CHROMATOGRAPHY

Chromatography Separation is the process by which a homogenous mixture or mass is

resolved into its constituent or individual units. Separation technique is used for
purification, qualitative identification or quantitative determination. “Chromatography
may be defined as the method for separating a mixture of components into
individual components through equilibrium distribution between two phases”.
According to IUPAC “Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases. One of which is
stationary (stationary phase) while the other is mobile (mobile phase) which involves in a
definite direction.”

The technique of chromatography is based on the differences in the rate at which the
component of the mixture move through a porous or solid medium called the stationary
phase under the influence of some solvent or gas called the mobile phase.
The chromatographic method of separation in general involves the following steps:
1. Retention of a substance or substances on the stationary phase.
2. Separation of the retained species by the mobile phase.
3. Recovery of the separated substance by continuous flow of mobile phase (elution).
4. Qualitative and quantitative analysis of eluted substances.

Classification of Chromatography
1. Classification on the basis of mobile phase
a. Gas Chromatography
i. Gas solid Chromatography(GSC)
ii. Gas liquid Chromatography(GLC)

b. Liquid Chromatography
i. Liquid – liquid Chromatography(LLC)
ii. Liquid – solid Chromatography(LSC)
iii. Ion – exchange Chromatography(IEC)
iv. Size – exclusion or Gel Chromatography

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 2. Classification on the basis of stationary phase
i. Planer Chromatography
ii. Column Chromatography

3. Based on the mode of separation

i. Adsorption Chromatography
ii. Partition Chromatography
iii. Ion exchange Chromatography
iv. Size – exclusion Chromatography

4. Various types of chromatographic Techniques:-

S.N Technique Stationary Phase Mobile Phase

1. Column Solid Liquid
Chromatography
2. Partition Liquid Liquid / Gas
Chromatography
3. Paper Liquid Liquid
Chromatography
4. Gas – liquid Liquid Gas
Chromatography
5. Gas – solid Solid Gas
Chromatography
6. Thin layer Solid Liquid
Chromatography
7. Ion – exchange Solid Liquid
Chromatography
8. Size Chromatography Liquid Liquid

MODES OF SEPERATION IN CHROMATOGRAPHY

i. Adsorption Chromatography
Based on adsorption equilibrium between solid stationary phase and liquid mobile
phase.
ii. Partition Chromatography
Based on partition equilibrium between liquid stationary phase and liquid / gas
mobile phase.

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iii. Ion exchange Chromatography
Based on ion exchange equilibrium between resin (stationary phase) and an
electrolytic mobile phase.

iv. Size exclusion Chromatography

Based on equilibrium between a liquid phase inside a porous structure or
molecular sieve (stationary phase) and liquid mobile phase.

Distribution coefficient / Partition coefficient

It is defined as the ratio of concentration of solute in stationary phase to the
concentration of the same solute in mobile phase.
CS
i.e KD =
CM
Where, KD = Distribution coefficient
CS = Concentration of the solute in stationary phase
CM = Concentration of the solute in mobile phase
It is temperature dependent constant.

Retention time (tR)

The time taken by a mobile phase to convey the solute from the point of injection through
column to the director is known as retention time (tR).

Dead volume (Vo)

It is the dead space, void volume or the hold by volume of the column i.e empty volume
of column.

Retention volume (VR)

It is the volume of mobile phase necessary to convey (carry) a solute band from the point
of injection through the column to the detector is called retention volume (VR).
VR = tR x FC
Where, tR = Retention time
FC = Volumetric flow rate

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 The flow rate (FC) in terms of column parameters is as follows,
Pi d2 L
FC = * * E total
4 to
2
Where, (Pi d / 4 ) = cross sectional area of the column
( L / to ) = average linear velocity
L = length of column
To = Dead Stop
E total = Total porosity of the column

Vcol * E total
Fc =
to
The average normal flow rate [U] of the mobile phase is L / t0
i.e U = L / t0

Adjusted Retention Volume (V’R)

It is the difference between retention volume and dead volume.
i.e V‟R = VR – V0
Average linear rate of migration of solute
V = L /tR, U = L/t0

Capacity factor / Retention factor (k)

It is related to the migration rate of solute and is defined by
K‟ = CS VS / CM VM
K‟ = KD * VS // VM eqn (1)
Where CS = Concentration of solute in stationary phase
VS = Concentration of solute in mobile phase
VS , VM = Volume of sample in stationary phase and in mobile phase
respectively
The volumetric phase ratio is often denoted by symbol “ Beta ”. Hence, equation (1)
can be written as:
K‟ = KD / “Beta”
Value of K‟is 0.5 < K< 20
When K‟ > 0.5, early eluting impurities are less like to overlap on analytic band.
When K‟ <20 , excessive broadening of the peak and too long run time or analysis time is
avoided.

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Selectivity factor / Relative retention (alpha)
The relative retention or selectivity factor (alpha) of two solute, where solute 1 elutes
before solute 2, is given by:
Alpha = K2‟ / K1‟ = KD2 / KD1 = tR2 / TR1 = VR2 / VR1
The relative retention is dependent on:
i) The nature of stationary and mobile phases.
ii) The column operating temperature.
The optimum value of „α‟ is between 1.05– 2.0. An increase in the value of „α‟ from
1.05 to 1.10 improves the resolution by four times for same value of N. (N = no. of
theoretical plates)
- When α is close to 1 it becomes impractical to operate because the column length
and column inlet pressure becomes difficult to achieve.
- If the value of α is above 2 the run time or analysis time become too long.

