Chromatography

Presented by : k. jayalakshmi
D/o k. kristaiah
1st yr M.pharm, pharmaceutical analysis
Raghavendhra institute of pharmaceutical education and research
[RIPER], AUTONOMOUS
Chromatography

Chromatography is a physical method of
separation in which the components to be
separated are distributed between two
phases ,one of which is stationary phase
while the other mobile phase moves in a
definite directions.

Classification of chromatography
1. Based upon nature of stationary phase and mobile phase
 Gas-solid chromatography
 Gas-liquid chromatography
 Solid-liquid chromatography
 Liquid-liquid chromatography
2. Classification based on instruments
a)Column chromatography
 Adsorption Column chromatography stationary phase is solid
based on absorption chromatography.
 Partition column chromatography stationary phase is liquid.
b)Paper chromatography
 Asending Paper chromatography
 Desending Paper chromatography
 Circular Paper chromatography
 Two dimensional Paper chromatography

c)Thin layer chromatography
Normal Thin layer chromatography
Two dimensional Thin layer chromatography
Continuous development Thin layer chromatography
High performance Thin layer chromatography
d)Gas chromatography
Gas liquid chromatography
Gas solid chromatography
Capillary Gas chromatography
e)High performance liquid chromatography
Normal phase High performance liquid chromatography
Reverse phase High performance liquid chromatography
Gradient High performance liquid chromatography
Isocratic High performance liquid chromatography
f)Super critical fluid chromatography
g)Ultra high pressure chromatography
h)Electro phoretic chromatography

3. Based on the principle of separation
Adsorption
Column chromatography
Thin layer chromatography
Normal phase High performance liquid chromatography
Gas solid chromatography
Partition
Paper chromatography
Reverse phase High performance liquid chromatography
Gas liquid chromatography
Ion exchange chromatography
Ion exchange resins are used
Affinity chromatography
Complex formation
Chiral chromatography
Chiral active stationary phase

Types of chromatographic techniques
 Thin layer chromatography
 High performance Thin layer chromatography
 Ion exchange chromatography
 Column chromatography
 Gas chromatography
 High performance liquid chromatography
 Ultra High performance liquid chromatography
 Affinity chromatography
 Gel chromatography

Techniques Stationary phase Mobile phase
Column/adsorption
chromatography
solid liquid
Partition
chromatography
liquid liquid
Paper chromatography liquid liquid
Thin layer
chromatography
Liquid/solid liquid
Gas liquid
chromatography
liquid Gas
Gas solid
chromatography
solid Gas
Ion exchange
chromatography
solid liquid

Thin layer chromatography
TLC is one of the simplest, fastest, easiest and least expensive of
several chromatographic techniques used in qualitative and
quantitative analysis to separate organic compounds and to test the
purity of compounds.
TLC is a form of liquid chromatography consisting of:
A mobile phase (developing solvent) and
A stationary phase (a plate or strip coated with a form of silica gel)
Analysis is performed on a flat surface under atmospheric
pressure and room temperature.
Thin Layer Chromatography can be defined as a method of
separation or identification of a mixture of components into
individual components by using finely divided adsorbent solid /
(liquid) spread over a glass plate and liquid as a mobile phase.
• Synonyms: Drop, strip, spread layer, surface chromatography
and open column chromatography

Adsorption or retention or partition or both or any other
principle of a substance (s ) on the stationary phase
 Separation of the adsorbed substances by the mobile phase
 Recovery of the separated substances by a continuous flow of
the mobile phase (elution)
 Qualitative and quantitative analysis of the eluted substances
Principle of TLC
It is based on the principle of adsorption chromatography or
partition chromatography or combination of both, depending on
adsorbent, its treatment and nature of solvents employed
The components with more affinity towards stationary phase
travels slower.
Components with less affinity towards stationary phase travels
faster
In TLC, a solid phase, the adsorbent, is coated onto a solid
support (thin sheet of glass, plastic, and aluminum) as a thin
layer (about 0.25 mm thick). In many cases, a small amount of a
binder such as plaster of Paris is mixed with the absorbent to
facilitate the coating.

Separations in TLC involve distributing a mixture of two or
more substances between a stationary phase and a mobile phase
1.The stationary phase: is a thin layer of adsorbent (usually silica
gel or alumina) coated on a plate.
2.The mobile phase: is a developing liquid which travels up the
stationary phase, carrying the samples with it.
Selection of Stationary Phase
 The choice of the stationary phase for a given separation
problem is the most difficult decision in TLC
The chose of stationary Phase in following characters
considered.
The chemical composition of the stationary Phase and in
particular that of its surface, must be suitable for the task. To
obtain satisfactory separation efficiency, the mean particle size,
the particle size distribution and the morphology of the particle
must be considered

Selection of mobile Phase
The choice of mobile phase is largely empirical but general rules can
be formulated. A mixture of an organic solvent and water with the
addition of acid, base or complexing agent to optimize the solubility
of the components of a mixture can be used. For example, good
separations of polar or ionic solutes can be achieved with a mixture
of water and n-butanol. Addition of acetic acid to the mixture allows
more water to be incorporated and increases the solubility of basic
materials, whilst the addition of ammonia increases the solubility of
acidic materials. If the stationary phase is hydrophobic, various
mixtures of benzene, cyclohexane and chloroform provide
satisfactory mobile phases.
Selection of adsorbents
Solubility of compound e.g, hydrophilic or lipophilic
 Nature of substance to be separated i.e. whether it is acidic, basic
or amphoteric
Adsorbent particle size
Adsorbent should not adhere to glass plate
Reactivity of compound with the solvent or adsorbent
Chemical reactivity of compounds with binders

PREPARATION OF CHROMATOPLATES
• Glass plates or flexible plates are commonly used for adsorbent.
Size used depends on type of separation to be carried out, the type
of chromatographic tank and spreading apparatus available.
• The standard sizes are 20 x 5 cm, 20 x 10 cm or 20 x 20 cm .
• The surface should be flat without irregularities.
• The standard film thickness is 250um
Methods for application of adsorbent
Pouring
Dipping
Spraying
Spreading.

Pouring:
The adsorbent of finely divided and homogeneous particle size is
made into slurry and is poured on a plate and allowed to flow over
it so that it is evenly covered.
Dipping:
This technique is used for small plates by dipping the two plates at
a time, back to back in a slurry of adsorbent in chloroform or other
volatile solvents. Exact thickness of layer is not known and
evenness of layer may not be good.
Spraying:
Slurry is diluted further for the operation of sprayer. But this
technique is not used now a days as it is difficult to get uniform
layer.
Spreading:
All the above methods fail to give thin and uniform layers. Modern
methods utilize the spreading devices for preparation of uniform
thin layers on glass plates.
Commercial spreaders are of two types
(a) Moving spreader,
(b) Moving plate type.
It gives layer thickness from 0.2 to 2.0 mm.

ACTIVATION OF PLATES
• After spreading plates are allowed to dry in air and further dried
and activated by heating at about 1000c for 30 mins.
• By removing the liquids associated with layer completely, the
adsorbent layer is activated.
Solvent Systems
• The solvent system performs the following main tasks:
• To dissolve the mixture of substances,
• To transport the substances to be separated across the sorbent
layer,
• To give hRf values in the medium range, or as near to this as
possible,
• To provide adequate selectivity for the substance mixture to be
separated.
• They should also fulfill the following requirements:
• Adequate purity,
• Adequate stability,
• Low viscosity,

• Linear partition isotherm,
• A Vapor pressure that is neither very low nor very high,
• Toxicity that is as low as possible.
• The choice of the mobile phase is depends upon the following
factors:
1. Nature of the substance to be separated
2. Nature of the stationary phase used
3. Mode of chromatography ( Normal phase or reverse phase)
4. Separation to be achieved- Analytical or preparative.
 The organic solvent mixture of low polarity is used Highly polar
solvents are avoided to minimize adsorption of any components
of the solvent mixture.
 Use of water as a solvent is avoided as it may loosen the
adhesion of a layer on a glass plate.
 Solvents with an increasing degree of polarity are used in liquid-
solid or adsorption chromatography. The solvents listed in elutropic
series are selected.

