable during reaction

z Dissolving power and selectivity can be controlled by selection of suitable

pressure/temperature combination.

Modifiers or co-solvents

SFC applications are performed on comparatively polar stationary phases

having CO2 modified with an organic solvent and sometimes even other highly
polar components, called additives such as acids and bases.

Methanol is widely used modifier and most polar modifiers which is completely
miscible with CO2.

Advantage of methanol as modifier

z It is widely available and inexpensive.

z It has low UV cut-off.

z It has relatively low toxicity.

z It is completely misciblewith CO2.

Physical properties of supercritical fluid

Density

Density of supercritical fluid lies between that of a gas and liquid, but it is
closer to that of a liquid. Density increases with increase in pressure at constant
temperature. However, as the temperature increases with constant pressure,
density of supercritical fluid decreases. It is a better carrier than gases due to
its higher density.

Viscosity

Viscosity of supercritical fluids is almost same as a gas and one-tenth of that

of a liquid. They are less resistant than liquids towards component flowing
through.

Diffusivity

Supercritical fluid has a diffusivity of 100x that of a liquid and 1/1000 to 1/

10000 x less than a gas. Liquid has less diffusivity than supercritical fluid.
Diffusivity is directly proportional to temperature and inversely to pressure.
Higher the diffusivity, faster will be the chances of carrier for analytical
applications.

ADVANTAGE OF SFC OVER NORMAL HPLC

z HPLC is comparatively expensive than SFC as it requires high purity

solvent as mobile phase and further its purification but no such thing is
required for CO2.

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z Due to high diffusivity of the supercritical fluid it has much greater Chromatography
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resolving power and shorter column lengths can be used as more
interactions can occur in shorter span. Supercritical fluids have low
viscosities which allows high flow rate with low pressure and faster analysis
(5 to 10 X faster).

z It produces less toxic and flammable waste.

ADVANTAGE OF SFC OVER GC

z It allows fast analysis of thermally labile compounds.

z It has wide range of detectors as compared to GC.

z Supercritical fluids have greater solvating power so SFC has larger

molecular ranges which involve non-volatile molecules, polar to be
analyzed.

z Compounds with high molecular weight can be analyzed.

Instrumentation

Supercritical fluid chromatography (SFC) instrument consist of a mobile phase

container which usually is a pressurized gas cylinder, mobile phase, a modifier,
a pump, an injector, an oven, a column in a thermostatic compartment, a
microprocessor, and detector (fig4).

Figure 4: Different components of SFC

Stationary phase

Open tubular columns and packed columns are two most common columns
used in SFC. It has internal coating of cross-linked siloxanes material as a
stationary phase. Length of columns ranges from 10 to 20 m.

Mobile phase

The most common supercritical fluid which is used in SFC is carbon dioxide
because of critical temperature and pressures are easy to approach.Other than
carbon dioxide, ethane, n-butane, ammonia, NO2, THF can be used.

Modifier

A second fluid which is called modifier fluid such as (alcohol, chloroform,

cyclic ethers etc.) improves the solvating ability of supercritical. It increases
the column efficiency for highly retained nonpolar solute.
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Separation Techniques Columns

There are two types of analytical columns used in SFC i.e. packed and
capillary.Packed columns are those which contain small deactivated
substancesthat adheres to thestationary phase.. Capillary columns are the open
tubular columns having small internal diameter and are made of fused silica.

Pumps, Injectors and Ovens

A pump is needed for the stable transfer of liquid CO2. Incoming CO2 and
pumps head should be kept cold to maintain CO2 in a liquid state. Syringe
pumps are used for packed columns. Supercritical fluid is injected by switching
of the content of a sample loop into the carrier fluid at the entrance of a column
using auto sampler. For precise temperature control of the mobile phase to
achieve stable extraction thermo stated column ovens are used.

Microprocessor

It is used for control pumping pressure, oven temperature and detector

performance.

Detectors

Flame ionization detector normally present in GC can be used in SFC. It can

also be coupled with mass spectrometry, an UV-visible spectrometer or an IR
spectrometer.

DISADVANTAGE OF SFC

z There is limited choice of mobile phases and unwanted reactions with

mobile phase.
z SFC is applicable only for highly polar compounds and proteins.
z Maintaining pressure is difficult.
z High-pressure vessels are expensive and bulky.
z High capital investment is required for equipment.
z It is not suitable for water-soluble analytes.
z Cleaning of instrument is time consuming.

