Chromatography

1.1 What is Chromatography?

Chromatography is the technique for the separation, purification, and testing of compounds.
The term “chromatography”  is derived from Greek, chroma meaning, “colour,” and graphein meaning
“to write.”
In this process, we apply the mixture to be separated on a stationary phase (solid or liquid) and a pure
solvent such as water or any gas is allowed to move slowly over the stationary phase, carrying the
components separately as per their solubility in the pure solvent.

1.2 classification of chromatography

1) On the basis of physical state of mobile and stationary phase

Stationary Mobile Basis of

Technique phase phase separation Notes

compound spotted
*Paper polarity of directly on a cellulose
chromatography solid (cellulose) Liquid molecules paper

*Thin layer glass is coated with thin

chromatography solid (silica or polarity of layer of silica on which is
(TLC) alumina) Liquid molecules spotted the compound

*Liquid column solid (silica or polarity of glass column is packed

chromatography alumina) Liquid molecules with slurry of silica

Size exclusion solid Liquid size of small molecules get

chromatography (microporous molecules trapped in the pores of
beads of silica) the stationary phase,
while large molecules
 Stationary Mobile Basis of
Technique phase phase separation Notes

flow through the gaps

between the beads and
have very small retention
times. So larger
molecules come out first.
In this type of
chromatography there
isn’t any interaction,
physical or chemical,
between the analyte and
the stationary phase.

molecules possessing the

opposite charge as the
resin will bind tightly to
the resin, and molecules
having the same charge
ionic charge as the resin will flow
Ion-exchange solid (cationic of the through the column and
chromatography or anionic resin) Liquid molecules elute out first.

Affinity solid (agarose Liquid binding affinity if the molecule is a

chromatography or porous glass of the analyte substrate for the enzyme,
beads on to molecule to it will bind tightly to the
which are the molecule enzyme and the unbound
immobilized immobilized analytes will pass through
molecules like on the in the mobile phase, and
enzymes and stationary elute out of the column,
antibodies) phase leaving the substrate
 Stationary Mobile Basis of
Technique phase phase separation Notes

bound to the enzyme,

which can then be
detached from the
stationary phase and
eluted out of the column
with an appropriate
solvent.

samples are volatilized

and the molecule with
gas lowest boiling point
(inert comes out of the column
gas like first. The molecule with
argon boiling point the highest boiling point
Gas liquid or solid or of the comes out of the column
chromatography support helium) molecules last.

2) On the basis of mechanism of separation

 Adsorption Chromatography: Separation is based mainly on differences between the adsorption

affinities of the sample components for the surface of an active solid.
 Partition Chromatography: Separation is based mainly on differences between the solubilities of
the sample components in the stationary phase (gas chromatography), or on differences
between the solubilities of the components in the mobile and stationary phases (liquid
chromatography).
 Ion-Exchange Chromatography: Separation is based mainly on differences in the ion exchange
affinities of the sample components.
Note: Present day ion-exchange chromatography on small particle high efficiency columns and
usually utilizing conductometric or spectroscopic detectors is often referred to as Ion
Chromatography (IC).
 Exclusion chromatography: Separation is based mainly on exclusion effects, such as differences
in molecular size and/or shape or in charge. The term Size-Exclusion Chromatography may also
be used when separation is based on molecular size. The terms Gel Filtration and Gel-
 Permeation Chromatography (GPC) were used earlier to describe this process when the
stationary phase is a swollen gel. The term Ion-Exclusion Chromatography is specifically used for
the separation of ions in an aqueous phase.
 Affinity Chromatography: This expression characterizes the particular variant of
chromatography in which the unique biological specificity of the analyte and ligand interaction is
utilized for the separation.

1.3 Column Chromatography

 What Is Column Chromatography?