THEORIES OF CHROMATOGRAPHY
Two theories have been put forward regarding the rate of migration of solute and
development of peaks in chromatogram. They are:
1. Plate theory
2. Rate theory

1. Plate theory:
According to this theory, a chromatographic column consists of a series of discrete
continuous horizontal layers which are formed as theoretical plates. Equilibrium of
the solute between the stationary phase and the mobile phase takes place at each of
these plates. Migration of solute occurs by series of stepwise transfer between one
plate to other immediately below. The efficiency of separation in a
chromatographic column increases as the number of theoretical plates increases.
This is because the number of equilibration will also correspondingly increase.
The number of theoretical plates (N) is a measure of column efficiency. If the
length of column is “L” and equivalent height of a theoretical plate is “H”, then,
N=L/H
The height equivalent of a theoretical plate (HETP) is the height of layer of
column such that the solution leaving. The layer is in equilibrium with the average
concentration of the solute in the stationary phase throughout the layer.

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 2. Rate theory:
The rate theory is able to explain the effect of variables such as mobile phase
velocity and absorbability‟s which determine the width of an elution band. It
relates the effects of these variables on the time taken by a solute to elute at the
end of column. Migration of solute practices in the column occurs in a random
manner progressing in a stop and go sequence independent of any other molecule,
some molecules get immobilized temporarily on the column while others migrate
consequently; some molecules migrate rapidly where as other many lag behind.
Hence, width of zone increases as it migrates; the column zone width is directly
related to the residence or retention time on the column and inversely proportional
to mobile phase velocity.

SOURCES OF BRAND BROADENING

Theories of band broadening in liquid and gas chromatography are nearly identical. Plate
height expresses in simple terms the extent band broadening and factors affecting band
broadening. Chromatographic peaks are broadened by three kinetically controlled
processes – eddy diffusion, longitudinal diffusion and non-equilibrium mass transfer. The
magnitudes of these effects are determined by controlled variables such as flow rate,
practical size of packing, diffusion rate and thickness of stationary phase.

Van Deemter developed an equation that relates the efficiency of chromatographic

column to the extent to which this processes occur.
Van Deemter developed an equation for gas – liquid chromatography is:
H = A + (B/µ) + Cvd

a. Eddy diffusion :
Band broadening from eddy diffusion is the result of the multitude of pathways by
which a molecule can find its way through a packed column. The lengths of these path
ways differ. Thus, the residence time in the column for molecules are of the same
species are variable. Solute molecules thus do not emerge simultaneously from the
column and broadening of the elution band occurs.
The quantity A in Van Deemeter equation describes the effect of eddy diffusion and
can be related to practical size, geometry and tightness of packing of the stationary
phase.
The quantity can be minimized by uniform packing of small size particles in the
column.

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 b. Longitudinal diffusion:
Longitudinal diffusion results from the tendency of the molecules to migrate from the
concentrated centre part of the band towards more dilute regions on either side. This
type of diffusion can occur both in mobile and the stationary phase causing further
band broadening. Longitudinal diffusion is more important when the mobile phase is
a gas because diffusion rates in gases are several times greater than those in liquids.
The amount of diffusion increases with time. Thus, the extent of broadening increases
as the flow rate decreases. Longitudinal diffusion term, B is inversely proportional to
flow and is significant only at low mobile phase velocities.

c. Non – equilibrium mass transfer:

Chromatographic peaks are broadened because the flow of mobile phase is so rapid
that true equilibrium between phases cannot be reached. For example, at the front of
the zone, where the mobile phase encounters fresh stationary phase equilibrium is not
instantly achieved, and solute is hence carried farther down the column than would be
expected under true equilibrium conditions. Similarly at the end point of the zone,
solutes in the stationary phase encounter fresh mobile phases. The net effect is
broadening of peak at both ends of the solute band.
The effect of non equilibrium mass transfer becomes smaller as the flow rate is
decreased because more time is available for equilibrium to be approached. Further,
true equilibrium can be expected if the channels through which the mobile phase
flows are narrow so that the molecules cannot move far to diffuse in order to reach the
stationary phase.

Paper Chromatography

Cellulose filter paper is used as a stationary phase in paper chromatography. Since, it is

hydrophilic; it is usually covered with a film of layer. Hence, it is regarded as liquid –
liquid chromatography.
Special papers which are highly purified and reproducible as to porosity and thickness are
used. Such paper contains sufficient absorbed water; other liquids can be made to
displace the water, hence, providing a different type of stationary phase. For example,
paper treated with silicone or paraffin oil permits reverse phase chromatography. Special
papers containing an absorbent or an ion – exchange resin are also available to permit
absorption or ion exchange chromatography.

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Principle
It is a type of partition chromatography in which the substances are distributed between
two liquids i.e the stationary liquid (usually water) which held in the fibers of paper
called stationary phase and the other is the moving liquid or developing solvent called the
mobile phase. The components of the mixture to be separate migrate at different points
on paper.
In this technique, a drop of test solution is applied as a small spot on a filter paper and
then dried. The paper is kept in a close chamber and the edge of the filter paper is dipped
into a solvent, which is called the developing solvent, which rises up past the applied spot
by capillary action when the solvent has run its prescribed length.
The paper is removed, dried and the spots are rendered visible by appropriate visualizing
reagents or locating reagents. The movement of substances relative to the solvent is
expressed in terms of Rf values.

