it consists of notes for hplc , ion exchange chromatography, gas chromatography principle , instrumentation, applications

1. CHROMATORGRAPHY
 Chromatography is a non-destructive procedure for resolving a mult-
component mixture of trace, minor, or major constituents into its
individual fractions.
 It may be defined as “a method of separating a mixtureof components
into individual componentsthrough equilibrium distributionbetween
two phases”.
 The technique of chromatography is based on the differences in the rate at
which the components of a mixture move through a porous medium
(called stationary phase) under the influence of some solvent or gas (called
moving phase).
The chromatographic method of separation, in general, involves the
following steps:
 Adsorption or retention of a substanceor substances on the stationary
phase.
 Separation of the adsorbed substances bythe mobile phase.
 Recovery of the separated substances by a continuous flow of the mobile
phase; the method being called elution.
 Qualitative and quantitative analysis of the eluted substances.
 In chromatography, the stationary phase may be a solid or a liquid and the
mobile phase may be liquid or a gas.
Stationary phase:
 The substanceon which adsorptionof the analyte (the substanceto be
separated during chromatography) takes place.
 It can be a solid, a gel, or a solid liquid combination.
Mobile phase:
 Solvent which carries the analyte ( a liquid or a gas).
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 High-performance liquid chromatography (HPLC; formerly referred
to as high-pressure liquid chromatography) is a technique in analytical
chemistry used to separate, identify, and quantify each componentin a
mixture.
 It relies on pumps to pass a pressurized liquid solvent containing the
sample mixture through a column filled with a solid adsorbentmaterial.
 Each componentin the sample interacts slightly differently with the
adsorbentmaterial, causing different flow rates for the different
components and leading to the separation of the components as they flow
out of the column.

2.  High Performance Liquid Chromatography (HPLC) is a form of column
chromatography that pumps a sample mixture or analyte in a solvent
(known as the mobile phase) at high pressure through a column with
chromatographic packing material (stationary phase).
 The sample is carried by a moving carrier gas stream of helium or
nitrogen.
 HPLC has the ability to separate, and identify compounds that are present
in any sample that can be dissolved in a liquid in trace concentrations as
low as parts per trillion.
 Because of this versatility, HPLC is used in a variety of industrial and
scientific applications, such as pharmaceutical, environmental, forensics,
and chemicals.
PRINCIPLE:
 Liquid chromatographyinvolves:
 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 componentwhich has more affinity towards the adsorbent , travels
slower.
 The componentwhich 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 5micron (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 bya 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 off this tube
(column) by a flow-through device (detector) that measures their amount.
 The output from the detectoris called a liquid chromatogram/

3.  In principle, LC and HPLC work the same way except the speed,
efficiency sensitivity and ease of operation of HPLC is vastly superior.
INSTRUMENTATION:
1. 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 migrated according to the non-
covalent interaction of the compound with the column.
2. Pumps:
 The role of the pump is to force a liquid (mobile phase) through the
liquid chromatography at a specific flow rate, expressed in millilitres
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(400
to 600 bar).
 During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
3. Column:
 Considered the “heart of 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.

4.  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 gives us a good separation.
4. Detector:
 The detector can detect the individual molecules that elute from the
column and covert the data into an electrical signal.
 A detector serves to measure the amount of those molecules.
 The detector provides an output a recorder or computer that results in
the liquid chromatogram.
 Detector is selected based on the analyte or the sample under
detection.
 Common detectors are,
 ultraviolet (UV):
 It is mainly used to separate and identify the principal
active components of a mixture.
 It cannot be used for testing substances that are low in
chromophores (colourless) as they cannot absorb light at
low range.
 Refractive Index (RI) detector:
 It 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.
 Eg: sugar, alcohol, fatty acid and polymers.
5. Data processing unit (computer):
 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.
APPLICATIONS:
 The column may be reused. It is especially useful for separating the high
molecular weight compounds which have either a low vapour pressure or
undergo pyrolysis when subjected to the higher required temperatures of
gas chromatography.
 Reverse phase partition HPLC is particularly useful for separation of
polar compounds suchas drugs and their metabolites, peptides, vitamins,
polyphenols and steroids.