In chemistry, Column chromatography is a technique which is used to separate a single chemical

compound from a mixture dissolved in a fluid. It separates substances based on differential
adsorption of compounds to the adsorbent as the compounds move through the column at
different rates which allow them to get separated in fractions. This technique can be used on
small scale as well as large scale to purify materials that can be used in future experiments. This
method is a type of adsorption chromatography technique.

 Column Chromatography Principle

When the mobile phase along with the mixture that needs to be separated is introduced from
the top of the column, the movement of the individual components of the mixture is at different
rates. The components with lower adsorption and affinity to stationary phase travel faster when
compared to the greater adsorption and affinity with the stationary phase. The components that
move fast are removed first whereas the components that move slow are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the
movement of the components is expressed as:
Rf = the distance travelled by solute/ the distance travelled by solvent
Rf is the retardation factor.
 Column Chromatography Diagram

 Column Chromatography Procedure

Before starting with the Column Chromatography Experiment let us understand the different
phases involved.
 Mobile phase – This phase is made up of solvents and it performs the following functions:

 It acts as a solvent – sample mixture can be introduced in the column.

 It acts as a developing agent – helps in the separation of components in the sample to form
bands.
 It acts as an eluting agent – the components that are separated during the experiment are
removed from the column
 Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone,
water, acetic acid, pyridine, etc.

 Stationary phase – It is a solid material which should have good adsorption property and meet
the conditions given below:

 Shape and size of particle: Particles should have uniform shape and size in the range of 60 –
200μ in diameter.
 Stability and inertness of particles: high mechanical stability and chemically inert. Also, no
reaction with acids or bases or any other solvents used during the experiment.
 It should be colourless, inexpensive and readily available.
 Should allow free flow of mobile phase
 It should be suitable for the separation of mixtures of various compounds.
  Column Chromatography Experiment

 The stationary phase is made wet with the help of solvent as the upper level of the mobile phase
and the stationary phase should match. The mobile phase or eluent is either solvent or mixture
of solvents. In the first step the compound mixture that needs to be separated, is added from
the top of the column without disturbing the top level. The tap is turned on and the adsorption
process on the surface of silica begins.
 Without disturbing the stationary phase solvent mixture is added slowly by touching the sides of
the glass column. The solvent is added throughout the experiment as per the requirement.
 The tap is turned on to initiate the movement of compounds in the mixture. The movement is
based on the polarity of molecules in the sample. The non-polar components move at a greater
speed when compared to the polar components.
 For example, a compound mixture consists of three different compounds viz red, blue, green
then their order based on polarity will be as follows blue>red>green
 As the polarity of the green compound is less, it will move first. When it arrives at the end of the
column it is collected in a clean test tube. After this, the red compound is collected and at last
blue compound is collected. All these are collected in separate test tubes.

 Column Chromatography Applications

 Column Chromatography is used to isolate active ingredients.

 It is very helpful in Separating compound mixtures.
 It is used to determine drug estimation from drug formulations
 It is used to remove impurities.
 Used to isolation metabolites from biological fluids.

 Types of Column Chromatography:

1. Adsorption column chromatography – Adsorption chromatography is a technique of separation,

in which the components of the mixture are adsorbed on the surface of the adsorbent.
2. Partition column chromatography – The stationary phase, as well as mobile phase, are liquid
in partition chromatography.
3. Gel column chromatography – In this method of chromatography, the separation takes place
through a column packed with gel. The stationary phase is a solvent held in the gap of a solvent.
4. Ion exchange column chromatography – A chromatography technique in which the stationary
phase is always ion exchange resin.

1.4 Thin Layer Chromatography

 What Is Thin Layer Chromatography?

Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The

experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated
with a thin layer of adsorbent material. The material usually used is aluminium oxide,
cellulose, or silica gel.
On completion of the separation, each component appears as spots separated vertically.
Each spot has a retention factor (Rf) expressed as:
Rf = dist. travelled by sample / dist. travelled by solvent
The factors affecting retardation factor are the solvent system, amount of material
spotted, absorbent and temperature. TLC is one of the fastest, least expensive, simplest
and easiest chromatography technique.