APPLICATION OF SFC

z It is used in quality control of chiral drugs.

z Polynuclear aromatic hydrocarbons in automobile exhaust, polyole-finic
antioxidants polyethoxylatedaklyphenols are analyzed successfully using
SFC.
z It is used for separation of thermally labile pesticides without resorting to
sample derivatization.
z It has beneficial application in forensic science as it used for identification
and analyses of explosive containing nitrate ester, nitramines, and drugs
of abuse like amphetamines, cocaine, and other related compounds.
62
z Organo-metals of thermally labile category, metal chelates, heavy metals, Chromatography
Techniques
can be separated using SFC.
z It is used for separation of the oligomers in a sample of the nonionic surface
TritonX100.
z Separation of components widely varying in their boiling points.
z It is used for extraction of fat from food products.
z It is used for photo-resist cleaning.
z It is used for fractionation and purification of polymers e.g. removal of
unchanged monomers from polymers.

Table 2: Critical pressure and critical temperature of selected substances, and

their notableproperties
(https://www.waters.com/waters/en_IN/PreparativeSFC/nav.htm? cid=134932612
andlocale=en_IN)

Substance Critical temp. Critical pressure Notable

(ºC) (bar) Properties

Carbon dioxide 31 74 Physical state

easily changed

Water 374 221 Extreme

conditions needed

Methanol 240 80 Extreme temperature

needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally
unfriendly

Nitrous Oxide 37 73 Oxidizing agent

n-Butane 152 38 Highly flammable

Figure 5: Working principle of SFC

63
Separation Techniques
SAQ 5
What do you mean by critical temperature and critical pressure of particular
solvent?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
SAQ 6

What is modifier or co-solvent?

.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

3.5 APPLICATION OF CHROMATOGRAPHIC

TECHNIQUE IN ENVIRONMENTAL
MONITORING
There are many applications of chromatography that help to monitor the
environment. It helps to keep our food safe and air and water clean.

z HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC) helps

to determine the presence of Fluazinam in soil. Fluazinam is pesticide
widely used against the fungal disease in potato cultivation. A specific
HPLC method using a diode array detector (DAD) helps to measure the
presence of Fluazinam in soil.

z Gas chromatography helps in measuring the ubiquitous pollutants in the

environment.

z HPLC technique is used for analysis air and water pollutants and for
monitoring pesticide level in environment. HPLC is used by federal and
state regulatory agencies to survey food and drug products.

z Gas chromatography is used for the determination of the volatile organic

compound(VOCs) in air. Volatile organic compound are a cause of concern
for human health due to their increased presence in the indoor environment.
They are responsible for a phenomenon called as a sick building
syndrome(SBS). Air monitoring method is used for assessing the indoor
pollution.

z There are numerous gases found in the industrial process and are harmful
for plants and human being such as sulphur dioxide, hydrogen sulphide.
Gas chromatography is used for the detection of these types of gases present
in the air.

z Gas chromatography is most widely used for the determination of trace

organic compounds present in environment.

z Both natural and synthetic food colours are added to foods to improve
64
 their acceptability and to make them more popular. Paper chromatography Chromatography
Techniques
has been primarily used for analysis of food colours in ice creams, sweets,
drinks and beverages, to ensure that only edible colours are added and no
non-permitted colouring agents are added to the foods.

z Certain organic compounds such as carbohydrates and amino acids are

identified or detected from a complex mixture of organic compounds with
the help of paper chromatography. It is used to separateanions and amino
acids.

z Thin layer chromatography is used for the determination of the various

plant extract. With TLC we also investigated the flavour components of
the juices of citrus fruits.

z Column chromatography is used for identification and determination of

non-volatile or strongly polar compounds in air and in surface, waste, and
drinking waters.

z Size exclusion chromatography is used for the removal of DOC(dissolved

organic carbon) from the drinking water.

z In size exclusion chromatography a gel Filtration-Based Method is used

for the Purification of Infectious Rotavirus Particles for Environmental
Research Applications.

z Soil and water measurement contamination of soil and water can come
from many areas, including acid rain, pesticides industrial waste and raw
sewage. These all are detected by GC-MS technique.