Storage of solvents
Storage of solvents is unnecessary if they are used in a TLC
chamber immediately after they have been prepared.
However, it is sometimes that certain solvent systems can be stored
for several months.
In this case, the best advice is to store them in a dark bottle in a
cool place. The “daily quota” of a solvent system should also be kept
cool in the summer, e.g. if laboratory temperatures exceed 25 °C.
Care must be taken to adjust the temperature to room temperature
before the development.
Some type of solvents
1 n- Heptane 2 n-Hexane 3 n-Pentane 4 Cyclohexane 5 Toluene
6 Chloroform 7 Dichloromethane 8 Diisopropylether 9
tertButanol 10 Diethyl ether 11 Isobutanol 12 Acetonitrile 13
Isobutyl methyl ketone 14 2Propanol 15 Ethyl acetate 16 1-
Propanol 17 Ethylmethylketone 18 Acetone 19 Ethanol 20
1,4-Dioxan 21 Tetrahydrofuran 22 Methanol 23 Pyridine

• n-Hexane
• Cyclohexene
• Toluene
• Benzene
• Diethyl ether
• Chloroform
• Dichloromethane
• 1,2 dichloroethane Increasing polarity
• Acetone
• Ethyl acetate
• Acetonitrile
• Propanol
• Methanol
• Acetic acid
• Water.

APPLICATION OF SAMPLE
• Sample solution in a non polar solvent is applied.
• The concentration of a sample or standard solution has to be
minimum of a 1% solution of either standard or test sample is
spotted using a capillary tube or micropipette.
• The area of application should be kept as small as possible for
sharper and greater resolution.
Sample Application (spotting)

How to Run Thin Layer Chromatography
• Step 1: Prepare the developing container
• Step 2: Prepare the TLC plate
• Step 3: Spot the TLC plate
• Step 4: Develop the plate
• Step 5: Visualize the spots
Preparation of the developing container
• It can be a specially designed chamber, a jar with a lid, or a
beaker with a watch glass on the top
• Pour solvent into the chamber to a depth of just less than 0.5 cm.
• To aid in the saturation of the TLC chamber with solvent vapors,
you can line part of the inside of the beaker with filter paper.
• Cover the beaker with a watch glass, swirl it gently.
• Allow it to stand while you prepare your TLC plate.

Preparation of the TLC plate
1. Pouring, Dipping, Spraying, Spreading
2. TLC plates used are purchased as 5 cm x 20 cm sheets. Each
large sheet is cut horizontally into plates which are 5 cm tall by
various widths;
3. Handle the plates carefully so that you do not disturb the coating
of adsorbent or get them dirty. Measure 0.5 cm from the bottom
of the plate.
4. Using a pencil, draw a line across the plate at the 0.5 cm mark.
This is the origin: the line on which you will spot the plate. Take
care not to press so hard with the pencil that you disturb the
adsorbent.
5. Under the line, mark lightly the samples you will spot on the
plate, or mark numbers for time points. Leave enough space
between the samples so that they do not run together; about 4
samples on a 5 cm wide plate is advised.

Spot the TLC plate
• Prepare 1% solution of drug dissolving in volatile solvents like hexanes,
ethyl acetate, or methylene chloride.
• Dip the microcap or micro capillary into the solution and then gently
touch the end of it onto the proper location on the TLC plate.
• Don't allow the spot to become too large - if necessary, you can touch it to
the plate, lift it off and blow on the spot. If you repeat these steps, the wet
area on the plate will stay small.
• This example plate has been spotted with three different quantities of the
same solution and is ready to develop.
Thin Layer Chromatography Column Development
• Place the prepared TLC plate in the developing beaker, cover the beaker
with the watch glass, and leave it undisturbed on your bench top.
• The solvent will rise up the TLC plate by capillary action. Make sure the
solvent does not cover the spot.
• Allow the plate to develop until the solvent is about half a centimeter
below the top of the plate.
• Remove the plate from the beaker and immediately mark the solvent front
with a pencil

Visualize the spots
• If there are any colored spots, circle them lightly with a pencil.
• Most samples are not colored and need to be visualized with a UV
lamp.
• Hold a UV lamp over the plate and circle any spots you see.
• Make sure you are wearing your goggles and do not look directly into
the lamp. Protect your skin by wearing gloves.
TLC Developing Chambers
• Ascending development,
• Descending development,
• Horizontal development.
Vertical Development
1. Solvent in Liquid-Vapour equilibrium
2. Solvent in Vapour adsorbs on the layer
3. Solvent migrating in the layer vaporizes

Development of Thin-Layer Chromatograms
1. One-dimensional development
 Single development
-vertical
- horizontal, in one direction
-horizontal, in opposite directions
-circular
-ant circular
 Multiple development
-separate runs over the same migration distance
-stepwise, increasing
-stepwise, decreasing
-automated multiple development, stepwise with solvent
gradient
2. Two-dimensional development
 Two dimensions, one solvent system
 Two dimensions, two solvent systems
 Separation in 1st dimension chemical reaction separation in 2nd
dimension

One-Dimensional Development
Most thin-layer chromatograms are produced in one dimension,
and in fact even today it is very difficult to obtain quantitative results
from plates developed in more than one dimension.
All present-day commercially available TLC scanners therefore
operate on the principle of a one-dimensional chromatographic lane.
Two-Dimensional Development
 More complete separation of sample components can be achieved
by two dimensional development. In this process, the plate is
developed normally and following complete drying, it is turned 90o
and the development of the plate is continued.
 This second development is performed using a different mobile
phase with very different selectivity (otherwise little further
separation would result).

Applications of TLC
• It is used for separation of all classes of natural products and is
established as an analytical tool in modern pharmacopoeias. - E.g.
Acids, alcohols, glycols, alkaloids, amines, macromolecules like
amino acids, proteins and peptides, and antibiotics -for checking
the purity of samples - as a purification process - examination of
reaction -for identifying organic compounds.
• Extensively used as an identification test and test for purity.
• As a Check on process – checking of distillation fractions and for
checking the progress of molecular distillation.
Applications of TLC for separation of Inorganic Ions – Used for
separating cationic, anionic, purely covalent species and also some
organic derivatives of the metals.
• Separation of Amino Acids- two dimensional thin – layer
chromatography.
• Separation of vitamins – vitamin E, Vitamin D3, vitamin A.
• Application of TLC in quantitative analysis.

High performance thin layer chromatography
• HPTLC (High performance thin layer chromatography) is the
automated, sophisticated form and improved method of TLC.
• It is a powerful analytical method equally suitable for qualitative
and quantitative analytical tasks.
• It is also known as planer or flat bed chromatography.
HPTLC is very popular for many reasons such as
• Visual chromatogram,
• Multiple sample handling,
• Enables the most complicated separation,
• Detection limit in nanogram range with UV absorption detection and
in pictogram range with fluorimetric detection.
• Large no of theoretical plates in minimum area of plates .
• Analysis time is greatly reduced in HPTLC due to shorter migration
distant.
• Higher efficiency due to smaller particle size(5 μm).

Principle
Same theoretical principle of TLC (Adsorption
chromatography) i.e. the principle of separation is
adsorption.
 Mobile phase flow by capillary action effect .
And component move according to their affinities
towards the adsorbent.
 The component with higher affinity toward adsorbent
travels slowly.
 And the component with lesser affinity towards the
stationary phase travels faster.
 Thus the components are separated on a
chromatographic plate according to their affinity and
separation also based on their solubility in mobile
phase.

Difference between TLC and HPTLC

Steps involving in HPTLC
1.Sample preparation
2.Selection of chromatographic layer
3.Plates
4.Pre-washing
5.Conditioning
6.Sample application
7.Pre-conditioning
8.Mobile phase
9.Chromatographic development
10.Detection of spots
11.Scanning & documentation

Sample preparation
1.For normal phase chromatography using silica gel / alumina pre-
coated plates, solvents – non polar
2.RP chromatography , usually polar solvents
Selection of Chromatographic layer
» Depends on the nature of material to be separated Commonly
used materials are Silica gel 60F, Alumina, Cellulose etc
Pre-washing
» To remove water vapors
» volatile impurities Which might get trapped in the plates To avoid
this, plates are cleaned by using methanol as solvent by
ascending or descending etc.
Conditioning
Plates activated by placing them in an oven at 120°C for 15 to 20
minutes

Sample Application
Application of 1.0 - 5µl for HPTLC
 Application carried out by Linomat applicator on the plates which
give uniform, safe & std. results
Usual concentration of applied samples 0.1 to 1 µg / µl for
qualitative Analysis and quantity may vary in quantization based on
UV absorption 1 to 5 µl for spot and 10 µL for band application.
 Manual , semi-automatic , automatic application
Manual with calibrated capillaries
Semi and auto-application through applicators
 Applicators use spray on or touch and deliver technique for
application.
Manual Sample Applicator
 The Nanomat serves for easy application of samples in the form of
spots onto TLC and HPTLC layers .
 The actual sample dosage performed with disposable capillary
pipettes , which are precisely guided by the capillary holder.