 Thin Layer Chromatography Principle

Like other chromatographic techniques, thin layer chromatography (TLC) depends on

the separation principle. The separation relies on the relative affinity of compounds
towards both the phases. The compounds in the mobile phase move over the surface of
the stationary phase. The movement occurs in such a way that the compounds which
have a higher affinity to the stationary phase move slowly while the other compounds
travel fast. Therefore, the separation of the mixture is attained. On completion of the
separation process, the individual components from the mixture appear as spots at
respective levels on the plates. Their character and nature are identified by suitable
detection techniques.
  Thin Layer Chromatography Procedure

Before starting with the Thin Layer Chromatography Experiment let us understand the
different components required to conduct the procedure along with the phases involved.

1. Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert
and stable. The stationary phase is applied on its surface in the form of a thin layer. The
stationary phase on the plate has a fine particle size and also has a uniform thickness.
2. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible
to keep a steady environment inside which will help in developing spots. Also, it prevents
the solvent evaporation and keeps the entire process dust-free.
3. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and
consists of a solvent mixture or a solvent. This phase should be particulate-free. The higher
the quality of purity the development of spots is better.
4. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is
moistened in the mobile phase.

 Thin Layer Chromatography Experiment

o The stationary phase that is applied to the plate is made to dry and stabilize.
o To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.
o Apply sample solutions to the marked spots.
o Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened
filter paper in the mobile phase.
 o Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample
faces the mobile phase.
o Immerse the plate for development. Remember to keep the sample spots well above the level
of the mobile phase. Do not immerse it in the solvent.
o Wait till the development of spots. Once the spots are developed, take out the plates and dry
them. The sample spots can be observed under a UV light chamber.

 Thin Layer Chromatography Applications

1. The qualitative testing of Various medicines such as sedatives, local anaesthetics, anticonvulsant
tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
2. TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical
metabolites from its blood plasma, urine, body fluids, serum, etc.
3. Thin layer chromatography can be used to identify natural products like essential oils or volatile
oil, fixed oil, glycosides, waxes, alkaloids, etc
4. It is widely used in separating multicomponent pharmaceutical formulations.
5. It is used to purify of any sample and direct comparison is done between the sample and the
authentic sample
6. It is used in the food industry, to separate and identify colours, sweetening agent, and
preservatives
7. It is used in the cosmetic industry.
8. It is used to study if a reaction is complete.

 Disadvantages Of Thin Layer Chromatography:

1. Thin Layer Chromatography plates do not have longer stationary phase.

2. When compared to other chromatographic techniques the length of separation is limited.
3. The results generated from TLC are difficult to reproduce.
4. Since TLC operates as an open system, some factors such as humidity and temperature can be
consequences to the final outcome of the chromatogram.
5. The detection limit is high and therefore if you want a lower detection limit, you cannot use TLC.
6. It is only a qualitative analysis technique and not quantitative.

1.5 Paper Chromatography

 What Is Paper Chromatography?

Chromatography technique that uses paper sheets or strips as the adsorbent being the
stationary phase through which a solution is made to pass is called paper chromatography. It is
 an inexpensive method of separating dissolved chemical substances by their different migration
rates across the sheets of paper. It is a powerful analytical tool that uses very small quantities of
material. Paper chromatography was discovered by Synge and Martin in the year 1943.

 Paper Chromatography Principle

The principle involved can be partition chromatography or adsorption chromatography.

Partition chromatography because the substances are partitioned or distributed between liquid
phases. The two phases are water held in pores of the filter paper and the other phase is a
mobile phase which passes through the paper. When the mobile phase moves, the separation of
mixture takes place. The compounds in the mixture separate themselves based on the
differences in their affinity towards stationary and mobile phase solvents under the capillary
action of pores in the paper. Adsorption chromatography between solid and liquid phases,
wherein the solid surface of the paper is the stationary phase and the liquid phase is the mobile
phase.