5.  The technique is particularly widely used in clinical and pharmaceutical
work as it is possible to apply biological fluids such as serum and urine
directly to the column, preferably using a guard column.
 The separation of some highly polar compounds,suchas amino acids,
organic acids and the catercholamines which are difficult to resolve,
adequately, by reverse phase chromatography.
GAS CHROMATOGRAPHY
 It is a process ofseparating component 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 an inert solid.
Two major types:
 Gas –solidchromatography:
 Here, the mobile phase is a gas while the stationary phase is a
solid.
 Used for separation of low molecular gases, eg., air components,
H2S, CS2 , rare gases, CO and oxides of nitrogen.
 Gas-liquid chromatography:
 The mobile phase is a gas while the stationary phase is a liquid
retained on the surface as an inert solid by adsorptionor chemical
bonding.
PRINCIPLE:
 The principle of separation in GC is “partition”.
 The mixture of componentto be separated is converted to vapour and
mixed with gaseous mobile phase.
 The componentwhich is more soluble in stationary phase travel slower
and eluted later.
 The componentwhich is less soluble in stationary phase travels faster and
eluted out first.
 No two components have same partition coefficient conditions.
 So the components are separated according to their partition coefficient.
 Partition coefficient is “the ratio of solubility of a substancedistributed
between two immiscible liquids at a constant temperature.

6. INSTRUMENTATION:
Sample injection system:
 Septum type injectors are the most common.
 These are composedofa glass tube vaporization of the sample takes
place.
 The sample is introduced into the injector through a self-sealing
silicone rubber septum.
 The carrier gas flows through the injector carrying vaporized solutes.
 The temperature of the injector should be adjusted so that flash
vaporization of solutes occurs.
 If the temperature of the injector is not high enough (at least 50 degrees
above highest boiling component) ,band broadening will take place.
Automatic Sampler:
 Sample vials are glass, throw type with vapour tight septum caps.
 The sampler flushes the syringe with new sample to remove traces of
previous sample.
 Pumps new sample to wet the syringe to remove any bubbles, takes in a
precisely measured sample and pumps in to the gas chromatograph.
Head space technique:
 It involves analysis of volatile components in a complex and viscous
mixture containing high proportion of non-volatile components.
 For quantitative analysis calibration of the volatiles in the vapour is
necessary.
 To reach this state the sample is placed in a glass vial and thermostatted.
 When equilibrium is achieved, an aliquot of the gas phase above the
sample is rapidly transferred onto the GC column.
 All the devices that are commonly used for gas sampling may be applied
to headspace analysis, including gas-tight syringes and gas sampling
valves.

7. Carrier Gas:
 The carrier gas plays an important role, and varies in the GC used.
 Carrier gas must be dry, free of oxygen and chemically inert mobile-
phase employed in gas chromatography.
 Helium is most commonly used because it is safer than, but comparable
to hydrogen in efficiency, has a larger range of flow rates and is
compatible with many detectors.
 Nitrogen, argon, and hydrogen are also used depending upon the desired
performance and the detector being used.
 Both hydrogen and helium, which are commonly used on most
traditional detectors such as Flame Ionization(FID), thermal conductivity
(TCD) and Electron capture (ECD), provide a shorter analysis time and
lower elution temperatures of the sample due to higher flow rates and low
molecular weight.
Columns:
 The column in chromatography is the heart of the technique.
 A column can either be a packed or open tubular.
 The packed columns are fabricated from glass, stainless steel, copper, or
other suitable tubes.
 Stainless steel is the most widely used because it is most inert and easy ot
work with.
 Open tubular or capillary columns are of two types:
 Wall coated open tubular (WCOT)is <1mm thick liquid coating on inside
of silica tube.
 Supported coated open tubular (SCOT)is 30 mm thick coating of liquid
coated supporton inside of silica tube.
 The most frequently used capillary column, is the fused silica open
tubular column (FSOT), which is a WCOT column.
Temperature control:
 A temperature programming facilitates controlled increase of even
temperature during an analysis.
 Thus, the latter peaks also become sharp and emerge quickly.
 Thus in temperature programming the components of a wide boiling
range mixture may be resolved efficiently.
 In generally the operation with linear temperature programme is more
common.