The nanomat is suitable for
• Conventional TLC plates including self coated Plates up to 20 ×
20cm
• HPTLC plates 10 × 10 cm and 20 × 10 cm
• TLC and HPTLC sheets up to 20 × 20 cm
Semi automatic sample applicator
• The instrument is suitable for routine use for medium sample
throughout . In contrast to the Automatic TLC sampler , changing the
sample the Linomat requires presence of an operator.
• With the linomat , samples are sprayed onto the chromatographic
layer in the form of narrow bands.
• During the spraying the solvent of the sample evaporates almost
entirely concentrating the sample into a narrow band of selectable
length.
Automatic Sample Applicator
• Samples are either applied as spots through contact transfer (0.1-5
micro lit) or as bands or rectangles (0.5->50 micro lit) using the spray
on techniques.
• Application in the form of rectangles allow precise applications of
large volume with out damaging the layer.
• ATS allows over spotting.

Chromatographic development
Ascending, descending, horizontal, continuous, gradient, multidimensional…
HPTLC – migration distance of 5-6mm is sufficient, after development, plates
removed & dried.
Common problems encountered during chro. Development are as follows…
1.Tailing:
due to the presence of traces of impurities, this can be reduced by buffering
the M.P
2. Diffusion :
This is seen as zones on chromatographic plates. This may arise due to non-
uniformity of M.P
DEVELOPING CHAMBER
Twin trough chamber
• Low solvent consumption:
 20 mL of solvent is sufficient for the development of a 20x20cm plate.
This not only saves solvent , but also reduces the waste disposal problem
• Reproducible pre –equilibrium with Solvent vapor:
For pre-equilibration, the TLC plate is placed in the empty trough opposite
the trough which contains the pre-conditioning solvent.
Equilibration can be performed with any liquid and for any period of time.
• Start of development :
 It is started only when developing solvent is introduced into the trough
with the plate.

Automatic developing chamber (ADC)
• In the ADC this step is fully automatic and independent of
environmental effects.
• The activity and pre-conditioning of the layer , chamber saturation
developing distance and final drying can be pre-set and
automatically monitored by ADC.
Detection of spots
Detection can be done by iodine vapor in iodine chamber. Visual
inspection at 254nm of UV region in UV cabinet.
Scanning & Documentation
1.HPTLC plates are scanned at selected UV regions WL by the
instrument & the detected spots are seen on computer in the form
of peaks.
2.The scanner converts band into peaks & peak height or area is
related to the concentration of the substance on the spot.

Detector consists of following
•Lamp selector
• Entrance lens slit
•Monochromator entrance slit
• Grating
•Mirror
•Slit aperture disc
• Mirror
• Beam splitter
• Reference photo multiplier
•Measuring photo multiplier
• Photo diode for transmission measurements.
Factor affecting HPTLC
• Types of stationary phase.
• Mobile phase
• Layer thickness
• Temperature
• Mode of development
• Amount of sample
• Dipping zone, etc.

Applications of HPTLC
• Pharmaceutical industry- Quality control,identity purity test
etc.
• Food Analysis- : Quality control , additives , pesticides ,stability
testing etc.
• Clinical Applications- Metabolism studies , drug screening
,stability testing etc
• Industrial Applications- Process development and optimization
etc.
• Forensic- Poisoning investigations
• Biomedical Analysis- Separation of gangliosides
• Environment Analysis-Pesticides in drinking water etc.
• Cosmetics- Hydrocortisone & cinchocaine in lanolin ointment
etc.
• Natural products ,plant ingredients- Glycosides in herbal
drugs, Piperine in piper longum etc.
• Finger print Analysis-Finger prints for identification of liquorice,
ginseng etc.
• Analysis of drugs in blood

COLUMN CHROMATOGRAPHY
• Column chromatography is one of the most useful methods for
the separation and purification of both solids and liquids.
• This is a solid - liquid technique in which the stationary phase is
a solid & mobile phase is a liquid.
PRINCIPLE
• Adsorption
• Mixture of components dissolved in the M.P is introduced in to
the column. Components moves depending upon their relative
affinities.
• Adsorption column chromatography, the adsorbent, packed in a
glass column, and a solvent, the mobile phase, that moves slowly
through the packed column. A solvent used as a mobile phase is
called an eluent.
• A compound attracted more strongly by the mobile phase will
move rapidly through the column, and elute from, or come off, the
column dissolved in the eluent.
• In contrast, a compound more strongly attracted to the
stationary phase will move slowly through the column.

Experimental aspects of column chromatography:
• Adsorbents: The usual adsorbents employed in column
chromatography are silica, alumina, calcium carbonate, calcium
phosphate, magnesia, starch, etc.,
• Alumina is generally suitable for chromatography of less polar
compounds. Silica gel gives good results with compounds containing polar
functional groups.
Adsorbent in C.C should meet following criteria
 Particles should be spherical in shape & uniform in size
 Mechanical stability must be high
 They shouldn’t react chemically
 It should be useful for separating for wide variety of compounds
 It should be freely available & inexpensive
(The particle size of the commercially available grade is in the range 50 –
200 µm).
Selection of Stationary Phase
• Success of chromatography depends upon proper selection of S.P, it
depends on the following:
1. Removal of impurities
2. No. of components to be separated
3. Length of the column used
4. Affinity differences b/w components
5. Quantity of adsorbent used

Mobile Phase
• They act as solvent, developer & eluent. The function of a mobile
phase are:
• As developing agent
• To introduce the mixture into the column – as solvent
• To developing agent
• To remove pure components out of the column – as eluent
• The choice of the solvent is depend on the solubility
characteristics of the mixture. The solvents should also have
sufficiently low boiling points to permit ready recovery of eluted
material.
• However, polarity as seen the most important factor in adsorption
chromatography.
• Different mobile phases used: ( in increasing order
of polarity)
• Petroleum ether, carbon tetrachloride, Cyclohexane, ether,
acetone, benzene, toluene, esters, water, etc
• It can b e used in either pure form or as mixture of solvents

COLUMN CHARACTERISTICS
The main function of all the columns is to support the stationary
phase.
The material of the column is mostly good quality neutral glass
since it shouldn’t be affected by solvents. An ordinary burette can
also be used as column for separation.
 Column dimensions - length & diameter ratio (10:1,30:1 or 100:1)
 Various accessories are attached to the top and bottom of the
column for maintenance of the elution process.
The length of the column depends upon:
• Number of compounds to be separated
• Type of adsorbent used
• Quantity of the sample
• Affinity of compounds towards the adsorbent used
• Better separation will be obtained with a long narrow column than
short thick column because number of plates will be more.

PREPARATION OF THE COLUMN
• It consists of a glass tube with bottom portion of the column – packed
with glass wool/cotton wool or may contain asbestos pad.
» Above which adsorbent is packed
» After packing a paper disc kept on the top, so that the adsorbent layer is
not disturbed during the introduction of sample or mobile phase.
Packing techniques in C.C
• There are two types of preparing the column, they are:
• i. Dry packing / dry filling
• Ii. Wet packing / wet filling
• The column should be free from impurity, before using column, it should
be washed properly and dry it.
• Before filling column with stationary phase, cotton/glass wool is kept.
• It should be uniformly filled.
Dry Packing Technique
Adsorbent is packed in the column in dry form.
 Fill the solvent, till equilibrium is reached.
DEMERIT: Air bubbles are entrapped b/w M.P & S.P→ cracks appear in the
adsorbent layer. • After filling tapping can be done to remove void spaces.