Migration Parameters:
The positions of migrated spots on the chromatogram are indicated by different terms
such as RF, RX and RM. These parameters are qualitative and characteristics of the
substance.

a. Retention Factor
Retention factor is related to the migration of the solute front relative to the solvent front
and is defined as:
Distance travelled by the solute front from the origin line
RF =
Distance travelled by the solvent front from the origin line

The RF value is a function of partition coefficient, it is constant for given substances,

provided the chromatographic conditions are kept constant with respect to temperature,
type of paper, duration and direction of development, nature and the shape and size of the
wide used in case of radial chromatography, the amount of liquid in the reservoir,
humidity etc.
The RF value defines the movement of the substance relative to the solvent front in a
given chromatographic system. The RF value of substance depends upon a number of
factors such as,
1. The solvent employed.

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 2. The medium used for separation i.e the quality of paper used in case paper
chromatography.
3. The nature of the mixture.
4. The temperature.
5. The size of the vessel in which the operation carried out.

b. RX:
In some cases, the solvent front runs off the end of the paper. The movement of substance
in such cases is expressed as RX but not RF.
Distance travelled by the substance from the origin line
RX =
Distance travelled by the standard substance from the origin line

c. RM:
The RM value is an additive value to which every functional group in the molecule
contributes and is a constant for a given solvent system and the type of paper used:
The RM values is defined as,
RM = log [(1/RF) – 1]

Types of paper chromatography:

i. Ascending paper chromatography
ii. Descending paper chromatography
iii. Ascending – Descending paper chromatography
iv. Radial or circular paper chromatography
v. Two dimensional paper chromatography

v. Two dimensional paper chromatography:

In this technique, a mixture is developed in one direction as in ascending
chromatography. After drying, the paper is again developed with another solvent in
direction at right angles to the first. This uses paper to maximum efficiency and allows
greater separation of the sample mixture. A spot is applied near one corner of the paper
which is rolled into a cylinder. After its first development, the cylinder is removed, dried
and again rolled into a cylinder such that the spots now will be developed at right angles

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to their previous direction of flow. After this development, the substances can be
identified by appropriate means.

Selection of filter paper

The choice filter paper depends upon whether it is being used for:
i. Quantitative or qualitative analysis
ii. Analytical or preparative chromatography
iii. Hydrophilic or lipophilic / neutral or charged species

The choice of filter paper depends upon the type of separation. Generally, coarser and
faster papers are used for separation of substance having sufficiently apart RF values.
Slow paper facilitate better resolution of substances and hence suitable for substances
having close RF values. Heavy papers like whatmann 3mm are used for preparative
purposes.
Whatmann filter papers used for chromatographic purposes have a content of 99 % of α-
cellulose or higher.

Characteristics of whatmann chromatographic papers.

For the efficient separation of polar substances, the exchange capacity of paper is
increased by increasing the carboxyl content (1- 4%) by partial oxidation.
The capillarity of the paper is increased by partial hydrolysis which is achieved by
soaking. The filter paper for 24 hrs in 7% HCl followed by washing with water of
ethanol.
Cellulose papers can also be used as a support for various absorbents like alumina, silica,
zirconium oxide etc, which gets precipitated in the pores of filter paper to produce a thin
sheet of this adsorbent with the flexibility of the paper but having adsorbent
characteristics of the precipitate. They can also be impregnated with powered or liquid
ion exchangers to produce ion – exchange papers.

Selection of developing solvent

The choice of developing solvent depends upon the simple fact that the RF values should
be different for different constituents present in a mixture.
The general criteria for a good solvent system are:
i. The RF value of the sample should lie between 0.05 and 0.85 in the system.
ii. The difference between the RF values of any two components must be 0.05, the
minimum values to separate any two components.

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 iii. The solvent should not undergo chemical reaction with any of the components
of sample mixture.
iv. The solvent should not interfere with the detection of the spots on the
developed chromatogram.
v. The composition of the solvent system should not alter with time.

Detection of spots
Two methods:
1. By chemical method
Chemical treatment is capable of imparting colour to colourless spots on the
paper. The reagents used for this purpose are known as visualizing reagents and
are applied by spraying the solution on the paper.
Some commonly used locating reagents are iodine, potassium permanganate,
dilute sulphuric acid, ninhydrine etc.

2. By physical methods:
Some colourless spots fluoresce under UV light and thus can be visualized.

Thin layer chromatography (TLC)

The technique of TLC is similar to paper chromatography in which the substances are
separated by the differential migration that occurs when a solvent flows along a thin layer
of fine powder coated on a glass or plastic plate. However, for most substances TLC
offers a faster and more efficient separation than paper chromatography and the majority
of paper chromatographic separations are superseded by the TLC procedure.

Advantages of TLC over paper chromatography

1. Short development time:
TLC requires less than an hour for good separation whereas paper chromatography
requires several hours to days.

2. Separation effects :
Separation by TLC is superior to paper chromatography.

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 3. Easy visualization of separated compounds:
Detection of Fluorescence compound is easier than on paper because the inorganic
background does not fluoresce.

4. Sensitivity:
TLC produces sharp and compact spots. The usual spry reagent can detect much
smaller quantities than with the more diffused by spots obtained in paper
chromatography. TLC is about10 – 100 times more sensitive than paper
chromatography.

5. Variable thickness of layers :

The thickness of adsorbent layer in TLC can be varied, thinner layer are used for
analytical separation and thicker for preparative separation which is not possible
with paper chromatography.