z Gum Arabic is a polysaccharide widely used in food industry as a viscosity

modifier or gelling agent.The physical properties and processability of
these water-soluble polymers are related to their molecular weight
distribution, which can be determined by gel permeation chromatography.

z GC has been used for differentiation of petroleum-type pollutant and to

study the fate of petroleum-type pollutants in underground and surface
waters.

z GC-MS technique is used to study of the bioremediation potential of

microorganisms for crude oil biodegradation.

z In different samples of water (ground water, surface water and drinking

water), the content of non-polar organic compounds, which are detectable
by high performance thin layer chromatography (HPTLC).

z Column chromatography helps in determination and separation of cardiac

glycosides in digitalis leaf.

z Column chromatography is best method to separate active component from

plant materials.

z Volatile samples, human breathe, blood, saliva and other secretions

containing large amounts of organic volatiles can be easily analyzed using
65
Spectroscopic Methods GC. The knowledge of the amount of compound in a given sample gives a
huge lead in studying the effects on human health and on the environment
as well.

z Air samples can be analyzed using GC. Mostly, GC coupled with FID is
used by air quality control units in order to determine the components of a
given air sample. Though other detectors are also useful, FID appears to
be the most appropriate due to its sensitivity and resolution and also for its
ability to detect very small molecules as well.

SAQ 7

What are the different applications of Gas chromatography to monitoring

the environment?
....................................................................................................................
....................................................................................................................
....................................................................................................................

3.6 LET US SUM UP

Chromatographic techniques are used for the quantitative and qualitative
analysis in various pharmaceuticals, food and dyes industries. Column
chromatographyhas various disadvantages including manual setup, time
consuming and accuracy. To overcome the problems, various advanced
techniques have been developed like gas liquid chromatography, HPLC Super
critical liquid chromatography and their application which are being employed
not only in industries but also for monitoring the environment. GLC is an
advanced techniquecompared to conventional chromatography.The separation
of compounds present in a mixture is carried out between a ‘gas’ mobile phase
and a ‘liquid’ stationary phase in GLC.However, in column chromatography,
mobile phase is liquid and stationary phase is solid. In GLC temperature of
gas phase can be controlled, but in column chromatography there is no way of
controlling any temperature of mobile phase. Like this HPLC is more advanced
than GLC in the manner that HPLC works under pressure of around (50-350
bar), unlike liquid chromatography which relies on the force of gravity to pass
through the mobile phase. Due to this small sample amount can be separated.
Column dimensions are 2.1-4.6 mm diameter and 30-250 mm length, and are
made with smaller adsorbent particles (2µm-50µm), this gives it a superior
resolving power (ability to distinguish compounds). HPLC is comparatively
expensive than SFC as it requires high purity solvent as mobile phase and
further its purification but no such thing is required for CO2. Due to high
diffusivity of the supercritical fluid it has much greater resolving power and
shorter column lengths can be used as more interactions can occur in shorter
span. Supercritical fluids have low viscosities which allows high flow rate
with low pressure and faster analysis (5 to 10 X faster). Nowadays many
applications of chromatographic techniques are being used to monitor the
environmental conditions. It helps to keep our food safe and air/ water clean.

66
 Molecular Spectroscopy
3.7 TERMINAL QUESTIONS
1. What are the advanced chromatography techniques used by pharmaceutical
industries and researchers?

2. What is the working principle of Gas Liquid Chromatography?

3. What is the advantage of GLC over column chromatography?

4. What is the advantage of HPLC over GLC?

5. What is the advantage of UPLC over HPLC?

6. What are the different applications of advanced chromatography

techniques?

ANSWERS
Self-Assessment Questions

1 Correctly fixing up, requires some mechanical expertise and physical

modification, time consuming. It requires constant care monitoring while
performing the experiment.

2. Stationary phase is a non- volatile liquid layer coated on a solid surface

and mobile phase is carrier gas and which is inert in nature i.e. unreactive
gas such as N2, He and Ar.

3. Inject port, column, detector and recorder.

4.