Wet Packing Technique
» ideal & common technique
The material is slurried with solvent and generally added to the column in portions.
S.P settles uniformly & no crack in the column of adsorbent.
» solid settle down while the solvent remain upward.
» this solvent is removed then again cotton plug is placed.
Introduction of the Sample
• The sample which is usually a mixture of components is dissolved in minimum
quantity of the mobile phase.
• The entire sample is introduced into the column at once and get adsorbed on the
top portion of the column.
• From this zone, individual sample can be separated by a process of elution.
• Development technique ( Elution)
• By elution technique, the individual components are separated out from the
column.
The two techniques are:
(i) Isocratic elution technique : in this elution technique , same solvent
composition or solvent of same polarity is used throughout the process of separation.
E.g: chloroform only
(ii) Gradient elution techniques: ( gradient – gradually)
 Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of
separation.
E.g; initially benzene, then chloroform, then ethyl acetate then chloroform

DETECTION OF COMPONENTS
• If the compounds separated in a column chromatography procedure are
colored, the progress of the separation can simply be monitored visually.
• If the compounds to be isolated from column chromatography are
colorless. In this case, small fractions of the eluent are collected
sequentially in labelled tubes and the composition of each fraction is
analyzed by TLC.
• Eluting the sample: Components a, b, and c separate as column
progresses.
• Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer
flasks.
Analyzing the fractions:
• Analyze the fractions by thin-layer chromatography
FACTORS AFFECTING COLUMN EFFICIENCY
1. Dimension of the column: column efficiency has been improved by
increasing length/width ratio of the column.
2. Particle size of column packing: separation to be improved by decreasing
the particle size of the adsorbent.
3. Activity of the adsorbent
4. Temperature of the column: The speed of the elution increases at higher
temperatures.
5. Packing of the column
6. Quality of solvents: solvents having low viscosities is giving better
results.

APPLICATIONS OF C.C
Separation of mixture of compounds
 Purification process
Isolation of active constituents
Estimation of drugs in formulation
 Isolation of active constituents
 Determination of primary and secondary glycosides in digitalis
leaf.
 separation of dia stereomers
Advantages of C.C
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of Mobile Phase
» Automation is possible
Disadvantages of C.C
» Time consuming
» more amount of Mobile Phase are required
» Automation makes the techniques more complicated & expensive

Ion Exchange Chromatography
 Ion exchange chromatography is the process by which a mixture of similar
charged ions can be separated by using an ion-exchange resin which
exchanges ions according to their relative affinities.
 The most common properties of all ion exchangers are:
● They are almost insoluble in water and organic solvents such as
benzene,carbon tetrachloride,ether etc.
● They are complex in nature ,ie infact they are polymeric.
● They have active or counter ions that will exchange reversible with other
ions in a surrounding solution without any substantial change in the
material.
PRINCIPLE

CLASSIFICATION
ACCORDING TO THE SOURCE,
1) Natural : Cation- Zeolytes,Clay
Anion- Dolomite
2) Synthetic : Inorganic & organic resins
ACCORDING TO THE CHEMICAL NATURE,
1) Strong cation exchange resin( Sulfonic acid group)
2) Weak cation exchange resin ( Methacrylate polymer)
3) Strong anion exchange resin ( Quaternary ammonium)
4) Weak anion exchange resin ( Methyl amino group)
ACCORDING TO THE STRUCTURE,
(a)Pellicular type with ion exchange film:
● Particle size of 30-40 µ with 1-2 µ film thickness.
● Ion exchange efficiency is 0.01-0.1 meq /g of ion exchange resin
(b)Porous resin coated with exchange beads:
● Particle size of 5-10 µ.
● Ion exchange efficiency is 0.5-2 meq/g of ion exchange resin.
(c) Macroreticular resin bead:
● Not highly efficient.
● Very low exchange capacity.

(d) Surface sulfonated and bonded electrostatically with anion exchange:
● Less efficient and low exchange capacity.
● Ion exchange capacity is 0.02meq/g.
APPARATUS OF ION EXCHANGE CHROMATOGRAPHY
PRACTICAL REQUIREMENTS
1)Column material & Dimensions:
Material: Glass(laboratories),High quality stainless steel or
polymers(industries) Dimensions: Length:diameter ratio of 20:1 to 100:1
2)Type of ion exchange resin & Physical characteristics:
(a)Type of ions to be separated- Cations or Anions
(b)Nature of ions to be separated- Strong or Weak
(c)Efficiency of the resin- Measured by ion exchange capacity
(d)Particle size of the resin- 50-100 mesh or 100-200 mesh is used
(e)Structural type of resin- Porous,pellicular etc
(f)Amount of crosslinking agent present-Which decides swelling of the
resin
3) Stationary phase:
● It is composed of two structural elements; the charged groups which
are involved in the exchanger process and the matrix on which the charged
groups are fixed.
● Sever matrix materials are- Cellulose, Silica, Polyacrylamide, Acrylate
co-polymer, Coated silica

4) Mobile phase:
● Generally, eluents which consist of an aqueous solution of a suitable
salt or mixtures of the salt with a small percentage of an organic solvent
are used in which most of the ionic compounds are dissolved.
● There are some eluent additives which have been used in ion exchange
chromatographyEDTA,Polyols,Glycerol,Glucose,Detergents,Lipids,Organic
solvents,Urea.
BUFFERS
● In ion exchange chromatography,PH value is an important parameter for
separation & can be controlled by means of buffer substances.
● For Cation exchange chromatography- Citric acid, Lactic acid, Acetic
acid, Formic acid
●For Anion exchange chromatography-Piperazine,N-Methyl
piperazine,Triethanolamine,Ethanolamine
5)Sample Preparation:
● For sample preparation, the sample must be soluble in eluent & should
ideally be dissolved in the mobile phase itself.
● To protect the column from possible damage, samples are filtered before
use to remove particulates.

6)Packing of the column:
● Wet packing method is the ideal technique of column packing in ion
exchange chromatography.
● Slurry is prepared by mixing silica(10-20g) and least polar solvent in a
beaker & poured in the column.
● When the packing is complete, the eluent is allowed to pass through column
for certain time.
7)Development of the chromatogram & elution:
● After introduction of the sample, development of the chromatogram is done
by using different mobile phases.
● There are two elution techniques-Isocratic & Gradient elution.
8)Analysis of the elute:
Different fractions collected with respect to volume or time is analysed for
their contents by several methods.
● Spectrophotometric method
● Polarographic method
● Conductometric method
● Radiochemical method
8)Regeneration of the ion exchange resin:
● Regeneration refers to the replacement of exchangeable cations or anions
present in the original resin.

● Regeneration of cation exchange resin is done by charging the column with
strong acid like hydrochloric acid.
● Regeneration of anion exchange resin is done by using strong alkali like
sodium hydroxide or potassium hydroxide.
INSTRUMENTATION
(a) BATCH METHOD
● This involves a single step equilibrium.
● The resin & the solution are mixed in vessel until the equilibrium is
attained & the solution is then filtered off.
● The batch method is used for softening of water & production of
demineralised water.
● Softening of water involves an exchange of calcium & magnesium ions,
which cause hardness by sodium ions. The sodium form of sulphonic acid is
generally used.
● Demineralised water is prepared by treating water with a cation exchanger
in the acid or hydrogen forms.
● The water is then treated with an anion exchanger in the basic or
hydroxide form.
(b) COLUMN METHOD:
● The apparatus used in the column method, consist of a glass column fitted
with a glass wool plug or a sintered glass disc at a lower end.
● A slurry of resin is made in distilled water and any fine particles are
removed by decantation.
● The slurry is then slowly poured into the column.

● The slurry is then slowly poured into the column.
● To ensure that no air bubbles remain in the column and that the
resin is uniformly distributed , the column is backwashed with
distilled water.
● The flow of water is stopped and the resin is allowed to settle.
● The excess water is then drained off.
● The level of water must never be allowed to fall below that of the
surface of the resin as otherwise the resin may dry up and channels
may be formed in the resin bead.
ION EXCHANGE TECHNIQUES
1)Frontal Analysis:
● In Frontal analysis, an incomplete separation of ion is obtained.
2) Displacement Development:
● Displacement development of the column is accomplished by
means of a substance which has a very
strong affinity for the exchanger.
3) Elution development:
● When the elution development is performed, the components of a
mixture separate and move down the column individually at
different rates depending on the affinity of the ion for exchanger.