8. Detector:
 The detector is the device located at the end of the column which
provides a quantitative measurement of the components of the mixture as
they elute in combination with the carrier gas.
 Each detector has two main parts that when used together they serve as
transducers to convert the detected property changes into an electrical
signal that is recorded as a chromatogram.
 The first part of the detectoris the sensorwhich is placed as close the
column exit as possiblein order to optimize detection.
 The second is the electronic equipment used to digitize the analog signal
so that a computer may analyze the acquired chromatogram.
 Most commonly used detectors are flame ionization detector, thermal
conductivity detector, electron capture detector, mass spectrometer.
 The detector sends as signal to the chart recorder which results in a peak
on the chart paper.
 The componentthat is detected first is recorded first/ the component that
is detected last is recorded last.
APPLICATIONS:
 GC analysis is used to calculate the content of a chemical product, for
example in assuring the quality of products in chemical industry or
measuring toxic substances in soil, air or water.
 Gas chromatography is used in the analysis of:
a) Air-borne pollutants
b) Performance-enhancing drugs in athlete’s urine samples.
c) Oil spills
d) Essential oils in perfume preparation.
 GC is very accurate if used properly and can measure picomoles of a
substancein a 1ml liquid sample, or parts-per-billion concentrations in
gaseous samples.
 Gas chromatography is used extensively in forensic science.
 Disciplines as diverse as solid drug dose(pre-consumption form)
identification and quantification, arson investigation, paint chip analysis
and toxicology cases, employ GC to identify and quantify various
biological specimens and crime scene evidence.
 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 identifying these compounds by
other methods like UV, IR, NMR.
 Quantitative analysis: area under a single componentelution peak is
proportional to the quantity of the detected component / responsefactor
of the detectors.

9. ION EXCHANGE CHROMATOGRAPHY
 Ion exchange may be defined as a reversible reaction in which free
mobile ions of a solid called ion exchange for different ions of
similar charge present in solution.
 Most ion exchangers for practical use consist of an insoluble
organic polymer into which a charged group has been introduced in
some manner.
 The backboneis generally a styrene-divinyl-benzene copolymer.
 Copolymers of acrylic acid derivates divinyl-benzene are also
frequently used.
 For use with biological macromolecules, it is usual to introduce
charged groups into cellulose fibers.
 The most common properties of all ion exchanges as follows:
a) They are almost insoluble in water and organic solvents such as
benzene, carbontetrachloride, ether etc.,
b) They are complex in nature, i.e., in fact they are polymeric.
c) They have active or counter ions that with other ions in a
material.
PRINCIPLE:
 The underlying principle is the attraction between oppositely
charged particles.
 Ion-exchange separations are mainly carried out in columns packed
with an ion-exchanger.
 There are two types of ion exchanger, namely cation and anion
exchanger .
 Cation exchangers posses negatively charged groups and these will
attract positively charged molecules
 These exchangers are also called acidic ion-exchange materials
since their negative charges result from the protolysis of acidic
groups.
 Anionic exchangers has positively charged groups which will attract
negatively charged molecules
 These are also called basic ion exchange materials since positive
charges generally result from the association of protons with basic
groups.
 The actual ion-exchange mechanism is composed offive steps:
a) Diffusion of the ion to the exchanger surface. It occurs very
quickly in homogeneous solutions.
b) Diffusion of the ion through the matrix structure of the
exchanger to the exchange site. This process controls the rate
of the whole ion exchange process.