6. Chemical inertness of stationary phase:

The inert nature of stationary phase used in TLC affords vigorous means of
detection including application of heat and corrosive reagents like conc. sulphuric
acid which is not possible in paper chromatography.

7. Wide choice of stationary phase:

TLC may be employed for adsorption partition, ion exchange or molecular
exclusion.

Experimental technique:
1. Selection of coating material:
A large number of coating materials are produced commercially for TLC purpose.
The selection of coating material depends primarily on the nature of substances to
be separated and hence anode of separation. Some examples of coating material and
their mode of separation are:
S.N Coating Substance Mechanism / mode
of separation
1. Silica or alumina (activated) Adsorption
2. Diethylaminoethyl cellulose, polyphosphate Io exchange
cellulose
3. Cellulose or silica (inactivated) Partition

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 4. Paraffin oil or silicone oil coated on cellulose or Reverse – phase partition
silica
5. Sephandex Molecular exclusion
Other adsorbent used in TLC are calcium phosphate, magnesium tricillate,
polyamide, silica gel – alumina (1:1), acetylated cellulose, ferric oxide hydrate etc.

Generally, t adsorbents do not adhere satisfactorily to glass surface and hence

binders like gypsum, starch, hydrated silicon oxide etc. are added to the adsorbent.
Gypsum is the most widely used binder and when it is used with silica gel. It is
represented as silica gel G.

Generally, inert fluorescent indicator such as Zinc silicate is added to coating

material. It helps in detection of non – fluorescent but UV absorbing substances as
dark spots on green fluorescent background produced by this additive.

2. Preparation of TLC plates:

The next step is to apply a thin and uniform layer of coating material on a plastic or
glass plate. Applications are available for this purposes and the coating material is
applied in the form of a suspension or slurry in same liquid.

The various mode of preparing layers as follows:

i. Pouring :
Slurry is poured on the plate and then moved back and forth to spread the slurry
uniformly over the surface.

ii. Dipping :
Prepared by dipping the plate in chloroform or chloro – e – form methanol
slurries of the adsorbant.

iii. Spraying:
A small point sprayer is used for distribution of the slurry on the glass plate.

iv. Spreading :
Slurry is placed in an applicator which is moved over the stationary plate or it is
held stationary and the plate is pushed by or pulled through.

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 Instead of preparing the TLC plates manually ready to use precoated TLC plates can
be used. The thickness of precoated plates varies from 0.1 to 0.2mm.

3. Activation of plates:
After preparation of the plates, the liquid associated with the thin layer has to be
removed, this is done by drying the plates in an oven at 1100 C for 30 minutes.

4. Purification of silica gel G layer:

Silica gel G contains iron as impurity, which cause distortion of the chromatograph.
Hence, it is purified by preliminary development of the air dried plates with
methanol – conc Hcl. Iron migrates with the solvent front to the upper edge of the
plate. The plates are again dried and activated at 110 0 C.

5. Sample application:
Samples are applied to the plates by means of capillary tube for qualitative work but
must be applied by micro-syringe for quantitative analysis.

6. Selection of mobile phase

Appropriate mobile phase for the separation of the components of sample is selected
by hit trial taking into account the polarity of sample and mobile phase component.

7. Development of plate:
After application of sample, the plates are dried and then developed in a twin –
through chamber saturated with the appropriate mobile phase. The developing
chamber has to be saturated to avoid unequal solvent evaporation losses from the
edges of the TLC plate which results in lack of reproducibility in RF values called
the “edge effect”.

8. Detection of components:
The coloured components can be detected visually coloured spots where as the
colourless spots can be detected by physical and chemical methods similar to that of
paper chromatography.

However, even non – fluorescent and UV absorbing substances can be detected by

TLC unlike paper chromatography which detects only fluorescent compounds

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 provided some fluorescent substance like Zinc Silicate or Zinc Cadmium Sulfide is
mixed with the coating material. Such substances appear as dark spot against
fluorescent background.

Application of TLC
It can be used in the separation and purification of alkaloids, amino acids, antibiotics and
other drugs.
The various applications of TLC are as follows:
1.Purity of sample:
Purity of sample is routinely carried out. For this,direct comparison of the sample and authentic sample is
done; if purity is present,it shows extra spots and this can be detected very easily.
2.Examination of reaction:
The reaction mixture is examined by TLC to assess whether the reaction is complete or otherwise.This
method is also used in checking other separational processes and purification process like distillation,
molecular distillation.
3.Identification of compounds:
TLC is increasingly employed in isolation,purification and identification of various class of organic
compounds. In photochemistry it is used for identification of natural products like volatile oils, fixed
oils,waxes,terpeines, alkaloids, glycosides,steroids etc.

4.Biochemical analysis:
TLC is extremely useful in separation of biochemical metabolite or constituent from its body fluids,blood
plasma,serum,urine etc.
5.In chemistry:
TLC methodology is increasingly used in chemistry for the separation and identification of compounds
which are closely related to each other. It is also used for identification of cations and anions in
inorganic chemistry.
6.In pharmaceutical industry:
Various pharmacopoeias have adopted TLC technique for detection of impurity in a pharmacopoeial
drug or chemical. More than 130 drugs are tested by TLC methods for detecting impurity as per B.P.
7.Various drugs like hypnotics, sedatives, tranquillisers, anticonvulsants, antihistaminics, analgesics,
local anaesthetics, steroidal drugs have been tested qualitatively by TLC method.
8.One of the most important application of TLC is in separation of multicomponent pharmaceutical
formulation.