CHROMATOGRAPHIC PARAMETERS
1)Rate of ion exchange process:
● The rate of ion exchange process depends on the rate of diffusion
which is the slowest state on ion exchange process
2)Flow rate:
● Due to differences in rates of exchange and the fact that they may
vary significantly for different kinds of separation, flow rates are to
be controlled (0.5-5 ml/min).
3)Mechanical strength:
● Polystyrene bead would have little mechanical strength and upon
adding functional group such as sulphonic acid to the polymer,
solubility is greatly enhanced.
● If the polymer is cross linked by the incorporation of divinyl
benzene, mechanical strength is imparted to the resin.
4)Swelling:
● Swelling is due to the tendency of particles to hydrate and
electrostatic repulsion of fixed ions with like charges
● Swelling is continued until an equilibrium is reached between the
osmotic pressure in the system and the opposing elastic force of the
flexible hydrocarbon chains.

5)Partcle size:
● Large surface area and small particles will increase the rate of ion
exchange process.
6)Porosity:
● High porosity offers a large surface area covered by charged groups and
so provides a high binding capacity.
7)Selectivity:
● The ion exchange in solutions is a selective process.
● At the absolute concentration of the solution decreases, polyvalent ions
are adsorbed better than monovalent ions, while at higher concentrations,
monovalent ion is adsorbed.
8)Total capacity:
● Total capacity of a resin is determined by taking a weighed sample of
resin, placing it in a column and passing through a solution of Kcl through
the column in excess.
9)Exchange capacity:
● It depends on the quality of the ions extracted from water by one gram of
air dry ion exchanger.
10)Cross linking:
● As the cross linking in the resin decreases, the resin swelling increases.
● Divinyl benzene is the most commonly employed cross linking agent and
imparts strength to the polymer by joining the chains together at various
positions. 18

FACTORS AFFECTING RESOLUTION
1) NATURE OF EXCHANGING IONS
2) NATURE OF ION EXCHANGE RESIN
3) CHEMICAL VARIABLES
4) PHYSICAL VARIABLES
5) ION EXCHANGE CONSTANT
6) SURFACE AREA
7) TEMPERATURE
8) COMPOSITION OF CATION EXCHANGE RESIN
9) LENGTH OF THE COLUMN
10) IONIC STRENGTH

APPLICATIONS OF ION EXCHANGE
CHROMATOGRAPHY
1)Total content of cation in a solution
2)Concentration of traces of an electrolyte
3)Conversion of salts to acids or bases
4)Separation of amphoteric metals from non-
amphoteric metals
5) Separation of metals, alloys & high alloy steels
6) Analysis of natural & industrial water
7) Separation of complex mixtures of biochemical
compounds
8) Production of analytical concentrates
9) Radiochemistry

Gas Chromatography
 Gas chromatography – It is a process of separating component(s)
from the given crude drug by using a gaseous mobile phase.
 It involves a sample being vaporized and injected onto the head of
the chromatographic column. The sample is transported through the
column by the flow of inert, gaseous mobile phase. The column itself
contains a liquid stationary phase which is adsorbed onto the
surface of an inert solid.
 Two major types
• Gas-solid chromatography (stationary phase: solid)
• Gas-liquid chromatography (stationary phase: immobilized liquid)
Components of Gas chromatography
Carrier gas
He (common), N2, H2, Argon
 Sample injection port
micro syringe
Columns
2-50 m coiled stainless steel/glass/Teflon

Detectors
 Flame ionization (FID)
 Thermal conductivity (TCD)
 Electron capture (ECD)
 Nitrogen-phosphorus
 Flame photometric (FPD)
 Photo-ionization (PID)
Advantages of Gas Chromatography
The technique has strong separation power and even complex
mixture can be resolved into constituents
The sensitivity of the method is quite high
 It gives good precision and accuracy
 The analysis is completed in a short time
The cost of instrument is relatively low and its life is generally
long
The technique is relatively suitable for routine analysis

Schematic diagram of a Gas Chromatograph

Carrier gas
 The carrier gas must be chemically inert.
 Commonly used gases include nitrogen, helium, argon, and carbon dioxide.
The choice of carrier gas is often dependant upon the type of detector which
is used.
 The carrier gas system also contains a molecular sieve to remove water and
other impurities.
P inlet 10-50 psig
F=25-150 mL/min packed column
F=1-25 mL/min open tubular column
Sample injection- Direct Injection
Direct injection :into heated port (>T oven) using micro syringe
(i) 1-20 uL packed column
(ii) 10-3 uL capillary column
Sample injection- rotary sample valve with sample loop
 Split injection: routine method
0.1-1 % sample to column
remainder to waste
Split less injection: all sample to column
best for quantitative analysis
only for trace analysis, low [sample]

On-column injection:
-for samples that decompose above boiling Point ( no heated injection port)
-column at low temperature to condense sample in narrow band
-heating of column starts chromatography.
Gas Chromatography – Columns
There are two general types of column, packed and capillary (also known as
open tubular).
 Packed columns contain a finely divided, inert, solid support material (
diatomaceous earth) coated with liquid stationary phase. Most packed
columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.
 Capillary columns have an internal diameter of a few tenths of a millimeter.
They can be one of two types; wall-coated open tubular (WCOT) or support-
coated open tubular (SCOT).
- Wall-coated columns consist of a capillary tube whose walls are coated with
liquid stationary phase. In support-coated columns, the inner wall of the
capillary is lined with a thin layer of support material such as diatomaceous
earth, onto which the stationary phase has been adsorbed.
- SCOT columns are generally less efficient than WCOT columns. Both types
of capillary column are more efficient than packed columns.

Gas Chromatography – Common Stationary phases

G C – DETECTORS
 There are many detectors which can be used in gas chromatography.
 Different detectors will give different types of selectivity.
 Detectors can be grouped into concentration dependant detectors and
mass flow dependant detectors.
The signal from a concentration dependant detector is related to the
concentration of solute in the detector, and does not usually destroy the
sample Dilution of with make-up gas will lower the detectors response.
 Mass flow dependant detectors usually destroy the sample, and the signal
is related to the rate at which solute molecules enter the detector. The
response of a mass flow dependant detector is unaffected by make-up gas.
G C – IDEAL DETECTOR
Sensitive (10-8-10-15 g solute/s)
 Operate at high T (0-400 °C)
 Stable and reproducible
 Linear response
 Wide dynamic range
 Fast response
 Simple (reliable)
 Nondestructive
 Uniform response to all analytics.

Applications of Gas Chromatography
 Qualitative Analysis – by comparing the retention time or volume of
the sample to the standard / by collecting the individual components
as they emerge from the chromatograph and subsequently identifying
these compounds by other method
 Quantitative Analysis- area under a single component elution peak
is proportional to the quantity of the detected component/response
factor of the detectors.
Volatile Oils, official monograph gives chromatography profile for
some drugs. E.g. to aid distinction between anise oil from star anise
and that from Pimpinelle anisum
Separation of fatty acids derived from fixed oils
 Miscellaneous-analysis of foods like carbohydrates, proteins, lipids,
vitamins, steroids, drug and pesticides residues, trace elements
 Pollutants like formaldehyde, carbon monoxide, benzene, DDT etc
 Dairy product analysis- rancidity
 Separation and identification of volatile materials, plastics, natural
and synthetic polymers, paints, and microbiological samples
 Inorganic compound analysis

High-performance liquid chromatography
•HPLC stands for “High-performance liquid chromatography”(sometimes
referred to as High- pressure liquid chromatography).
•High performance liquid chromatography is a powerful tool in analysis, it
yields high performance and high speed compared to traditional columns
chromatography because of the forcibly pumped mobile phase.
•HPLC is a chromatographic technique that can separate a mixture of
compounds
•It is used in biochemistry and analytical chemistry to identify, quantify
and purify the individual components of a mixture.
PRINCILPE;
•To understand the principle of HPLC , we must first look at the principle
behind liquid chromatography
•Liquid chromatography is a separation technique that involves:
•the placement (injection) of a small volume of liquid sample
•into a tube packed with porous particles (stationary phase)
•where individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity.
•The main principle of separation is adsorption .

