Sathyabama University Department of Biomedical Engineering

SBMX1009 ANALYTICAL INSTRUMENTATION

UNIT V CHROMATOGRAPHY

Prepared by : Dr T Sudhakar Page 1 of 21

Definition

Chromatography is a physical method of separation of the components of a mixture

by distribution between two phases of which one is stationary bed and other a fluid phase that
percolates though along the stationary phase.

Chromatography was first reported by Russian Botanist classification.

Retention time (tr): The total time that a compound spends in both a mobile phase
and stationary phase. In other words, the time between sample injection and a analyte peak
reaching detector at the end of the column is termed as retention time. It is generally
expressed in mts.

Dead time (tm): The dead time is a time that a non retained compound spends in a
mobile phase, which is also the amount of the time the non-retained compounds spend in the
column.

Adjusted retention time (t’R): The adjusted retention time is the time that a
compound spends in the stationary phase. The adjusted retention time is the difference
between dead time and the retention time for a compound retention time for a compound.

T¢r = tr – tM

Capacity Factor / Partition Ratio (k¢)

The capacity factor is the ratio of the mass of the compound in the stationary phase
relative to the mass of the compound in the mobile phase.

K = (tr – tM)/ TM

Distribution Constant

Ratio of the concentration of a compound in the stationary phase relative to the

concentration of the compound in the mobile phase.

Concentration of a CPD in stationary phase

KD =
Concentration of a compound in mobile phase

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Linear velocity (µ)

The linear velocity is the speed not which the carried gas (or) mobile phase travels
through the column. Linear velocity expressed in centimeters per second.

µ = L/tM

Definition

Paper chromatography is defined as the technique in which the analysis of unknown

substances is carried out mainly by the flow of solvents on specially designed filter paper.

Types of Chromatography

i) Paper adsorption, ii) Paper partition.

Paper adsorption: Paper impregnated with silica act as adsorbent (stationary phase)
and solvent as mobile phase.

Paper partition: Moisture / water present in the pores of cellulose fibres present in
filter paper act as stationary phase and solvent as mobile phase.

Principle

The principle of separation is mainly partition rather than adsorption. Cellulose layers
in filter paper contains moisture which act as stationary phase. Organic solvents (or) buffers
are used as mobile phases.

Requirements

∑ Stationary phase and paper used.

∑ Application of sample
∑ Mobile phase
∑ Development technique
∑ Detecting and visualizing agents

Stationary phase and paper used

Whattman filter paper of different grade like no.1, 2, 3. Choice of filter paper depends
upon thickness, flow rate and purity. Eg: Modified filters: Acid (or) base washed paper, filter
paper.

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Application of Sample

The sample to be applied is dissolved in the mobile phase and applied using capillary
(or micropipette.

Concentration of sample must be low.

Mobile Phase

Pure solvents, buffer solutions (or) mature of solvents are used. Eg:Hydropholic
mobile phases, n-butanol, glacial acetic acid.

Development technique

Ascending development: The solvent flour against gravity. The spots are kept at the
bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom.

Detecting (or) visualizing agents: After the development of chromatogram the spot
should be visualized.

Detecting colored spots can be done visuality.

But for detecting colorless spot any one of the technique can be used.

A) Non specific methods

1) Iodine chamber method (brown colored spots are observed. When iodine
crystals are applied.
2) UV – chamber for fluorescent compounds.

B) Specific methods

Specific spray reagents (or) detecting agents (or) visualizing agents are used to find
out the nature of compound. Eg: ninhydrin in acetone for aminoacids, radioactive compound
– autoradiography is used.

Quantitative analysis

1) Direct method: Densitometer is an instrument which measures the density of

spots.
2) Indirect method: In this technique the spots are cut in to portions and eluted with
solvents – solutions can be analysed by spectrophometry.