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9.In food and cosmetic industry,TLC method is used for separation and identification of
colors,preservatives, sweetening agents,and various cosmetic products.

APPLICATIONS OF HPTLC:
1.In pharmaceutical products:
HPTLC is used in analyzing the purity and efficacy of many pharmaceutical preparation and dosage
forms. Examples:HPTLC method was successfully used to analyze fixed dose tablets samples of
Lamivudine, nevirapine, stavudine.
HPTLC method used for analysis of paracetamol, diclofenac potassium,and famotidine both as bulk drug
and in tablet formulation.
It is useful for determining “uniformity of content” test,which is prescribed for drug formulation with a
small quantity of the drugs.

2.In natural products:

HPTLC not only confirm but also establish the identity of natural products.It is also an ideal screening
tool for adulterations and is highly suitable for evaluation, monitoring,harvesting,extraction
processes,and testing of stability.
3.In forensic science:
HPTLC is useful in detecting chemicals of forensic concern,including abuse drugs, poisons, adulterations,
chemical weapons,and illict drugs.
4.HPTLC is used for purity,control of chemicals,pesticides, steroids,and water analysis.
5.It is widely used for analysis of vitamins,water soluble food dyes,pesticides in fruits,vegetables,and
other food stuffs.
-
-

Problems in TLC
 Over – large spots:
Sample spots made using TLC capillaries should be no larger than 1- 2 mm in
diameter, because component spots in the developed plate will be no smaller than,
and will usually be larger than 2mm in diameter, then components with similar RF
values may not be resolved because their spots will be so large that they will overlap
considerably and may appear to be one large spot. Small initial spots on the other
hand, maximize the potential of complete separation of components.

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  Uneven Advance of solvent Front:
A common problem in TLC is uneven advance of solvent along the plate. Instead of
a straight line, the solvent front may appear to bow either up or down in the center.
Uneven advance of solvent leads to uneven advance of substance spots, and
inaccurate RF values result. A frequent cause of uneven solvent advance is the use
of a developing chamber that does not have flat bottom. Glass bottles usually have
bottoms that curve upward from the edge to the center. If the bottom of the TLC
plate is placed on this curved surface, the shape of the solvent advance line may
mirror the shape of container bottom. It is therefore important to use flat bottomed
developing tanks TLC. A bowed solvent front may also result if too little developing
solvent is placed in the chamber, if the plate is cut improperly, so that the sides are
not exactly perpendicular to the bottom edge, and if the slide is excessively tighter
in the chamber. Care in choosing and using a developing chamber is the best
defense against the curved solvent front.
Water is seldom used as a developing solvent because it has a tendency to produce a
dramatically curved front. This may be due to its unusually high surface tension.

 Streaking :
Sometimes a substance will move along a TLC plate as a long streak, rather than as
a single discrete spot. This is the result of spotting the plate with too much
substance, more than the moving solvent can handle the solvent moves as much
substance as it can, but a substantial amount of substance is left behind. The
substance is dragged along by the solvent leaving a trial of the substance that may
sometimes span the entire distance between the starting line and the solvent front
streaking can be eliminated by systematically diluting the spotting solution until
development and visualization show the substances moving as single spots, rather
than elongated streaks.

Ion –Exchange Chromatography:

The principle underlying ion – exchange chromatography is the attraction between

oppositely charged particles. Many compounds have ionizable groups and hence they
carry net positive or negative charge which can be utilized in separating mixtures of such

17
compounds. The net charge exhibited by such compounds is dependent on their PKa and
pH of the solution.
Ion exchange separations are carried in columns packed with an ion exchanger. There are
two types of ion exchangers: Cation and Anion exchangers. Cation exchanger posses
negatively charged groups and they attract positively charged species. These exchangers
are also called acidic ion exchange materials, since their negative charges result from the
photolysis of acidic groups. Anion exchanger posses positively charged groups and they
attract negatively charged species. These exchangers are also called basic ion exchange
materials since positive charge results from the association of protons with the basic
groups.
The capacity of an ion to undergo exchange depends upon the charge and the size of the
hydrated ion in the solution. Under, similar conditions, the capacity increases with the
increase in the charge on the ion and decrease in the size of hydrated ion. The ion
exchange affinity for exchangers is as follows:
1. The ion with the highest charge has the highest affinity:
i.e Na+ < Ca2+ < Al3+ < Th4+ ( for cation exchangers)
& I- < SO42- < PO43- (for anion exchangers)

2. The ion with the greatest size (i.e least hydrated ions ) and charge has the highest
affinity
i.e Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ < Sn2+ (for cation exchanger)
& F- < OH- < Cl- < CN- < Br- < NO3- < I- < SO42- < PO43- (for anion exchangers)

Cation exchangers
Cation exchanger is a high molecular weight, cross – linked polymer having sulfonic,
carboxylic, phenolic etc group as an integral part of the resin and an equivalent amount of
cation. Thus, a cation exchanger is a polymeric anion to which active cations are
attached. In cation exchangers, the hydrogen ion are mobile and exchangeable other
cations which the anions ( - COO- , - SO3- & O- ) remains attached to the resin network.
The general exchange reaction for cation exchanger can be represented as:
HnR + Nm+ MnR + nH+

Sulfonate exchanges are strongly acidic exchangers and are completely dissociated. Their
exchange properties are independent of the pH of the mobile phase. Hence, they are
strong cation exchangers.
e.g: sulfonated polyster resins.

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A carboxylate group carried by an exchanger permits the exchange of cationic species
only when the pH is sufficiently high to permit dissociation of the carboxylic group.
Hence, they are weak cation exchangers.
e.g:carboxylic polymethacrylate.

