PART II

Separation Techniques

6. Chromatography

02/14/2025 CHROMATOGRAPHY 1
 INTRODUCTION TO CHROMATOGARAPHY

History
◦ Russian botanist Tswett M. (1872–1919)

◦ Used a column packed with a stationary phase of calcium

carbonate to separate colored pigments
from plant extracts.
◦ The sample was placed at the top of the

column and carried through the stationary

phase using a mobile phase of petroleum ether.
◦ Chroma-color, graphy-writing
02/14/2025 CHROMATOGRAPHY 2
 INTRODUCTION….

 Chromatography is a separation process that is achieved by

distributing the components of a mixture between two
phases,
 a stationary phase and
 a mobile phase.
 Those components held preferentially in the stationary
phase are retained longer in the system than those that are
distributed selectively in the mobile phase.

 solutes are eluted from the system in the order of their

increasing distribution coefficients with respect to the
stationary phase
02/14/2025 CHROMATOGRAPHY 3
 INTRODUCTION…

• Mobile phase – is a moving phase that continuously

flows through the stationary phase and carries the
analyte.
• Stationary phase – the fixed or non-moving phase,
immobile phase.

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 Introduction …
Separates components in mixture:
Based on
- polarity
- boiling point
- ionic strength
- size
There are different types of chromatography

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 INTRODUCTION…

Chromatographic techniques
Planar Chromatography Column chromatography
plane support SP SP-in column
◦ Paper chromatography
Liquid SP-soaked in cellulose paper ◦ Gas chromatography
◦ Thin layer chromatography
◦ High performance liquid
adsorbent (Al2O3 or SiO2, usually)
chromatography
coating a sheet of plastic or glass

Separated cpds-appear as spot

Separated cpds-appear
as a peak
02/14/2025 CHROMATOGRAPHY 11
 INTRODUCTION…
• Detection of separated
compounds in PC and TLC is
made either
– By its natural color
– By scanning under UV
fluorescence
– By spraying with reagents
• Characterization of the
compounds is made by
measuring retardation factor
(Rf)

02/14/2025 CHROMATOGRAPHY 12
 INTRODUCTION…

Column chromatography:-

• This is the most common type of chromatography

 The progress of a chromatographic separation is monitored

with a suitable detector situated at the end of the column.

 A plot of the detector’s signal as a function of time or

volume of eluted mobile phase is known as

chromatogram and consists of a peak for each of the

separated solute bands.

02/14/2025 CHROMATOGRAPHY 13
 INTRODUCTION…
• The chromatogram can be
characterized by its retention
time and peak width
• There are d/t terms
associated with a
chromatogram
• The time taken for the
mobile phase to pass through
the column is called tM.

02/14/2025 CHROMATOGRAPHY 14
 INTRODUCTION…
• Chromatographic Resolution
– The goal of chromatography is to separate a sample into
a series of chromatographic peaks, each representing a
single component of the sample.
– Resolution is a quantitative measure of the degree of
separation between two chromatographic peaks, A and
B, and is defined as

15
02/14/2025 CHROMATOGRAPHY
 INTRODUCTION…
• Capacity factor/retention factor/
– is often used to describe the migration rate of an
analyte on a column
– When an analyte’s retention factor is less than one,
elution is so fast that 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.

02/14/2025 CHROMATOGRAPHY 16
 INTRODUCTION…
• Column Selectivity
– The relative selectivity of a chromatographic column
for a pair of solutes is given by the selectivity factor,
α, which is defined as

– The selectivity factor is always greater than one

(species A elutes faster than species B).

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 INTRODUCTION…
• Column efficiency
– Separation power of the column
– Efficiency is determined by the number of
theoretical plates
– Separate equilibrations of the sample between
the stationary and mobile phase occur in these
"plates”

02/14/2025 CHROMATOGRAPHY 18
 INTRODUCTION…
• Column efficiency…
– The number of theoretical plates that a real
column possesses can be found by
examining a chromatographic peak after
elution;

where w1/2 is the peak width at half-height.

02/14/2025 CHROMATOGRAPHY 19
 INTRODUCTION…
Resolution can be increased by increasing
• Capacity factor
• Decreasing T0 in GC
• Decrease solvent strength in LC
• Increasing the volume of stationary phase

• Selectivity factor – by changing the Sp and Mp

• Number of theoretical plates - decreasing particle
size of the stationary phase

02/14/2025 CHROMATOGRAPHY 20
 INTRODUCTION…
Examples
1. In a chromatographic analysis of lemon oil a peak for
limonene has a retention time of 8.36 min with a
baseline width of 0.96 min. gama-Terpinene elutes at
9.54 min, with a baseline width of 0.64 min. What is
the resolution between the two peaks

2. In a chromatographic analysis of low-molecular-

weight acids, butyric acid elutes with a retention time
of 7.63 min. The column’s void time is 0.31 min.
Calculate the capacity factor for butyric acid.
02/14/2025 CHROMATOGRAPHY 21
 INTRODUCTION…
Examples…

3. In the same chromatographic analysis for low-molecular-

weight acids considered in Example 2, the retention time
for isobutyric acid is 5.98 min. What is the selectivity factor
for isobutyric acid and butyric acid?