•When a mixture of components are introduced into the column . various
chemical and/or physical interactions take place between the sample
molecules and the particles of the column packing .
•They travel according to their relative affinities towards the stationary
phase. The component which has more affinity towards the adsorbent,
travels slower.
The component which has less affinity towards the stationary phase
travels faster.
•Since no two components have the same affinity towards the stationary
phase, the components are separated
•HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample into a tube packed with
tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)
•where individual components of the sample are moved down the packed
tube (column) with a liquid (mobile phase) forced through the column by
high pressure delivered by a pump.
•These components are separated from one another by the column packing
that involves various chemical and/or physical interactions between their
molecules and the packing particles.
•These separated components are detected at the exit of this tube (column)
by a flow-through device (detector) that measures their amount. The
output from the detector is called a liquid chromatogram

In principle, LC and HPLC work the same way except the speed , efficiency,
sensitivity and ease of operation of HPLC is vastly superior.
TYPES OF HPLC
I.BASED ON MODE OF SEPERATION
1.Normal phase chromatography - stationary phase is polar (hydrophilic)
and mobile face is non-polar (hydrophobic).
2.Reverse phase chromatography- stationary face is non-polar
(hydrophobic) and mobile face is Polar (hydrophilic).
•Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than
Polar-Non Polar bonds.
•Reverse phase chromatography is more commonly used as drugs are
usually hydrophilic.
II.BASED ON PRINCIPLE OF SEPERATION
1.Absorption Chromatography
•In the Absorption Chromatography solute molecules bond directly to the
surface of the stationary phase
•the component which has more affinity towards mobile phase elutes first
& the component which has less affinity towards stationary phase elutes
later.
No two components have same affinity towards mobile phase & stationary
phase.

2. Ion-exchange chromatography
•Ion exchange chromatography is a process that allows the separation of
ions and polar molecules based on their charge.
•It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids.
•Retention is based on the attraction between solute ions and charged sites
bound to the stationary phase. Ions of the same charge are excluded.
•The use of a resin (the stationary solid phase) is used to covalently attach
anions or cations onto it. Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by electrostatic forces.
3.Ion-pair chromatography
•It is a form of chromatography in which ions in solution can be "paired" or
neutralized and separated as an ion pair on a reversed-phase column.
•Ion-pairing agents are usually ionic compounds that contain a
hydrocarbon chain that imparts a certain hydrophobacity so that the ion
pair can be retained on a reversed-phase column.
4. gel permeation chromatography
•This type of chromatography lacks an attractive interaction between the
stationary phase and solute.
•The liquid or gaseous phase passes through a porous gel which separates
the molecules according to its size.

• The pores are normally small and exclude the larger solute molecules, but
allows smaller molecules to enter the gel, causing them to flow through a
larger volume. This causes the larger molecules to pass through the column
at a faster rate than the smaller ones.
5.Affinity Chromatography
• This is the most selective type of chromatography employed. It utilizes the
specific interaction between one kind of solute molecule and a second
molecule that is immobilized on a stationary phase.
• For example, the immobilized molecule may be an antibody to some
specific protein. When solute containing a mixture of proteins are passed
by this molecule, only the specific protein is reacted to this antibody,
binding it to the stationary phase. This protein is later extracted by
changing the ionic strength or pH.
6.Chiral chromatography
• It involves the separation of stereoisomer's. In the case of enantiomers,
these have no chemical or physical differences apart from being three-
dimensional mirror images. Conventional chromatography or other
separation processes are incapable of separating them. To enable chiral
separations to take place, either the mobile phase or the stationary phase
must themselves be made chiral, giving differing affinities between the
analytes.

III. BASED ON ELUTION TECHNIQUE
1.Isocratic elution
•A separation in which the mobile phase composition remains
constant throughout the procedure is termed isocratic elution.
•In isocratic elution, peak width increases with retention time
linearly with the number of theoretical plates. This leads to the
disadvantage that late-eluting peaks get very flat and broad.
• Best for simple separations
• Often used in quality control applications that support and are in
close proximity to a manufacturing process.
2.Gradient elution
•A separation in which the mobile phase composition is changed
during the separation process is described as a gradient elution.
•Gradient elution decreases the retention of the later-eluting
components so that they elute faster, giving narrower peaks . This
also improves the peak shape and the peak height.
• Best for the analysis of complex samples.
• Often used in method development for unknown mixtures.
• Linear gradients are most popular.

IV.BASED ON SCALE OF OPERATION
1.Analytical HPLC
No recovery of individual components of substance
2.Preparative HPLC
Individual components of substance can be recovered
V.BASED ON TYPE OF ANALYSIS
1.Qualitative analysis
Analysis of a substance in order to ascertain the nature of its
chemical constituents.
We can separate individual components but cannot assess the
quantity in this analysis.
2.Quantitaive analysis
Determining the amounts and proportions of its chemical
constituents .
Quantity of the impurity and individual components can be
assessed.

A.Solvent delivery system(mobile phase)
•The mobile phase in HPLC refers to the solvent being
continuously applied to the column or stationary phase
•The mobile phase acts as a carrier to the sample
solution
•A sample solution is injected into the mobile phase of
an assay through the injector port
•As a sample solution flows through a column with the
mobile phase, the components of that solution migrate
according to the non-covalent interaction of the
compound with the column
•The chemical interaction of the mobile phase and
sample , with the column , determine the degree of
migration and separation of components contained in
the sample

B.Pumps
•The role of the pump is to force a liquid (called the
mobile phase) through the liquid chromatograph at a
specific flow rate, expressed in milliliters per min
(mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min
range.
•Typical pumps can reach pressures in the range of
6000-9000 psi (400to 600-bar).
•During the chromatographic experiment, a pump can
deliver a constant mobile phase composition
(isocratic) or an increasing mobile phase composition
(gradient

Types of HPLC pumps
There are several types of pumps used for HPLC analysis, most
commonly used are reciprocating piston pump, syringe pump and
constant pressure pump.
1.Reciprocating piston pumps:
•Consists of a small motor driven piston which moves rapidly back
and forth in a hydraulic chamber that may vary from 35-400µL in
volume.
•On the back stroke , the separation column valve is closed , and
the piston pulls in solvent from the mobile phase reservoir.
•On the forward stroke, the pump pushes solvent out of the column
from the reservoir.
•A wide range of flow rates can be attained by altering the piston
stroke volume during each cycle , or by altering the stroke
frequency.
•Dual and triple head pump consists of identical piston chamber
units which operate at 180 or 120 degrees out of phase.

2.Syringe type pump
•These are most suitable for small bore columns because this
pump delivers only a finite volume of mobile phase before it has to
be refilled. These pumps have a volume between 250 to 500mL.
•The pump operates by a motorized lead screw that delivers mobile
phase to the column at a constant rate .The rate of solvent delivery
is controlled by changing the voltage on the motor.
3.Constant pressure pump
•In these types of pumps , the mobile phase is driven through the
column with the use of pressure from the gas cylinder
•.A low-pressure gas source is needed to generate high liquid
pressures
•The valving arrangement allows the rapid refill of the solvent
chamber whose capacity is about 70mL
•This provides continuous phase flow rates

C. Injector:
•The injector serves to introduce the liquid sample into the flow
stream of the mobile phase for analysis.
•It is equipped with six port valves so that a sample can be injected
into the flow path at continuous pressure
•For a manual injector, the knob is manually operated to deliver the
sample to the column
•The knob is set to LOAD position for sample injection using a
syringe , the sample is injected into the sample loop , which is
separated from the flow path
•The knob is turned to INJECT position and the eluent travels
through the loop from the pump and delivers the sample to the
column
•Typical sample volumes for manual injector are 5-to 20-microliters
(μL).
•The injector must also be able to withstand the high pressures of
the liquid system.
•An autos ampler is the automatic version for when the user has
many samples to analyze or when manual injection is not practical.
It can continuously Inject variable volume a of 1 μL – 1mL

D.Column
•Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest
using various physical and chemical parameters.
•It is usually made of stainless steel to withstand high pressure
caused by the pump to move the mobile phase through the column
packing other material include PEEK and glass.
•The small particles inside the column are called the “packing”
what cause the high back pressure at normal flow rates.
•Column packing is usually silica gel because of its particle shape ,
surface properties , and pore structure give us a good separation
•Other material used include alumina, a polystyrene-divinyl
benzene synthetic or an ion-exchange resin.
-Pellicular particle: original, Spherical, nonporous beads, proteins
and large biomolecular separation (dp: 5 μm).
-Porous particle: common used, dp: 3 ~ 10 μm. Narrow size
distribution, porous micro particle coated with thin organic film.