Anion – exchangers
An anion exchanger is a polymer having amine or quaternary ammonium exchanger is a
polymer having amine quaternary ammonium groups as integral part of the resin and an
equivalent amount of anions such as Cl-, OH- ions etc. These anions are mobile and
exchangeable. The anion exchange behavior of such exchanger may be represented as,

R(Cl) + nX- RXn + nCl-

Quaternary ammonium exchangers are strongly basic and are completely dissociated.
Their exchange property is independent of the pH of the mobile phase. Hence, they are
strong anion exchangers. E.g : Quaternary ammonium polystyrene.
A tertiary amine exchanger can exchange anion only in acidic medium and hence they are
weak anion exchanger E.g Polyamine polystyrene

The efficiency of the resin depends on the degree of cross linking. Greater the cross –
linking, higher the efficiency of the resin.

Ion – exchange capacity:

The exchange capacity of a cation – exchange resin is found by determining the number
of milliequivalents of Na+ ion which are exchanged by 1 gm of the dry resin in the
hydrogen form.
A solution of NaCl known concentration is passed through a column containing a known
weight of the resin and the acid eluting form the column is collected and titrated with the
standard solution of alkali. If V ml of an alkali solution of strength N in milliequivalents
per litre is required to titrate all the acid eluted from the resin weighing e.g., then,

Ion exchange capacity = (V * N) / W

Similarly, the exchange capacity of anion exchangers is determined by passing a solution

of sodium nitrate of known concentration through a column. The concentration of the Cl-

19
or OH- ion eluting from the column is found by titration with a standard solution of silver
nitrate or acid respectively.

APPLICATION OF ION – EXCHANGE CHROMATOGRAPHY

Ion – exchange chromatography can be used for the determination of pharmaceuticals
which are in ionic or salt forms such as:
- Determination of ephedrine sulphate
- Determination of diphenylhydration sodium capsules
- Separation of amino acids etc
It is used in the demineralization of water.
Ion exchange has wide range of application.some of them are given below:
1. Separation of similar ions:
The ion exchange chromatography is used for separation of similar ions ,as different ions
undergo exchange reactions to different extents e.g a mixture of Na+,H+,K+, can be
separated by using cation exchange resin.
2. For the separation and purification of many charged or ionizable molecules such as
proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc. from natural
sources or synthetic origin.
3. Most typical example of application is preparation of high purity water for power
engineering, electronic and nuclear industries; i.e. polymeric or mineralic insoluble ion
exchangers are widely used for water softening, water purification, water
decontamination, etc.
4. Application for ion exchange in domestic water treatment is the removal of nitrate and
natural organic matter.
5. Ion-exchange processes are used to separate and purify metals, including separating
uranium from plutonium and other actinides.
6. Ion exchangers are used in nuclear reprocessing and the treatment of radioactive waste.
7.

-
-

20
 GEL CHROMATOGRAPHY OR SIZE –
EXCLUSION CHROMATOGRAPHY

Exclusion chromatography is based on the ability of controlled porosity substrates for

sort and separate sample mixtures according to the size and shape of the sample
molecules. Compounds can be separated according to their molecular size by exclusion
chromatography, which is based on the diffusion or permeation of solute molecules into
the inner pores of the column packing. The shape and size of the molecules to be
separated govern their ability to enter the pore. The smaller molecules entering the pore
without the hindrance are the last to be eluted. Molecules that are too large enter the
pores are completely excluded and travel with the solvent system. Between these two
extremes, intermediate size molecules can penetrate some passage but not others and
consequently are retarded in their progress down the column and exit at intermediate
time. The selection of column packing, each with its corresponding exclusion limit,
permits separation of substances of wide molecular size ranges.

Column packings:
Column packings are either semi-rigid, cross – linked macromolecular polymers or rigid,
controlled pore size glasses or silica. The semi-rigid materials swell slightly and they can
withstand maximum pressure up to 300psi owing to bed compressibility.
The most widely used polymer is dextran (sephadex), which is formed by cross linking of
polysaccharide dextran with epichlorohdrin. The exclusion limits of sphadex varies
depending on the pore size which can be controlled by changing the amount of
epichlorydrin incorporated. The exclusion limits for various grades of sephadex are as
follows:

Type of sephadex Exclusion limit

3500 – 4500
3000 – 1000
40000 – 50000
100000
200000
21
Polyacryalimide polymer (Bio-Gel) formed by cross-linking of polyacrylamide with
methylene bis-acrylamine is another semi-rigid gel available in various grades p2, p6, p30,
p100 and p300 .

Styrene-divinylbenzene polymers (ultrastyragel) permits traditional of molecular weights

ranging from 100-500 milion Da.
Partially sulfonated polystyrene beads are compatible with aqueous systems and the non
– sulfonated with non aqueous systems.

Hydrophilic packing using polymer of 2-hydroxyethylmethacrylate and ethylene

dimethacrylate can withstand pressures up to 3000 psi and can be used with aqueous
systems as well as variety of polar organic solvents.

Porous glasses and silica cover a wide range of pore size diameters and can withstand
high pressure. These packings are chemically resistant at PH values less than 10 and can
be used with aqueous and polar organic solvents. With non polar solvents, the surface has
to be deactivated by silylation to avoid irreversible

Column Chromatography
Column Chromatography is the basic type of chromatographic procedure which was
developed during early stages of chromatography development. Column chromatography
basically is a type of adsorption chromatography technique. The separation of
components depends upon the extent of adsorption of component on the stationary phase.