4. A chromatographic analysis for the chlorinated pesticide Dieldrin

gives a peak with a retention time of 8.68 min and a baseline
width of 0.29 min. How many theoretical plates are involved in
this separation? Given that the column used in this analysis is 2.0
02/14/2025 CHROMATOGRAPHY 22
meters long, what is the height of a theoretical plate?
 7. GAS
CHROMATOGRAPHY (gc)

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 Gas Chromatography
Definition
• A method in which sample of gases or volatilized
components are separated due to their relative degree of
affinity for fixed stationary phase of the column and mobile
phase (carrier gas).
• Liquid or solid stationary phase and gas mobile phase are
used.
Classification
– Gas solid chromatography (GSC)
– Gas liquid chromatography
02/14/2025 (GLC)
CHROMATOGRAPHY 24
 Gas Chromatography…
Principles of GC
• Sample which must be volatile and thermally stable at the
operating temperature are introduced in to the gas flow
via an injection part located at the top of the column.
• A continuous flow of gas elutes the components from the
column in order of increasing distribution ratio from
where they pass through a detector connected to a
recording system.

02/14/2025 CHROMATOGRAPHY 25
Gas Chromatography…

• Most GC requires small amount of the sample and no

attempt is made to collect the separated compounds.
– Analytical GC
• However large columns are available which can separate
samples up to one gram.

• Receivers attached to the outside of the chromatography

can collect the individual compounds after passing
through the detectors.
– Preparative GC

02/14/2025 CHROMATOGRAPHY 26
Gas Chromatography…
What Types of Compounds are Suitable for GC Analysis?
• For a compound to be suitable for GC analysis, it must
possess appreciable volatility at temperatures below 350–
400 °C.
• The compound must be able to withstand high
temperatures and be rapidly transformed into a vapor
without degradation or reacting with other compounds.
• As a general rule, the greater the molecular weight or
polarity of a compound, the lower is its volatility.

• The presence of polar functionalities such as hydroxyl and

amine groups severely decrease compound volatility (Eg.
Sugars and amino acids)
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 Gas Chromatography…

What type of …
• As a rule, inorganic compounds are not suitable for GC
analysis. Metals and salts do not possess the required
volatility.
• Many organo-metallics have sufficient volatility for
analysis due to the high organic content of these
molecules.
• However, there are many exceptions. Many biomolecules
and pharmaceuticals are thermally sensitive and
degrade at the temperatures used in gas chromatography
• Overall, it has been estimated that only about 10% of all
compounds can be analyzed by GC.
02/14/2025 CHROMATOGRAPHY 28
 Gas Chromatography…
Advantages Disadvantages
• Gases diffuse at higher rate • Directly Applied to
than liquids-shorter analysis volatile compounds
time only
• Fast equilibration condition- • Samples should not be
higher efficiency thermo-labile
• Picogram (10-12) amount of • GC is expensive
sample can be detected-high
sensitivity
• Detectors specific for
elements like N, S &Cl

02/14/2025 CHROMATOGRAPHY 29
Gas Chromatography…

INSTRUMENTATION
• A gas chromatographic system is comprised of six
major components:
• Gas supply and Flow controllers
• Injector
• column
• Oven
• Detector and a data system.
• In most cases, the injector, detector and oven are
integral parts of the gas chromatograph; the column,
gases and recording device are separate items and are
often supplied by a different
02/14/2025
manufacturers.
CHROMATOGRAPHY 30
 Gas Chromatography…
Applications of GC

• Gas chromatography is widely used for the analysis of a

diverse array of samples in environmental, clinical,
pharmaceutical, biochemical, forensic, food science, and
petrochemical laboratories.
• Can be classified into
• Quantitative application
• Qualitative application

02/14/2025 CHROMATOGRAPHY 60
 Gas Chromatography…
A. Qualitative Application
• Gas chromatography can be used for qualitative purposes.
• When using an IR or a mass spectrometer as the detector,
the available spectral information often can be used to
identify individual solutes.
• With conventional non spectroscopic detectors, other
methods must be used to identify the solutes.
• One approach is to spike the sample by adding an aliquot of
a suspected analyte and looking for an increase in peak
height/area/.
• Retention times also can be compared with values measured
for standards, provided that the operating conditions are
identical.
02/14/2025 CHROMATOGRAPHY 61
 Gas Chromatography…
B. Quantitative Application
In a quantitative analysis, the height or area of an
analyte’s chromatographic peak is used to determine its
concentration.
Calibration curves are usually constructed by analyzing a
series of external standards and plotting the detector’s
signal as a function of their known concentrations.
As long as the injection volume is identical for every
standard and sample, calibration curves prepared in this
fashion give both accurate and precise results.
02/14/2025 CHROMATOGRAPHY 64
 8. High Performance Liquid
Chromatography
(HPLC)

02/14/2025 CHROMATOGRAPHY 65
 HPLC
Definition
 HPLC is type of column chromatography in which components of
mixture are separated by distributing them b/n liquid mobile phase
and either solid or liquid stationary phase.