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Rf value is the ratio of distance travelled by the solute to the distance travelled by the
solvent front

Distance travelled by salute

Rf =
Distance travelled by solvent front

Distance travelled by sample

Rx =
Distance travelled by standard

Applications

∑ Paper chromatography is more useful for the analysis of polar compounds like
aminoacids, sugars and natural products
∑ Separation of mixtures of drugs of chemical (or) biological origin, plant extracts.
∑ Separation of vitamins, antibiotics and proteins.
∑ Identification of foreign substances in drugs.
∑ Identification of decomposition products.
∑ Analysis of metabolites of drugs in blood, urine etc.

Identification of drug

Drug Mobile phase Detecting agent

Erythromycin Isobutyl methyl ketone Nutrient agar containing

Bacillus spp

Identification of related compound

Drug Mobile phase Detecting agent

Vitamin A Diaxn Methanol UV – 366 nm

70 : 15

Thin Layer Chromatography (TLC)

Principle:

The principle of separation is adsorption one or more compounds are spotted on a thin
layer of adsorbent coated on a chromatographic plate. The mobile phase solvent flows
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through because of capillary action (against gravitational force). The components move
according to their affinities towards the adsorbent. The component with more affinity
towards the stationary phase travels slower. The component with lesser affinity towards the
stationary phase travels faster. Thus the components are separated on a TLC based on the
affinity of the components towards the stationary phase.

Practical requirements

∑ Stationary phase (silica gel – G)

∑ Glass plates
∑ Preparation and activation of TLC plates
∑ Application of sample
∑ Development tank
∑ Mobile phase
∑ Development tank
∑ Mobile phase
∑ Development technique
∑ Detecting (or) visualizing agents

Stationary phases

Several adsorbents which can be used as stationary phases, their composition and
ratio in which they have to be mixed with H2O (or) solvents to form a slurry has to be
calculated. Eg. Silica gel – G, Silica +CaSO4 1: 2

Glass Plates

Size - 20 cm × 20 cm or 20 cm × 10 cm

Glass plates should be of good quality to withstand temperature for drying the plats.

Preparation and activation of TLC plates

After preparing the slurry

Ø
By spreading (TLC spreader) technique
Ø
Slurry is poured inside the reservoir of TLC spreader
Ø
The thickness of the adsorbent layer is adjusted by using a knob in spreader
Ø
Spreader is rolled only once on the plates
Ø
Plates are allowed for setting (air drying)
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Ø
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Plates are activated by keeping in an oven at 100oC for 1 hour.

Application of sample

Sample concentration 2 – 5µl

Sample is applied by using capillary tube (or) micropipette.

Spot should be kept at least 2 cm above the base of the plate and spotting area should
not be immersed in the mobile phase in the development tanks.

Development tank

Developing tank or chamber of different sizes to hold. The plates of standard

dimensions are used.

Mostly glass beakers, jars are used to avoid wastage of solvents.

It is better to use development tank.

Mobile Phase

Mobile phases depends upon various factors.

1) Nature of substances to be separated

2) Nature of stationary phase used. Eg: Petroleum ether, ethylacetate alcohols,
cyclohexane, chloroform.

Development technique

One dimensional development (vertical)

In this technique the plates are kept vertical and the solvent flows against gravity,
because of capillary action. Most separations done practically are of this type only.

Detecting (or) visualizing agents

After the development of TLC plates the spots should be visualized.

Detecting coloured spots can be done visuality

For colorless spots any one of the technique can be used.

Iron specific method

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Iodine chamber method: where brown (or) amber spots are observed when the TLC plates
are kept in a tank with few iodine crystals at the bottom.

Eg: 1. Sulphuric acid spray reagent, 2. UV chamber for fluorescent cpds.

Specific method

1) Ferric chloride for phenolic cpds ninhydrin – for aminoacids.

Qualitative analysis

Rf – retardation factor

Distance travelled by salute

Rf =
Distance travelled by solvent front

Rf value ranges from 0 to 1.

When the Rf value of a sample and reference compound is same, the compound is
identified by its standard.