The dimensions of the analytical column are usually -straight,
Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm), diameter of
particle(35 μm). Number (40 k ~ 70 k plates/m)
Guard column is used to remove particular matter and
contamination, it protect the analytical column and contains
similar packing its temperature is controlled at < 150 °C, 0.1 °C
As mention before , columns are divided into different types
according to their functions (see types of HPLC)

E.Detector:
•The detector can detect the individual molecules that elute from the
column and convert the data into an electrical signal
•A detector serves to measure the amount of those molecules
•The detector provides an output to a recorder or computer that
results in the liquid chromatogram
•Detector is selected based on the analyte or the sample under
detection
Commonly used detectors in HPLC
Ultraviolet (UV) •This type of detector responds to substances that
absorb light.
•The UV detector is mainly to separate and identify the principal
active components of a mixture.
•UV detectors are the most versatile, having the best sensitivity and
linearity.
•UV detectors cannot be used for testing substances that are low in
chromophores (colorless or virtually colorless) as they cannot absorb
light at low range.
•They are cost-effective and popular and are widely used in industry.

Fluorescence
•This is a specific detector that senses only those substances that
emit light. This detector is popular for trace analysis in environmental
science.
•As it is very sensitive, its response is only linear over a relatively
limited concentration range. As there are not many elements that
fluoresce , samples must be syntesized to make them detectable.
Mass Spectrometry
•The mass spectrometry detector coupled with HPLC is called
HPLCMS. HPLC-MS is the most powerful detector,widely used in
pharmaceutical laboratories and research and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing
and providing molecular identity of a wide range of components.
Refractive Index (RI) Detection The refractive index (RI) detector uses
a Monochromator and is
Refractive Index (RI) Detection The refractive index (RI) detector
uses a monochromator and is one of the least sensitive LC detectors.
•This detector is extremely useful for detecting those compounds that
are non-ionic, do not absorb ultraviolet light and do not fluoresce.
•e.g. sugar, alcohol, fatty acid and polymers.

F . Data processing unit (Computer)
•Frequently called the data system, the computer not only
controls all the modules of the HPLC instrument but it takes
the signal from the detector and uses it to determine the time of
elution (retention time) of the sample components (qualitative
analysis) and the amount of sample (quantitative analysis).
•The concentration of each detected component is calculated
from the area or height of the corresponding peak and
reported.

OPERATION
Switch on instrument
Check system set up
Prime the pump
Prepare the column
Set-up software(for system flushing)
Software set up(for sample run)
Sample injection
Chromatograph data acquisition

CALIBRATION
Calibration of HPLC is done to check the
performance of its instrument.
1.Flowrate(pump calibration)
2.Detector and injector linearity
3.System precision
4.Column oven temperature
5.Detector wavelength accuracy

1.Pump calibration
•Disconnect the column and connect the inlet and
outlet tubing’s with a union.
•Prime all the lines at 5 ml/min flow rate with water
and ensure that flow line is free from air bubbles.
•Set the flow rate at 1ml / min and collect the mobile
phase (water) in a dry preweighed beaker and collect the
mobile phase for 10 min. weigh the beaker to get the
weight of mobile phase.
•Calculate the flow rate by dividing the weight obtained
with weight per ml and 10 (run time).
•Calculate the corresponding flow rate. Carry out the
experiment in duplicate.
•Acceptance criteria
0.05ml for small quantities
0.1ml for larger quantities

2.Detector and Injector linearity
Column : ODS or C18
Mobile phase : milli Q water and acetonitrile (80:20)
Flow rate: 1ml/min
Temperature: 40 Centigrade
Detector wavelength:272 nm
Runtime:10min
Preparation of stock solution:
•Take 50mg of caffeine and transfer into 50ml of flask and make it
up to the mark with the diluents.
•Caffeine is used a it gives a multiwavelength response and is stable
•Prepare solutions of different PPM (100-600)
•Inject them and calculate the area
•Correlation coefficient r is used to check the detector linearity and
cannot be less than .999
• r = NƩXY-ƩXƩY/√ [NƩX²-(Ʃx) ²][NƩY²-(ƩY)²]
•The graph obtained between concentration and area is linear.

3.System precision
•From the stock solution pipette out 1ml of stock solution and
transfer into 10ml flask and make it upto the mark with diluent for
100PPM conc.
•Calculate the percentage of RSD of areas of 5 different injections.
•% RSD = standard deviation/average value of above 5 injection
areas 100
Where
SD = √ Ʃ(X-M) ²/n-1
N= # of injections
M=average area
X=area
For system precision % of RSD is not more than 1

4.Column oven temperature
1. Set the column oven temperature to 30 and leave it for 30
minutes.
2. Open the door of the column oven and keep the thermometer
and leave it for 30 min.
3. now note down the reading in the thermometer .
4. Similarly change the column oven temperature to 40 C and 50 C
and repeat the above procedure.
Acceptance criteria
2 c

5.Detector wavelength accuracy
Column : C18 Mobile phase: HPLC grade methanol Flow
rate:1ml/min.
Retention time:5 minutes Wavelength:272nm.
1. Inject the methanol and record blank
2. Inject 20 ml of standard solution at 268nm
3. Similarly inject the standard solution by increasing the nm
upto 278nm(increasing it 1 at a time).
Acceptance criteria
273nm 1

MAINTAINANCE
1.RESERVOIR
Possible Cause
Preventive Maintenance
1. Blocked inlet frit 1. a. Replace (3–6 months)
b. Filter mobile phase with
0.4 - 0.5 µm filter
2. Gas bubbles 2. Degas mobile phase,
sonification
Possible Cause Preventive Maintenance
1. Blocked inlet frit 1. a. Replace (3–6 months)
b. Filter mobile phase with
0.4 - 0.5 µm filter
2. Gas bubbles 2. Degas mobile phase,
sonification

2. PUMP
Possible Cause Préventive Maintenance
1. Air bubbles 1. Degas mobile phase , do not change mobile phase
during run
2. Pump seal failure 2. Replace (3 months),clean with 1 N acid
3. Check valve failure 3. Filter mobile phase; use inletline frit ; keep spare
4.Improper cleaning 4.Clean with Isopropyl alcohol , mobile phase
container must be cleaned
with mobile phase and other sections with solvent

3.INJECTOR
1.Washing 1.wash before and after use
2. Rotor seal wear 2. a. Do not overtighten
b. Filter samples
3.Syringe 3.Sterilize when fresh
sample is used

4.Column
1. Number of injections 1. 2000 or less
2. Blocked frit 2.. a. Filter mobile phase b.
Filter samples c. Use in-line
filter and/or guard column
3. Void at head of column 3.. a. Avoid mobile phase pH
>8
b. Use guard column c. Use
precolumn (saturator column

5.Detector
1.Lamp failure; decreased
detector
1.Replace (6 months) or keep
spare lamp
response; increased detector
noise
2. Bubbles in cell 2. a. Keep cell clean
b. Use restrictor after cell
c. Degas mobile phase

6. Software
Update frequently, around 6-12 months

Applications
HPLC is one of the most widely applied analytical separation techniques.
Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.
Environmental;
•Phenols in Drinking Water.
•Identification of diphenhydramine in sediment samples.
•Biomonitering of PAH pollution in high-altitude mountain lakes through
the analysis of fish bile.
•Estrogens in coastal waters - The sewage source.
•Toxicity of tetracycline's and tetracycline degradation products to
•environmentally relevant bacteria.
•Assessment of TNT toxicity in sediment..

Forensics;
•A mobile HPLC apparatus at dance parties - on-site identification
and quantification of the drug Ecstasy.
•Identification of anabolic steroids in serum, urine, sweat and hair.
•Forensic analysis of textile dyes.
•Determination of cocaine and metabolites in meconium.
•Simultaneous quantification of psychotherapeutic drugs in
human plasma.
Clinical;
•Quantification of DEET in Human Urine.
•Analysis of antibiotics.
•Increased urinary excretion of aquaporin 2 in patients with liver
cirrhosis.
•Detection of endogenous neuropeptides in brain extracellular fluids.

Food and Flavor;
•Ensuring soft drink consistency and quality.
•Analysis of vicinal diketones in beer.
•Sugar analysis in fruit juices.
•Polycyclic aromatic hydrocarbons in Brazilian vegetables and
fruits.
•Trace analysis of military high explosives in agricultural crops.
Stability of aspartame in the presence of glucose and vanillin.