Principle
When aqueous mixture of mobile phase and sample to be separated are introduced from
top of the column, the individual components of mixtures move with different rates.
Those with lower affinity and adsorption to stationary phase move faster and eluded out
first while those with greater affinity to stationary phase move slower and slower and
eluted out last.
The solute molecules absorb to the column in a reversible manner. The rate of movement
of the component is given as follows:

22
Selection of material:
The column chromatography requires a vertical column (preferably glass column) with a
knob at the bottom end. This is preferably a burette shaped cylindrical column without
graduation or readings.
Mostly used adsorbent material for stationary phase is silica gel. The particles of
stationary should be of uniform size and shape without contamination. Mobile phase,
preferably solvents of chromatography grade, either a single solvent or a mixture of
solvents are required for the separation.
Cotton used or asbestos pad, to plug the exit of column of the bottom and thereby hold
the column of stationary phase and let only escape of solvent and sample.

Preparation of column and working:

The stationary phase material is suitably moistened with mobile phase and packed
sufficiently in the column with a cotton or asbestos pad at the bottom. The extent material
or sample to be separated is placed on the top of packed stationary phase. The mobile
phase is poured into the column over the sample. A collecting beaker is placed at the
bottom of column near the end to collect the eluent.

Applications
i. Column chromatography is best suited to separate active principle front plant
material.
ii. In separation of compounds after organic synthesis to obtain desired molecule.
iii. To separate or purify natural compound mixture like alkaloids glycerides.

23
24
Introduction:
Gas chromatography is the technique of choice for separation of thermally stable and
volatile organic and inorganic compounds. It may subdivided into:
 Gas –liquid chromatography (GLC) accomplishes the separation by partitioning the
components of a chemical mixture between a moving (mobile) gas phase and a
stationary liquid phase.
 Gas –solid chromatography (GSC) having solid adsorbent as the stationary phase.

Principle:
A gaseous mobile phase flows under pressure through a heated tube either coated with a
liquid stationary phase or packed with liquid stationary phase coated onto a solid support.
The analyte is loaded onto the head of the column via a heated injection port where it
evaporates. It then condensed at the head of the column, which is at a lower temperature.
The oven temperature is then either held constant or programmed to rise gradually. Once
on the column separation of a mixture occurs according to the relative lengths of time
spent by its components in the stationary phase. Monitoring of the column effluent can be
carried out with a variety of detectors.

Application:
 The characterization of some unformulated drugs, particularly with regard to
detection of process impurities.
 Limit tests for solvent residues and other volatile impurities in drug substances.
 Sometimes used for quantification of drugs in formulations, particularly if the drug
lacks a chromophore.
 Characterization of some raw materials used in synthesis of drug molecules.
 Characterization of volatile oils ( which may be used as excipients in
formulations), proprietary cough mixtures and tonics, and fatty acids in fixed oils.
 Measurement of drugs and their metabolites in biological fluids.

Limitation:
 Only thermally stable and volatile compounds can be analyzed.
 The sample may require derivatisation to convert it to a volatile form, thus
introducing an extra step in analysis and, potentially, interferants.
 Quantitative sample introduction is more difficult because of the small volumes of
sample injected.
 Aqueous solution and salt cannot be injected into the instrument.

25
Instrumentation:
A gas chromatography essentially comprises of following vital components, namely:
a) Carrier gas regulator and flow meter,
b) Sample injection system
c) Separation column
d) Thermal compartment
e) Detectors,
f) Recording of signal current and
g) Integrator

Fig: schematic diagram of gas chromatography

Fig: Block diagram of GC

26
All the following conditions should be achieved for typical analysis:
 Constant flow of carrier gas.
 The introduction of sample vapor into the flowing gas stream.
 Appropriate length of stationary phase.
 Maintaining the column at the appropriate temperature.
 Detecting sample components as they eluted from the column.
 Readable proportional to the amount of each component.

Carrier gas:
The purpose of the carrier gas is to transport the sample through the column to the
detector. The carrier gas must be chemically inert. Commonly used gases include
nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependent
upon the type of detector which is used. The carrier gas system also contains a molecular
sieve to remove water and other impurities. Selecting the proper carrier gas is very
important because it affects both column and detector performance. The purity of the
carrier gas should be at least 99.99%. Impurities such as oxygen or water can cause
column and detector deterioration.

27
Sample introduction and injector system:
Sample introduction by syringe: The volumes injected in GC are routinely in the range
of 0.5 – 2 µL. the most commonly used type of syringe is shown in figure below. The
usual syringe volumes are 5 and 10 µL. a recommended technique for injection into a
capillary GC is to fill the syringe with about 0.5 µL of solvent and draw this solvent into
the barrel slightly before filling with sample. The sample is also drawn into the barrel to
leave an air gap below it. The syringe needle can then be introduced into the injector and
left for a couple of seconds to warm up before the plunger is depressed. The syringe is
then withdrawn immediately from the injection port.

Fig: typical micro syringe used in GC

Sample injection port (split /split less injector):

Fig: a) above left, injection chamber. The carrier gas enters the chamber and can leave by
three routes when the injector is in split mode. A portion of carrier gas (1) flows upward
and purges the septum, another (2) exits through the split outlet (a needle valve regulates
the split) and (3) finally a proportion passes onto the column. (b) Above right, cold
injection onto the column.