 Paper Chromatography Procedure

Below we have explained the procedure to conduct Paper Chromatography Experiment for easy
understanding of students.

 Selecting a suitable type of development: It is decided based on the complexity of the solvent,
paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are
easy to perform. Also, it is easy to handle, the chromatogram obtained is faster and the process
is less time-consuming.
 Selecting a suitable filter paper: Selection of filter paper is done based on the size of the pores,
and the sample quality.
 Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable
solvent (inert with the sample under analysis) used in making the mobile phase.
 Spot the sample on the paper: Samples should be spotted at a proper position on the paper by
using a capillary tube.
 Chromatogram development: Chromatogram development is spotted by immersing the paper
in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the
sample on the paper.
 Paper drying and compound detection: Once the chromatogram is developed, the paper is
dried using an air drier. Also, detecting solution can be sprayed on the chromatogram developed
paper and dried to identify the sample chromatogram spots.

 Paper Chromatography Applications

There are various applications of paper chromatography. Some of the uses of Paper

Chromatography in different fields are discussed below:

 To study the process of fermentation and ripening.

 To check the purity of pharmaceuticals.
 To inspect cosmetics.
 To detect the adulterants.
 To detect the contaminants in drinks and foods.
 To examine the reaction mixtures in biochemical laboratories.
 To determine dopes and drugs in humans and animals.

 Types of paper chromatography:

1. Ascending Paper Chromatography – The techniques goes with its name as the solvent moves in
an upward direction.
2. Descending Paper Chromatography – The movement of the flow of solvent due to gravitational
pull and capillary action is downwards hence the name descending paper chromatography.
3. Ascending – Descending Paper Chromatography – In this version of paper chromatography
movement of solvent occurs in two directions after a particular point. Initially, the solvent
travels upwards on the paper which is folded over a rod and after crossing the rod it continues
with its travel in the downward direction.
4. Radial or Circular Paper Chromatography – The sample is deposited at the center of the circular
filter paper. Once the spot is dried, the filter paper is tied horizontally on a Petri dish which
contains the solvent.
5. Two Dimensional Paper Chromatography – Substances which have the same r f values can be
resolved with the help of two-dimensional paper chromatography.
1.5 Ion Exchange Chromatography

 What is ion exchange chromatography

Ion-exchange chromatography is a type of chromatography that separates analytes based on

charge. A column is used that is filled with a charged stationary phase on a solid support, called
an ion-exchange resin. Strong cation-exchange chromatography preferentially separates out
cations by using a negatively-charged resin while strong anion-exchange chromatography
preferentially selects out anions by using a positively-charged resin. This type of
chromatography is popular for sample preparation, for example in the cleanup of proteins or
nucleic acid samples.

Ion-exchange chromatography is a two-step process. In the first step, the sample is loaded onto
the column in a loading buffer. The binding of the charged sample to the column resin is based
on ionic interactions of the resin to attract the sample of the opposite charge. Thus, charged
samples of opposite polarity to the resin are strongly bound. Other molecules that are not
charged or are of the opposite charge are not bound and are washed through the column. The
second step is to elute the analyte that is bound to the resin. This is accomplished with a salt
gradient, where the amount of salt in the buffer is slowly increased. Fractions are collected at
the end of the column as the elution occurs and the purified sample of interest can be recovered
in one of these fractions. Another technique, such as spectroscopy, may be needed to identify
which fraction contains the sample. Ion-exchange chromatography is especially useful in protein
studies, to isolate proteins of interest that have a specific charge or size, as size can determine
the number of interactions with the resin.

Ion-exchange chromatography is a more general separation technique than affinity

chromatography, which is also often used in preparing protein samples, where an antibody is
attached to a column to bind one specific analyte. A new affinity column must be purchased for
each analyte, while the same type of ion-exchange column, often with different eluting
conditions, can be used to clean up many proteins of the same charge. Ion-exchange
chromatography can also be used in conjunction with other types of chromatography that
separate based on other properties. For example, size-exclusion chromatography separates
based on size and could be used before ion-exchange chromatography to choose compounds of
only a given size.