ADVANTAGES OF HPLC
1. Separations fast and efficient (high resolution power)
2. Continuous monitoring of the column effluent
3. It can be applied to the separation and analysis of very complex
mixtures
4. Accurate quantitative measurements.
5. Repetitive and reproducible analysis using the same column.
6. Adsorption, partition, ion exchange and exclusion column
separations are excellently made.
7. HPLC is more versatile than GLC in some respects, because it has
the advantage of not being restricted to volatile and thermally
stable solute and the choice of mobile and stationary phases is
much wider in HPLC
8. Both aqueous and non aqueous samples can be analyzed with
little or no sample pre treatment
9. A variety of solvents and column packing are available, providing
a high degree of selectivity for specific analyses.
10. It provides a means for determination of multiple components in
a single analysis.

AFFINITY CHROMATOGRAPHY
• Affinity Chromatography is essentially a sample purification
technique, used primarily for biological molecules such as proteins.
• It is a method of separating a mixture of proteins or nucleic acids
(molecules) by specific interactions of those molecules with a
component known as a ligand, which is immobilized on a support. If
a solution of, say, a mixture of proteins is passed over (through) the
column, one of the proteins binds to the ligand on the basis of
specificity and high affinity (they fit together like a lock and key).
• The other proteins in the solution wash through the column
because they were not able to bind to the ligand.

PRINCIPLE
• Affinity chromatography is one of the most diverse and
powerful chromatographic methods for purification of a specific
molecule or a group of molecules from complex mixtures
• It is based on highly specific biological interactions between
two molecules such as interactions between enzyme and
substrate, receptor and ligand, or antibody and antigen.
• These interactions which are typically revesible are used for
purification by placing one of the interacting molecules referred
to as affinity ligand onto a solid matrix to create a stationary
phase while a target molecule is in the mobile phase.
• Many of the commonly used ligands coupled to affinity
matrices are now commercially available and are ready to use.

CHROMATOGRAPHIC MEDIA
• A matrix in its use here is a substance,usually in bead form to
which a specific ligand is covalently bound.
• In order to for the matrix to be effective it must have certain
characters:
1)It must be insoluble in solvents and buffers employed in the
process
2)It must be chemically and mechanically stable..
3)It must be easily coupled to a ligand or spacer arm onto which the
ligand can be attached.
4)It must exhibit good flow properties and have a relatively large
surface area for attachment

IMMOBILIZED LIGAND
• The ligand can be selected only after the nature of the macromolecule to
be isolated is known.
• When a hormone receptor protein is to be purified by affinity
chromatography, the hormone itself is an ideal candidate for the ligand.
• For antibody isolation ,an antigen or hapten may be used as ligand.
• If an enzyme is to be purified, a substrate analog, inhibitor,cofactor,or
effect or may be used as a the immobilized ligand.
ATTACHMENT OF LIGAND TO MATRIX
• Several procedures have been developed for the covalent attachment of
the ligand to the stationary phase. all procedures for gel modification
proceed in two separate chemical steps:
1)Activation of the functional groups on the matrix and
2)Joining of the ligand to the functional group on the matrix.
• A wide variety of activated gels is now commercially available. the most
widely used are described in the following:

SELECTION OF A GEL OR LIGAND
• Many type of matrix-ligand systems are commercially available and cost
are reasonable so time can be saved by purchasing preactivated gel for
direct attachment of ligand.
BUFFER
• Buffer is used for formation of complex between a matrix and ligand. as
slight change in ionic concentration weakens the interactions between
them.
AFFINITY ELLUTION
• In this method a selective substance added to the buffer causes
selective elution of bound macromolecule-ligand complex. resulting in
elution of desired macromolecule.
CHAOTROPIC AGENTS
• If gentle and selective elution methods do not release the bound
macromolecule then mild denaturing agents can be added to the buffer.
the most powerful agents are urea, guanidine.

APPLICATIONS
1)It is used for isolation and purification of all biological
macromolecule.
2)It is used to purify nucleic acid, antibodies, enzymes.etc
3)To notice which biological compounds bind to a particular
substance.
4)to reduce a amount of substance in a mixture.

Gel permeation chromatography
It is also called as Gel chromatography
or
Size exclusion chromatography
or
Gel filtration
Or
Molecular-sieve chromatography
This is a technique in which the separation of
components is based on the difference in molecular
weight or size, and is one of the effective methods used
to isolate and analyze the biomacromolecular
substances.

Principle of separation
 It’s a technique that separates dissolved molecules on the basis of
their size by pumping these molecules through specialized columns
containing a microporous packing material(gel).
 Stationary phase is a porous polymer matrix whose pores are
completely filled with the solvent to be used as the mobile phase.
 The pore size is highly critical, since the basis of the separation is
that molecules above a certain size are totally excluded from the
pores, and the interior of the pores is accessible, partly or wholly, to
smaller molecules.
 The flow of mobile phase will cause larger molecules to pass
through the column unhindered, without penetrating the gel
matrix, whereas smaller molecules will be retarded according to
their penetration of the gel.

Theory of separation
A column is made up of swollen gel particles and the solvent used to
swell the gel in a suitable tubular container.
An equation is given below:
Vt = V0 + Vi + Vm
where,
Vt = the total volume of the column (which can be measured),
V0 = the volume of liquid outside the gel matrix (known also void or dead
volume),
Vi = the volume of liquid inside the matrix,
Vm= the volume of the gel matrix

1. Stationary phase:
Composed of semi-permeable, porous polymer gel beads with well defined
range of pore sizes .
Properties of gel beads:
1- Chemically inert
2- Mechanically stable
3- Has ideal and homogeneous porous structure (wide pore size give low
resolution).
4- Uniform particle and pore size.
5- The pore size of the gel must be carefully controlled.
Examples of gel
Dextran(Sephadex) gel: An α 1-6-polymer of glucose natural gel
 Agarose gel: A 1,3 linked β-D- galactose and 1,4 linked 3,6-anhydro-α,
L-galactose natural gel
Acryl amide gel: A polymerized acrylamide, a synthetic gel
GPC components
1. Stationary Phase
2. The Mobile Phase
3. The Columns
4. The Pump
5. Detectors

2. The Mobile Phase
Composed of a liquid used to dissolve the biomolecules to make the mobile
phase permitting high detection response and wet the packing surface.

3. Columns
Commercially Available Columns include
 Analytical column- 7.5–8mm diameters.
 Preparative columns-22–25mm
 Usual column lengths-25, 30, 50, and 60 cm.
 Narrow bore columns- 2–3mm diameter have been introduced
4. The pump
Are either syringe pumps or reciprocating pumps with a highly constant
flow rate.
5. Detectors
Concentration sensitive detectors
Bulk Property Detectors- Refractive Index (RI) Detector
Solute Property Detectors- Ultraviolet (UV) Absorption Detector
Evaporative Detectors- Evaporative Light Scattering Detector (ELSD)
Molar mass sensitive detectors
1. Light Scattering Detectors
• Low Angle Light Scattering (LALS) Detectors
• Multiangle Light Scattering (MALS) detectors
2. Viscosity Detectors- Differential Viscometers

Separation procedure
1- Preparation of column for gel filtration which involves
 Swelling of the gel
 Packing the column
Washing:
After packing, several column volumes of buffer solution is passed
through the column to remove any air bubbles and to test the column
homogeneity.
2- Loading the sample onto the column using a syringe
3- Eluting the sample and detection of components
Advantages and disadvantages
Advantages:
Short analysis time.
Well defined separation.
Narrow bands and good sensitivity.
There is no sample loss.
Small amount of mobile phase required.
The flow rate can be set.

Disadvantages:
 Limited number of peaks that can be resolved within the short time scale
of the GPC run.
 Filtrations must be performed before using the instrument to prevent
dust and other particulates from ruining the columns and interfering with
the detectors.
The molecular masses of most of the chains will be too close for the GPC
separation to show anything more than broad peaks.
Applications of GPC
Proteins fractionation
Purification
Molecular weight determination.
Separation of sugar, proteins, peptides, rubbers and
others on the basis of their size.
This technique can be use to determine the quaternary
structure of purified proteins.

Chromatography

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