10. c) Exchange of ions at the exchange site and it is an equilibrium
process. Themore highly charged the molecule to be
exchanged, the tighter it binds to the exchanger and the less
readily it is displaced by other ions.
d) Diffusion of the exchanged ion through the exchanger to the
surface.
e) Selective desorption by the eluant and diffusion of the
molecule into the external solution.
 The rate of ion exchange is controlled by the law of massaction.
Cation exchangers:
 A cation exchanger is a high molecular weight, cross linked polymer
having sulphonic, carboxylic, phenolic etc., groups as an integral part of
the resin and an equivalent amount of cations.
 It is a polymeric anion to which active cations are attached.
 In this cation exchanger, the hydrogen ions are mobile and exchangeable
with the other cations and the anions remain attached to the resin
network.
Anion exchangers:
 An anion exchanger is a polymer having amine or quaternary ammonium
groups as integral parts of the resin and an equivalent amount of anions
such as CI- , SO4
2-, OH- ions, etc.
 These anions are mobile and exchangeable.
 The anions exchange behaviour of these materials may be represented as
follows:
WORKING PRINCIPLE:
Equilibration:
 The first step is equilibration of the stationary phase before the
initiation of chromatography.
 The stationary phase must be equilibrated to certain requirements
that depend on the experiment.
HnR + nNa+
↔ NanR + nH+
(Resin) (Solution) (Resin) (Solution)
M, (ResA⁺) B¯ + C¯ ↔ (ResA⁺) C¯ + B¯
Anion exchange Anionin Resin
Resin solution

11.  Once equilibrated, the charged ions in the stationary phase will be
attached to its oppositecharged exchangeable ions.
 Exchangeable ions such as chloride and sodium ions.
 After equilibration, the column must needs to be washed.
 The washing phase will help elute out all impurities that does not
bind to the matrix while the protein of interest remains bounded.
Sample introduction:
 Solution of the sample which is to be introduced is prepared.
 Pour on the top of the ion exchange resin using a micro syringe or
micro pipette.
Elution:
 Once the sample has been loaded on to the column and the column
has been washed with the buffer to elute out all non-desired
proteins, elution is carried out to elute the desired proteins that are
bound to the matrix.
 Bound proteins are eluted out by utilizing a gradient of linearly
increasing salt concentration.
 With increasing ionic strength of the buffer, the salt ions will
compete with the desired proteins in order to bind to charged groups
on the surface of the medium.
 This will cause desired proteins to be eluted out of the column.
 Proteins that have a low net charge will be eluted out first as the salt
concentration increases causing the ionic strength to increase.
 Proteins with high net charge will need a higher ionic strength for
them to be eluted out of the column.
 Proteins are desorbed relative to the number of charged groups on
their surface.
Two techniques are generally used to bring solutions in contactwith ion
exchange resins. They are
a) Batchmethod
b) Column method.

14. APPLICATIONS:
a) Separations ofsimilar ions form one another:
 Ion exchange chromatography is being used to separate similar
ions from one another because the different ions undergo exchange
reactions to different extents.
 Eg: a mixture of Li+, Na+ and K+ ion can be separated by passing
their solution through a cation exchanger.
b) Removalof interfering radicals:
 In the estimation of Ca2+ or Ba2+ ions by the oxalate or sulphate
method, phosphate ion is found to interfere.
 Thus the solution is passing through a sulphonic acid cation
exchanger.
c) Softening of hard water:
 Removal of Ca2+ or Ba2+which is the reason for hardness of water
is done by passing the hard water through a cation exchangers
charged with Na+.
 Thoseions are retained in the column whereas the Na+ ions pass
into solution.
d) Complete demineralisation of water:
 This requires removal of both cation and anion.
 Thus first passed through an acidic cation exchanger when the
metallic cations are exchanged by H+ ions.
 Then is passed through a basic anion exchanger when the anions
commonly present in the water are exchanged by OH- ions of the
exchanger.
e) Separation of lanthanides
f) Separation of actinides
g) Purification of organic compounds extracted in water
h) Separation of sugars
i) Separation of amino acids
j) Preparation of pure reagents