 Ion Exchange Chromatography Principle

Exchange of ions is the basic principle in this type of Chromatography. In this process, two types of ion-
exchange chromatography. They are i.e., cationic and anionic exchangers can be used.

 Cationic exchangers possess negatively charged groups, and these will attract positively charged
cations. These exchangers are also called “Acidic ion exchange” materials because their negative
charges result from the ionization of acidic groups.

Anionic exchangers have positively charged groups that will attract negatively charged anions.
These are also called “Basic ion exchange” materials.

 Types of Ion Exchange Resins

 Two main groups of materials are used to prepare ion exchange
resins: Polystyrene and Cellulose.

 Resins made from both of these materials differ in their flow properties, ion
accessibility and chemicals, and mechanical stability.

 Polystyrene resins are proposed by the polymerization reaction of styrene and divinylbenzene.

 A higher concentration of divinylbenzene produces higher cross-linkages.

 Polystyrene resins are very useful for separating small molecular weight compounds.

 Increasing the cross-linkage increases the rigidity, reduces swelling, reduces porosity & reduces
the solubility of the polymeric structure.

 Sulfonic acids are strong acids with good proton dissociation ability. By sulfonation process,
acidic functional groups are easily attached to nearly every aromatic nucleus.

 Resins substituted with sulfonic acid groups are strong cationic exchangers.

 To prepare weekly acidic exchanger, carbohydrate groups can be attached to the aromatic rings
instead of a sulfonic acid group.

 If basic functional groups are introduced, the resin can exchange anions rather than cations.
Strong anion exchangers are prepared with a tertiary amine, yielding a strongly basic quaternary
ammonium group. Weak anionic exchangers can be prepared with secondary amines, yielding a
weakly basic tertiary amine.

 Cellulose resins have much greater permeability to macromolecular polyelectrolytes and possess
a much lower charge density as compared to polystyrene exchangers.

 Carboxymethyl cellulose (CM-cellulose) – Cationic exchanger

 DEAE cellulose – Anionic exchanger

  Preparation of the exchange medium

There are three steps are of absolute importance:

1) Swelling Of Medium: (Pre-Cycling)

Swelling makes the functional groups to be exposed for ion exchange.

 The swelling of anion exchangers is usually carried out by treating it. first with an acid (0.5N HCl)
and then with base (0.5N NaOH).
 Exactly the reverse is the case with cationic exchangers. The matrix can be treated with EDTA for
impurity eliminations.

2) Removal Of Very Small Particles

These fines will decrease the flow rate and the unsatisfactory reaction. To remove fines, the exchanger
is repeatedly suspended in a large volume of water and after the larger polymers have settled down, the
slow sedimenting materials decanted.

3) Equilibration With Counter Ions

This is accomplished by washing the exchanger with different reagents depending upon the desired
counter ion to be introduced.

 NaOH –> counter ion to be introduced is “Na+”

 HCl –> counter ion to be introduced is “H+”

 NaNO3 –> counter ion to be introduced is “NO3”

Choice of Buffers
 Anionic exchange Chromatography should be carried out with cationic buffers.

 Cationic exchange Chromatography should be carried out with anionic buffers.

 The pK of the buffer should be as near as possible to the pH at which the system is buffered. This
results in high buffer capacity, which can withstand the local

 changes of pH in the column easily.

Buffers PH range
 Ammonium acetate 4 to 6

Ammonium formate 3 to 5

Pyridinium formate 3 to 6

Pyridinium acetate 4 to 6

Ammonium carbonate 8 to 10

Ion-Exchange Chromatography Procedure

 Ion exchange separations are carried out mainly in columns packed with an ion-exchanger.
These ionic exchangers are commercially available. They are made up of styrene and
divinylbenzene.

 DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic exchanger. The choice of the

exchanger depends upon the charge of the particle to be separated. To separate anions “Anionic
exchanger” is used, to separate cations “Cationic exchanger” is used.
  First, the column is filled with ion exchanger then the sample is applied followed by the buffer.
The tris-buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used.

 The particles which have a high affinity for ion exchanger will come down the column along with
buffers. In the next step using the corresponding buffer separates the tightly bound particles.

 Then these particles are analyzed spectroscopically.

Applications of Ion Exchange Chromatography

 It is extremely used in the analysis of amino acids. The amino acid “Autoanalyzer” is based on in
exchange principle.

 To determine the base composition of nucleic acids. Chargaff used this technique for established
the equivalence of Adenine and Thymine; Guanine and Cytosine.

 This is the most effective method for water purification. Complete deionization of water (or) a
non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute
anions for hydroxyl ions. This is usually achieved by the method is used for the softening of
drinking water.

 Proteins are also successfully separated by this technique.

 It is also used for the separation of many vitamins, other biological amines, and organic acids
and bases.
One of the main disadvantages of ion-exchange chromatography is its buffer requirement: because
binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the
stationary phase, IEX columns must be loaded in low-salt buffers.
For some applications, this restriction may require a buffer exchange step prior to ion exchange
chromatography.

1.6 van dimmter equation

The efficiency of a column is measured by theoretical plates, Nth, and can be normalized with the length
of the column to give the height equivalent theoretical plate, called HETP or H. The Van dimmter
equation describes the various factors influencing H, and is divided into eddy diffusion, longitudinal
diffusion, and mass transfer terms. The relative importance of these factors varies with mobile phase
velocity. Particle size and morphology contribute to H, along with a variety of other factors.
Understanding the van Deemter equation allows the determination of the optimum mobile phase
velocity.

The theoretical plate number Nth shows the relation between retention time and peak width
and describes column quality and separation power.

Factors affecting column efficiency (plate number)

 Column length
 Particle size
 Packing quality
 Linear velocity (flow)
 Instrument quality (dead volume)   
 Retention factor

Stationary phase particle size is one of the most important factors in the van Deemter equation.  For a
given column length, the plate number (N th) is inversely related to the particle size of the column
packing. The smaller the particles, the higher the plate number and the separation power.

The plate number is also dependent on the flow rate (F) of the mobile phase. There is a certain
velocity, the so-called optimum flow, at which the plate number is highest (and H is lowest). A
lower or a higher flow rate provides less plates (higher H).  In routine HPLC, columns are always
operated at velocities above the optimum. The reduced column efficiency is less significant than
the shorter analysis time at the higher than optimal flow rates.

To describe the contribution of the above factors, several so-called plate height equations have
been developed. A plate height equation expresses the correlation between plate height and
mobile phase velocity. Best known is the van Deemter equation, which describes the various
contributions to plate height (H).  In this equation the parameters that influence the overall
peak width are expressed in three terms:

H = HETP (plate height)

A = eddy diffusion term
B = longitudinal diffusion term
u = linear velocity
C = Resistance to mass transfer coefficient  

Peak height and peak broadening are governed by kinetic processes in the column such as
molecular dispersion, diffusion and slow mass transfer. Identical molecules travel differently in
the column due to probability processes. The three processes that contribute to peak
broadening described in the van Deemter equation are:

 A-term: eddy diffusion: The column packing consists of particles with flow channels in between.
Due to the difference in packing and particle shape, the speed of the mobile phase in the various
flow channels differs and analyte molecules travel along different flow paths through the
channnels.
 B-term: longitudinal diffusion: Molecules traverse the column under influence of the flowing
mobile phase. Due to molecular diffusion, slight dispersions of the mean flow rate will be the
result.
 C-term: resistance against mass transfer. A chromatographic system is in dynamic equilibrium.
As the mobile phase is moving continuously, the system has to restore this equilibrium
continuously. Since it takes some time to restore equilibrium (resistance to mass transfer), the
concentration profiles of sample components between mobile and stationary phase are always
slightly shifted. This results in additional peak broadening.