Quantitative analysis

∑ Direct method – the quantity of the individual spots can be determined by using
denistometric method

Indirect method

Quantitative analysis can be done after eluting the individual spots with solvent and
filtering off the stationary phase and concentration is measured by using UV
spectrophotometry.

Applications of TLC

1. Separation of mixtures of drugs of chemical plant extract etc.

2. Separation of carbohydrates, vitamins, antibiotics proteins.
3. Identification of drugs.
Drugs Stationary Phase Mobile Phase Detecting Agent
Chloropromazine Silica Gel G Ether : Ethtylacetate UV -254 nm

Column chromatography

Principle: When a column of stationary phase is use of the technique is called as

column chromatography. Based on the nature of stationary phase that is whether it is solid
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(or) liquid it is called as column adsorption chromatography or column partition

chromatography.

A solid stationary phase and a liquid mobile phase is used and the principle of
separation is adsorption. When a mixture of components dissolved in the mobile phase is
introduced into the column. The individual components move with different rates depending
upon their relative affinities.

The compound with lesser affinity towards the stationary phase (adsorbent) moves
faster and hence it is eluted out of the column first. One with greater affinity towards the
stationary phase (adsorbent) moves slower down the column and hence it is eluted later. Thus
the Cpds are separated.

Distance moved by the solute

R =
(rate of movement of Distance travelled by solvent front
movement)

Practical Requirements

1. Stationary phase (adsorbents)

i) Particles should have uniform size and distribution spherical shape
ii) Should have mechanical stability
iii) Should be inert and should not react with the solute and other
components.
iv) Insoluble in mobile phases
v) In expensive and should separate wide variety of compounds.
Eg:
Weak Medium Strong absorbent
Sucrose CaCO3 Activated Mg silicate
Starch Ca3 (PO4)2 Activated alumnia
Silica gel is commonly used as stationary phase

Mobile Phase

Mobile phase is very important and they act as solvent, developer and as eluent. Eg.
Petroleum ether, carbon tetra chloride, ether acetone, chloroform, alcohol and H2O.

Column Characteristics

The material of the column is mostly good quality neutral glass since it should not be
affected by solvents, acids (or) alkalies.
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An ordinary burette can also be used as column for separation.

Size -100 : 1(length : diameter for more efficiency).

Column depends upon

1. Affinity of compounds towards the adsorbent used.

2. Type of adsorbent used
3. Quantity of the sample

Preparation of the column

The bottom portion of the column is packed with cotton wool (or) glass wool, above
which the column of adsorbent is packed.

After packing the column with the adsorbent, a similar paper disc is kept on the top so
that the adsorbent layer is not disturbed, which will leads to the formation of irregular bands
in separation.

Column is prepared by wet packing technique.

Wet packing technique: This is the ideal technique. The required quantity of the
adsorbent is mixed with the mobile phase and poured into the column.

The stationary phase settles uniformly in the column and there is no entrapment of air
bubbles.

There will not be any crack in the column of adsorbent.

Thus the bands eluted from the column will be uniform and ideal for separation.

Sample application

The sample which is usually a mixture of components in dissolved in a minimum

quantity of mobile phase.

The entire sample is introduced into the column at once and gets adsorbed onto the
top portion of the column.

Development technique (elution)

The individual components are separated out from the column. The two techniques
are:

i) Isocratic elution technique

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In this elution technique the same solvent composition (or) solvent of same. Polarity
in used throughout the process of separation. Eg. Chloroform.

ii) Gradient elution technique

In this elution technique, solvents of gradually increasing polarity (or) increasing

elution strength are used during the process of separation.

Initially low polar solvent is used followed by gradually increasing the polarity, to a
more polar solvent. Eg: Initially benzene, then chloroform, ethyl acetate.

Detection of components

The detection of coloured components can be done visally.

Different coloured bands are seen moving down the column which can be collected
separately.

For detecting colourless compounds

i) UV – vis – detector
ii) Fluorescence detector.

Recovery of components

The best technique is to recover the components by a process called as elution. The
components are called as eluate, the solvent called as eluent and the process of removing the
components from the column is called as elution.