 It is a sophisticated form of liquid chromatography

 Fine column packing particles are used to increase the efficiency

 The decrease in particle size of the packing materials makes the
use of pumping system necessary
 It is often necessary to use samples less than 20 microgram.
 Under such conditions, sensitive detectors and data handling
systems are required to CHROMATOGRAPHY
02/14/2025
analyze the column effluent. 66
 HPLC…
Advantages over GC

 Only 10% of known compounds can be handled by

GC with out prior chemical modification.

 Some compounds may not be handled effectively by

GC
– They may be insufficiently volatile and hence can
not pass through the column
– They may be thermally unstable and hence might
decompose under the conditions of separation

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 HPLC…
Advantages over GC…
 HPLC on the other hand is not limited by sample
volatility or thermal stability.

 Thus HPLC is ideally suited for separation of

Macromolecules of biomedical interest

Labile Natural products
A wide variety of high molecular weight
compounds like Proteins, nucleic acids,
polysaccharides, synthetic polymers etc.
02/14/2025 CHROMATOGRAPHY 68
 HPLC…
Advantages over GC…
 Very difficult separations are often achieved by LC:

Reasons include
– Liquid chromatographic separations are the result of
interaction b/n sample molecules, stationary and
mobile phase
– Interaction with the mobile phase is absent in GC
– Presence of many variables assist in controlling and
improving separation.
– Greater variety of stationary phases are available for
HPLC
02/14/2025 CHROMATOGRAPHY 69
 HPLC…
Advantages over GC…

– Chromatographic separation is generally enhanced

at lowered temperature.

• Low temperature makes intermolecular

interactions more effective.

• This favors HPLC that is usually performed at

room temperature

02/14/2025 CHROMATOGRAPHY 70
 HPLC…
Advantages over GC…

Relative ease of isolate recovery

◦ Separated fractions can easily be collected in HPLC

by placing a flask at the end of the column

◦ Recovery of separated components in GC is also

possible but is generally less convenient and not
quantitative.

02/14/2025 CHROMATOGRAPHY 71
 HPLC…
Modes of HPLC

1. Normal phase HPLC- uses polar stationary

phase and non-polar mobile phase

2. Reversed phase HPLC- uses non-polar

stationary phase and polar mobile phase

02/14/2025 CHROMATOGRAPHY 72
 HPLC…
Principles of HPLC

 Mobile phase is filtered and pumped through a

chromatographic column

 The sample is injected and loaded onto the column

where the components of the mixture are separated.

 The effluent is monitored using a detector and

recorded as peaks.

02/14/2025 CHROMATOGRAPHY 73
 HPLC…
HPLC Instrumentation

Solvent Reservoirs

02/14/2025 CHROMATOGRAPHY 74
 HPLC…
A modern HPLC consists of
• A solvent unit with degassing system and gradient
mixers
• High pressure pump and pressure gauge that can
provide constant flow
• Injector/Injection port/
• Column
• High sensitivity detectors
• High speed data acquisition and recorder systems
• Low dispersion connecting tubes - connects valve to
column and column to detector
02/14/2025 CHROMATOGRAPHY 75
 HPLC…
APPLICATIONS OF HPLC IN PHARMACEUTICAL ANALYSIS

– Isolation of natural pharmaceutically active compounds

– Control of microbiological processes
– Assay of pure drugs and their dosage forms.

• A few typical examples are discussed below:

02/14/2025 CHROMATOGRAPHY 102

 HPLC…

a. ISOLATION OF NATURAL PHARMACEUTICALLY ACTIVE

COMPOUNDS
Some plant alkaloids and glycosides can be isolated as stated below :

02/14/2025 CHROMATOGRAPHY 103

 HPLC…
b. CONTROL OF MICROBIOLOGICAL PROCESSES
• The major areas of antibiotic production operations are:
• kinetics of the microbiological process
• monitoring of the on-going process
• isolation and purification of active ingredients
• purity control of active constituents and
• monitoring derivatization reactions of these compounds.

HPLC-controlled analysis of a microbiological process during Penicillin

Production :
Chromatographic conditions are as follows :
– Column : Size-25 cm × 4.6 mm ID ;
– Adsorbent : Lichrosorb-NH2
– (R) (10 μm) ;
– Mobile-phase : 0.005 M H2SO4 buffer (pH 4.4))/acetonitrile
02/14/2025(50 : 50) ; Flow rate : 3 ml min–1 ;
CHROMATOGRAPHY 104
 HPLC…

02/14/2025 CHROMATOGRAPHY 105

 THANK YOU

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