Normal phase Reversed phase

Stationary phase Polar (silica gel) Non-polar (C18)

Mobile phase Non-polar Polar(aqueousorganic)

(organic solvent)

Sample movement Non-polar fastest Polar fastest

Separation based on Differentpolaritie Differenthydrocarbon

(functionality) Content

5. Different component of HPLC

z The solvent reservoir or multiple reservoirs

z A high pressure pump

z A column

z Injector system

z Detector

6. The critical temperature of a substance is the temperature at and above

67
Separation Techniques which vapor of the substance cannot be liquefied, no matter how much
pressure is applied.

The critical pressure of a substance is the pressure required to liquefy a

gas at its critical temperature. Some examples are shown below.

7. SFC applications are performed on relatively polar stationary phases with

CO2 modified with an organic solvent and sometimes other highly polar
components, such as acids and bases, called additives. Methanol is widely
used modifier and most polar modifiers which is completely miscible with
CO2.

8. Soil and water measurement contamination of soil and water can come
from many areas, including acid rain, pesticides industrial waste and raw
sewage. These all are detected by GC-MS technique.

TERMINAL QUESTIONS

1. These are gas chromatography, gas liquid chromatography, HPLC, super

critical fluid chromatography.

2. The GLC is based on the partition of stationary phase and mobile phase.
Stationary phase is liquid and it is selected on the basis of polarity by the
rule of “like dissolves like” so the selected liquid solution is polar liquid
which is able to interact with the solute particles. The sample mixture
after passing through the stationary phase and mobile phase will separate
on the basis of partition coefficient.

Partition coefficient (Kg) = Conc. of solute in liquid / Conc. of solute in gas

3. Advantage of GLC over column chromatography:

z In GLC the process of separating the compounds in a mixture is carried

out between a gas mobile phase and a liquid stationary phase whereas
in column chromatography mobile phase is liquid and stationary phase
is solid.

z In GLC temperature of gas phase can be controlled but in column

chromatography there is no way of controlling any temperature of
mobile phase.

4. HPLC is preferred over other chromatography in following ways

z HPLC works under pressure of around (50-350 bar), unlike liquid

chromatography which relies on the force of gravity to pass through
the mobile phase. Due to this small sample amount can be separated.

z Column dimensions are 2.1-4.6 mm diameter and 30-250 mm length,

and are made with smaller adsorbent particles ( 2µm-50µm), this gives
it a superior resolving power (ability to distinguish compounds).

z This technique is fast

z Highly sensitive
68
 z Accurate Chromatography
Techniques
z Automated

5. HPLC is comparatively expensive than SFC as it requires high purity

solvent as mobile phase and further its purification but no such thing is
required for CO2.

Due to high diffusivity of the supercritical fluid it has much greater

resolving power and shorter column lengths can be used as more
interactions can occur in shorter span. Supercritical fluids have low
viscosities which allows high flow rate with low pressure and faster analysis
(5 to 10 X faster).

z It produces less toxic and flammable waste.

z Advantage of SFC over GC

z It allows fast analysis of thermally labile compounds.

6. Applications of chromatography techniques

z HPLC helps to determine the presence of Fluazinam in soil. Fluazinam

is widely used pesticide used against the fungal disease in potato
cultivation. A specific HPLC method using a diode array detector
(DAD) which helps to measure the presence of Fluazinam in soil.

z Gas chromatography helps in measuring the ubiquitous pollutants in

the environment.

z HPLC technique is used for analysis air and water pollutants and for
monitoring pesticide level in environment. Federal and state regulatory
agencies use HPLC to survey food and drug products.

z Gas chromatography is used for the determination of the volatile

organic compound (VOCs) in air. Volatile organic compound are a
cause of concern for human health due to their increased presence in
the indoor environment. They are responsible for a phenomenon called
as a sick building syndrome (SBS). Air monitoring is being used to
assess indoor pollution.

z There are numerous gases found in the industrial process and are
harmful for plants and human being such as sulphur dioxide, hydrogen
sulphide. Gas chromatography is used for the detection of these types
of gases present in the air.

z Gas chromatography is most widely used for the determination of

trace organic compounds present in environment.

z Both natural and synthetic food colours are added to foods to improve
their acceptability and to make them more popular. Paper
chromatography has been primarily used for analysis of food colours
in ice creams, sweets, drinks and beverages. To ensure that no non-
69
 permitted colouring agents are added to the foods, only edible colours
are permitted for use.

z Certain organic compounds such as carbohydrates and amino acids

are identified or detected from a complex mixture of organic
compounds with the help of paper chromatography. It is useful in the
separation of anions and amino acids.