Recovery is done by collecting as different fractions of mobile phase of equal volume

like 10 ml, 20 ml etc. (or) unequal volume. They can also be collected time wise fraction ever
10 to 20 mts. Fractions are measured by using UV-spectrophotometer.

Applications

1. Separation of mixture of Cpds

2. Isolation of bioactive constituents (components)
3. Isolation of metabolites from biological fluids
4. Estimation of drugs in formulations
5. Purification process

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Principle

Gel permeation chromatography, the separation is based on molecular size and shape.
The gel permeation column is packed with a stationary phase in the form of a gel which
contains pores of a specific size.

As the sample is carried through the column bed by the carried liquid, the sample
molecules penetrates through the pores in the gel depending upon the size and shape of the
molecules it is separated.

Large molecules do not penetrates the gel and are consequently quickly eluted,

Preparation of Sephadex G-250

1 gm of sephadex G – 250
Ø
Is dissolved in 100 ml of buffer
Ø
Allowed to swell overnight
Ø
Gel stars swelling in water
Supernatant Sediment
Ø
Sediment is washed twice (or) thrice with H2O
Ø
Sediment act as gel and packed in column

Packing of column

Column size - 75 cm
Gel - Sephadex G – 250 (3 – 5)
Buffer - Tris 10.5 N, pH 7.5) + 0.15 M NaCl
Sample - Serum

Sample Applications

Sample is applied carefully over the buffer which drags the sample to the gel bed to
enhance polymerization.

Since ptn has larger molecular weight it soon eluted out and other impurities with low
molecular weight will enters the gel beads and are later eluted out.

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After elution is completed by using elution buffer the fractions is collected by using test tube
subjected to UV – spectrophotometer and OD valve is taken at 280 nm.

To determine the concentration of ab’s.

Applications refer ion exchange and column

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.

Principle

The principle of separation is by reversible exchange of functional groups between the

ions present in the solution and those present in the ion exchange rein.

Cation exchange: (separation of cations using CER). The cations to be separated are
present in solution and exchange for similar ions present in cation exchange resin a solid
matrix.

It is represented: solid – H+ + M+Æ Solid – M+ + H+(solution)

The cations retained by the solid matrix of IER can be eluted by using buffers of different
strength and hence separation of cations can be effected.

Anion exchange (Separation of anions using anion exchange resin)

The anions to be separated are present in solution and exchange of similar ions
present in anion exchange resin – a solid matrix

Solid OH + A(solution) Æ Solid – A + OH (solution)

The anions retained by the solid matrix of IER can be eluted by using buffers of different
strength and hence separation of anions can be effected.

Ion Exchange resins (classification of resins)

1. Strong cation exchange resin

2. Weak cation exchange resin According to chemical nature
3. Strong anion exchange resin
4. Weak anion exchange resin

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Eg: Organic ion exchange resin are widely used organ (IER) are polymeric resin matrix
containing exchange sites.

The resin is composed of polystyrene and divinnyl benzene.

Poystyrene ‡ contains sites for exchange function groups

Divinyl benzene ‡ act as cross linking age (mechanical stability)

Eg: Functional groups present in different IER

Strength cation exchange resin - SO3H – sulphonic acid group

Weak cation exchange resin - COOH – Carboxnyl

Strong anion exchange resin - NR2 – 2o Amine

Weak anion exchange resin - NH2 Amine

Properties of rein

Particle size – 50 – 200 mesh, should allow free and uniform flow of mobile phase

Cross linking and swelling – optimum quantity of cross linking agent is required for
effective separation.

Practical requirements

Column material and dimensions

Column are made up of glass
Which are resistant to strong acids and alkalies
Size -20 : 1 to 100 : 1 for higher efficiency can be used.

Type of ion exchange resin

Packing of the column – Wet packing method is used.

The resin is mixed with the mobile phase and packed in the column uniformly. The
ample to be separated is dissolved in the mobile phase and introduced all at once in to the
column.