1.7 High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid
chromatography.

Instead of a solvent being allowed to drip through a column under gravity, it is forced through under
high pressures of up to 400 atmospheres. That makes it much faster.

All chromatographic separations, including HPLC operate under the same basic principle; separation of a
sample into its constituent parts because of the difference in the relative affinities of different molecules
for the mobile phase and the stationary phase used in the separation.

 
Types of HPLC
There are following variants of HPLC, depending upon the phase system (stationary) in the process :

1. Normal Phase HPLC

This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-
polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are
hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.

Polar samples are thus retained on the polar surface of the column packing longer than less polar
materials.

2. Reverse Phase HPLC

The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such
as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions
hence the more nonpolar the material is, the longer it will be retained.

3. Size-exclusion HPLC

The column is filled with material having precisely controlled pore sizes, and the particles are separated
according to its their molecular size. Larger molecules are rapidly washed through the column; smaller
molecules penetrate inside the porous of the packing particles and elute later.

4. Ion-Exchange HPLC

The stationary phase has an ionically charged surface of opposite charge to the sample ions. This
technique is used almost exclusively with ionic or ionizable samples.

The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the
longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are
used to control elution time.

 
Instrumentation of HPLC

As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump, injector,
column, detector and integrator or acquisition and display system. The heart of the system is the column
where separation occurs.

1. Solvent Resorvoir

Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is
usually a mixture of polar and non-polar liquid components whose respective concentrations are varied
depending on the composition of the sample.

2. Pump

A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column
and detecter. Depending on a number of factors including column dimensions, particle size of the
stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000
kPa (about 6000 psi) can be generated.

3. Sample Injector

The injector can be a single injection or an automated injection system. An injector for an HPLC system
should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to 4000 psi).
4. Columns

Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an
internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a
particle size of 3–10 µm.

Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally
the temperature of the mobile phase and the column should be kept constant during an analysis.

5. Detector

The HPLC detector, located at the end of the column detect the analytes as they elute from the
chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-
spectrometric and electrochemical detectors.

6. Data Collection Devices

Signals from the detector may be collected on chart recorders or electronic integrators that vary in
complexity and in their ability to process, store and reprocess chromatographic data. The computer
integrates the response of the detector to each component and places it into a chromatograph that is
easy to read and interpret.

Applications of HPLC
The information that can be obtained by HPLC includes resolution, identification and quantification of a
compound. It also aids in chemical separation and purification. The other applications of HPLC include :

Pharmaceutical Applications

1. To control drug stability.

2. Tablet dissolution study of pharmaceutical dosages form.
3. Pharmaceutical quality control

Environmental Application

1. Detection of phenolic compounds in drinking water.

2. Bio-monitoring of pollutants.

 
 Applications in Forensics

1. Quantification of drugs in biological samples.

2. Identification of steroids in blood, urine etc.

3. Forensic analysis of textile dyes.

4. Determination of cocaine and other drugs of abuse in blood, urine etc.

Food and Flavour

1. Measurement of Quality of soft drinks and water.

2. Sugar analysis in fruit juices.

3. Analysis of polycyclic compounds in vegetables.

4. Preservative analysis.

Applications in Clinical Tests

1. Urine analysis, antibiotics analysis in blood.

2. Analysis of bilirubin, biliverdin in hepatic disorders.

3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.

 Students Stories - The Learning Tree

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References :

1. Laboratory info
https://laboratoryinfo.com/hplc/
2. Chromedia analytical science http://chromedia.org/chromedia?
waxtrap=xqegzCsHqnOxmO1IecCbC&subNav=wnjedDsHqnOxmO1EcCz
3. Biochemistry den
https://www.biochemden.com/ion-exchange-chromatography/
4. BYJU'S – The Learning App
https://byjus.com/