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Mobile Phase

Only different strengths of acids, alkalis and buffers are used as eluting solvents. Eg:
0.1 N HCl, 1 N NaOH, acetate buffer, PoA buffer.

Development of the chromatogram and elution

After introduction of sample

Ø
Using acids, alkalis and buffers of different pH are used
Ø
To separate the compounds.

Elution: Isocratic elution technique, Gradient elution technique.

Isocratic elution technique – Same solvent composition is used. Same strength of acid,
alkali or buffer.

Gradient elution technique – Initially less acidic or basic character is used followed by
increasing the acidity or basicity of mobile phase.

Gradient elution is used for complex mixture fractions of the eluent is collected.
Volume wise or time wise and analysed.

Analysis of the elute

Fracture collection are analysed by using spectrophotometric method and

radiochemical method.

Regeneration of the ion exchange resin

Regeneration refers to the replacement of the exchangeable cations (or) anions present
in the original resin.

Regeneration of cation exchange resin

By charging the column with strong acid line HCl.

Regeneration of anion exchange resin

By using strong alkali like NaOH.

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Applications

1. Softening of H2O
2. Demineralisation (or) deionization of H2O
3. Purification of some solutions to be free from ionic impurities.
4. Separation of inorganic ions (cations and anions).
5. Organic separations Eg: aminoacids, proteins antibiotics vitamins fatty acids etc.
6. Biochemical separations like isolation and some drugs (or) metabolites of blood, urine
etc.

Gas Chromatography

Gas chromatography consists of a gas solid chromatography (GSC) and gas liquid
chromatography (GLC).

In both types gas is used as mobile phase and either solid (or) liquid is used as
stationary phase.

Principle

The principle of separation in GLC is partition gas is used as mobile phase. Liquid
which is coated onto a solid support is used as stationary phase. The mixture of components
to be separated is converted to vapour and mixed with gaseous mobile phase.

The component which is more soluble in the stationary phase travels slower and
eluted later. The component which is less soluble in the stationary phase travels faster and
eluted to out first.

The components are separated according to their partition coefficient.

Partition coefficient is the ratio of solubility of a substance distributed between two

immiscible liquids at a constant temperature. Criteria for compounds to be analysed by GC.

Two important criteria are

1) Volatility : Less a compound is volatile it cannot be mixed with mobile phase.

Hence volatility is important.
2) Thermostability : All the compounds will be in the form of vapour. There will
be solid as well as liquid samples.

Hence to convert them to a vapour form they have to be heated at a higher

temperature. At that temperature the compounds have to be thermostable they are not
thermostable the compounds can be analysed by GC. Since they will be decomposd.
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Practical requirements

1. Carrier gas
2. Flow regulators and flow meters
3. Injection device
4. Columns
5. Temperature control devices
6. Detectors
7. Recorders and integrators

Carrier gas

The choice of carrier gas determines the efficiency of chromatographic separation.

Most widely used carrier gases are: hydrogen, helium, nitrogen and argon

Hydrogen : Better thermal conductivity, it is useful in cases of thermal conductivity

detector a flame ionization detector.

Helium : Excellent thermal conductivity, thermal conductivity detector expensive.

Flow regulators and flow meters

As carrier gases are stored under high pressure flow regulators are used to deliver the
gas with uniform pressure (or) flow rate.

Flow methods are used to measure the flow rate of carrier gas. Eg. Rotameter and
soap bubble flow meter.

Injection devices: Sample for introducing into the column can be of any type that is gas,
liquid (or) solid in nature.

Gases – introduced into the column by valve devices

Liquid – most GS instruments have a high quality rubber septum through which
sample is injected.

Rubber is made up of good quality silicone rubber which can withstand high
temperature of preheating device and withstand repeated injections over a period of time.

Solid – Samples are dissolved in a suitable solvent and then they are injected through
a septum.

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Columns

Column is one of the important part of GC which decides the separation of efficiency.
Column are made up of glass ‡ inert – do not react with sample, high fragile difficult to
handle, or stainless steel ‡long file no fragility, but react with sample.

Column can be i) Analytical, ii) preparative

Analytical – 1 – 1.5 m,3 – 6mm (diameter) – only small amount of sample can be loaded
Preparative – 3 – 6 M - 6 - 9 mm – High amount of sample can be loaded.
Eg. Open tubular column (or) capillary (or) go lay column
30 – 90 m in length, 0.025 - 0.075 cm. Made up of stainless steel in the form of coil.
Inner wall of capillary is coated with stationary phase liquid in the form of thin film
Column more resistance to the flow of carrier gas

Temperature control device

Pre-heaters: are used in GC to convert the sample into its vapour form and mix them
with the mobile phase (or) carrier gas.

Preheaters are present along with injecting device. As soon as liquid samples are
injected they are converted into vapour form.

Thermostatically controlled Oven

Detectors which can detect the difference between a pure carrier gas and a eluted
component. Eg. Flame ionization detection.

The carrier gas used with this type of detector can be hydrogen.

This ionisation detectors are based upon the electrical conductivity of carrier gases.

When pure carrier gases alone passes there is not ionization and no current flows,
when a component emerges from the column number of samples converted.

Ions are produced because of ionization of the thermal energy of the flame.

This causes a potential difference and causes a flow of current which is amplified and
recorded as signal.

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Advantages

This detector is extremely sensitive and back ground noise is low hence µg quantities
of the solute can be detected. Linearity is excellent.

Stable and insensitive to small changes in the flow rate of carrier gas and water
vapour. Responds to most of the organic compounds.

Recorders: are used to record the responsive obtained from detectors after
amplification, they record the baseline concentration and all the peaks obtained with respect
to time.

Retention time for all the peaks can be found out from such recordings.

Integrators: They are improved various of recorders with data processing

capabilities.

They can record the individual peaks with RT, height and width of peak, peak area,
percentage of area.

Applications

1. Assay of drugs
2. Presence of foreign (or) related substance
3. Purity of compounds
4. Isolation and identification of drugs (or) metabolites in urine, plasma and serum.
5. Isolation and identification of mixture of components like amino acids, plant
extract etc.

High performance liquid chromatography (HPLC)

Introduction: The technique of HPLC is so called because of its improved

performance when compared to classical column chromatography.

It is also called as HPLC since high pressure is used when compared to classical
column chromatography. The development of HPLC from classical column chromatography
can be attributed to development of small particle sizes. Smaller particle size is important
since they offer more area over the conventional larger size particle.

Types of HPLC technique

Based on modes of chromatography – normal phase mode: In normal phase

technique, a polar charge is used and nonpolar mobile phase is used.
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Reverse phase mode: In reverse phase technique, a non-polar stationary phase and polar
mobile phase is used. Hence polar components gets eluted. First and nonpolar components
are retained for longer time. Since most of the drugs and pharmaceuticals are polar in nature
they are not retained for a longer time and eluted faster which is advantageous.

Based on principle of separation

1. Adsorption chromatography: The principle of separation is adsorption,

separation of components takes place because of the difference in affinity of
compounds towards stationary phase.
2. Ion-exchange chromatography: The principle of separation is ion exchange
which is reversible exchange of functional groups. In ion-exchange
chromatography, an ion exchange resin is used to separate a mixture of similar
charged ions for cations, a cation exchange resin is used. For anions, an anion
exchange resin is used.
3. Gel permeation chromatography: In this type of chromatography, a mixture of
components with different molecular sizes are separated by using gels. The gels
used acts as molecular sieve and hence a mixture of substances with different
molecular sizes are separated.
4. Affinity chromatography: uses the affinity of the sample with specific stationary
phases.

Based on elution technique

Isocratic technique: In this technique the same mobile phase combination is used
throughout the process of separation. The same polarity (or) elution strength is maintained
throughout the process.

Gradient separation: In this technique, a mobile phase combination of lower polarity

(or) elution strength is used followed by gradually increasing the polarity or elution strength.

Based on the scale of operation : 1) analytical HPLC (2) preparative HPLC.

Based on the type of analysis: Qualitative analysis – which is used to identity the
compound, detect the presence of impurities in this done by using retention time values.

Quantitative analysis: which is done to determine the quantity of the individual (or)
several components in a mixture this is done by comparing the path area of the standard of
sample.

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Principle of separation in HPLC

The principle of separation in normal phase made and reverse phase mode is
adsorption. When a mixture of components are introduced into a HPLC column, they travel
according to their relative affinities towards the stationary phase. The components which has
more affinity towards the adsorbent travels slower. The component which has less affinity
towards the stationary phases travels faster. Since no two components have the same affinity
towards the stationary phase, the components are separated.

Instrumental requirements

1. Pumps – solvent delivery system

2. Mixing unit, gradient controller and solvent degassing
3. Injector – manual (or) auto injection.
4. Guard column
5. Detectors
6. Recorders and integrators

Solvent delivery system: The solvents (or) mobile phases used must be passed
through the column oil high pressure at about 1000 – 3000 psi. This is because as the particle
size of stationary phase (sp) is few µ (5 – 10 µ) the resistance to the flow of solvent is high.
Hence such high pressure is recommended. The solvent used must be of high purity
preferably HPLC grade and filtered through 0.45 µ filter.

Pump: There are different types of pump available they are mechanical pump and
pneumatic pumps. Mechanical pumps operate with constant flow rate this is used in
analytical scale. Pneumatic pumps operate with constant pressure and use highly compressed
gas.

Mixing unit: Mixing unit is used to mix solvents in different proportions and pass
through the column mixing of solvents is done either with a static mixes which is packed with
beads (or) magnetic stirrer and operates under high pressure.

Gradient controller: (refer) – gradient elution. Gradient controller is used when two
(or) more solvent pumps are used for such separations.

Solvent degassing: When solvents are pumped under high pressure, gas bubbles are
formed which will interfere with the separation process, steady baseline and the shape of the
peak. Hence degassing of solvent is important which is done by using ultrasonication method.

Injector (Manual (or) autoinjector) eg. Rheodyne injects. It is the most popular
injector. This has a fixed volume loop like 20 µl (or) 50 µl or more. Injector has two modes

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that is load position when the sample is loaded in the loop and inject mode when the sample
is injected.

Analytical column: Analytical column is the most important part of the HPLC
technique which decides the efficiency of separation.

Column material: The column are made up of either stainless steel, polyethylene, peek
(polyether ether) all these withstand high pressure.

Column length – 5 cm – 30 cm
Column diameter - 2mm – 50 mm
Particle size – 1 – 20 µ
Particle nature – spherical, uniform size, porous material.

Surface area: 1 gm of S.P provides surface area ranging from 100 - 800 sq.m with an
average of 400 sq.m. eg: Silanol group, C18 octa decyl silane (ODS – column)

Detectors: used depends upon the property of the Cpds to be separated.

Eg. UV detector, this detector is based upon the light adsorption characteristics of the
sample. Two types of this detector are available one is the fixed wavelength detector which
operates at 254 nm where most drugs compounds absorb. The other is variable wavelength
detector which can be operated from 190 nm – 600 n.

Recorders and Integrators : Refer – GC

Applications of HPLC: HPLC is being more widely used in several fields. Apart
from the use in pharmaceutical field. It is used in chemical and petrochemical industry,
environmental applications, forensic applications, biochemical separations, biotechnology,
food analysis etc. In fact there is not field where HPLC is not being used. It is a versatile and
sensitive technique which can be used in several techniques.

1. Qualitative and quantitative analysis of a given sample.

2. Checking the purity of a CPD and presence of impurities.
3. Isolation of identification of drugs or metabolites in urine, plasma, serum etc.
4. Isolation and identification of mixture of Cpds.
5. Multicomponent analysis (or) determination of mixture of drugs.
6. Identification of related Cpds
7. Assay of drugs

B.Tech (Biomedical Engineering) 21