28
The injector can be used in one of two modes i.e. split or splitless. The injector contains a
heated chamber containing a glass liner into which the sample is injected through the
septum. The carrier gas enters the chamber and can leave by three routes (when the
injector is in split mode). The sample vaporizes to form a mixture of carrier gas,
vaporized solvent and vaporized solutes. A proportion of this mixture passes onto the
column, but most exits through the split outlet. The septum purge outlet prevents septum
bleed components from entering the column.

Column used in GC:

29
30
31
Here,
 Wall-coated open tubular column (WCOT): Liquid stationary phase on inside wall
of column.
 Support-coated open tubular column (SCOT): liquid stationary phase coated on
solid support attached to inside wall of column.
 Porous-layer open tubular column (PLOT): stationary phase on inside wall of
column.

32
Detector:
 A detector located at the exit of the separation column senses the presence of the
individual components as they leave the column.
 The detector volume must be small to prevent the remixing of components separated
on the column.
 The electrical analog output of the detector is amplified and then sent directly to a
strip chart recorder or converted to a digital signal and sent to a micro computer
system.

33
Classification of detectors:
a. Classification according to selectivity:
 A non-selective detector: responds to all compounds except the carrier gas.
 A selective-detector: responds to a range of compounds with a common
physical or chemical property.
 A specific detector: responds to a single chemical compound.

b. Classification according to destructive ability:

 Concentration dependent detectors: The signal from a concentration
dependent 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 dependent 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.

Some examples of detector:

1. Thermal conductivity detector (TCD): The detector consists of four identical
filaments, which make up the arms of a whetstone bridge. Two filaments are used as
references and are always immersed in the pure carrier gas. The other two, placed at
the exhaust of chromatographic column, detect the presence of sample component in
the carrier gas stream by a change in the thermal conductivity of the carrier gas and
sample mixture. A current passed through the bridge from a power supply causes the
filaments to heat up. When exposed to a pure carrier gas flow, the filaments soon
reach temperature equilibrium and a constant resistance, causing the bridge to be
electrically balanced. In this balanced condition there is no output signal, and hence
the recorder draws a straight base line. When a component of the sample is carried
past one of the filaments, the now changed thermal conductivity of the gas causes the
filament to either gain or lose heat. As the temperature of the filament change, the
resistance of the filament changes also, causing the bridge to become unbalanced and
a signal to be sent to the recorder.

34
Fig: a. thermal conductivity detector Fig: b circuit diagram of TCD

2. Flame ionization detector (FCD):

In this detector, the effluent from the column is mixed with H2 and air, and ignited.
Organic compounds burning in the flame produces ions and electrons which can
conduct electricity through the flame. A large electrical potential is applied at the
burner tip, and a collector electrode is located above the flame. The current resulting
from the pyrolysis of organic compounds is measured. FIDs are mass sensitive rather
than concentration sensitive, this gives the advantage that changes in mobile phase
flow rate does not affect the detector‟s response. The FID is a useful general detector
for the analysis of organic compounds; it has high sensitivity, a large linear response
range, and low noise. It is also robust and easy to use, but unfortunately, it destroys
the sample. The FID responds proportionately to the number of –CH2- groups
introduced into the flame, for example, the response to an equimolar amount of
butane is twice that to ethane. There is no response from fully oxidized carbons such
as carbonyl or carboxyl groups. If desired CO and CO2 can easily be converted to
CH4 by reduction with H2 over a nickel catalyst and then measured by the detector.

35
3. Nitrogen phosphorous detector (NPD):
Compared with the FID, this thermionic detector has a smaller flame in which the
catalytic decomposition of compounds containing nitrogen (N), or phosphorus (P)
yields, fairly specifically, negative ions which are received by a collector electrode.
As it appears on the representation of above fig (b), it comprises a small ceramic
cylinder doped with an alkaline salt (e.g. rubidium sulfate). A voltage is applied to
maintain small plasma (8000C) through the combustion of an air/hydrogen mixture. In
these conditions the nitrogen present in air does not yield ions. Detector sensitivity is
typically between 0.1 and 0.4 pg/s for nitrogen- or phosphorus-containing analytes,
with a linear range of five orders of magnitude.

4. Electron capture detector (ECD):

This selective detector is considered to be excellent for trace analysis when analytes
contain halogen atoms or nitro groups. A flow of nitrogen gas which has been ionized
by electrons generated from a low energy β-radioactive source passes between two
electrodes maintained at a voltage differential of around 100V. at equilibrium, a base
current I0 is generated, mainly due to free and very mobile electrons. If molecules
(M), containing an electrophore such as halogen (F,Cl,Br), cross the zone between the
two electrodes, they capture thermally excited electrons to form heavy negative ions,
which by consequence are much less mobile, leading to a decrease in the signal.


N 2  N 2  e 
M  e  M 
M   N 2  M  N 2

36
 The measure intensity decreases exponentially by following a law of type I=I 0 exp
[-KC].this non-destructive detector, well suited for compounds with high electron
affinity, is mainly used for analyses of chlorinated pesticides.

Fig: electron capture detector (ECD)

5. Photo- ionization detector (PID):

This detector is fairly selective but it has only a narrow range of application,
convenient for hydrocarbons as well as for sulphur or phosphorus derivatives. The
operating principle consists to provoke ionization of the analytes by irradiation with a
UV lamp emitting photons of high energy (of 8.4 to 11.8 eV). The photo-ionization
occurs when the energy of the photon is greater to that of the first ionization of the
compound. A photon of 9.6 eV can, for example, ionize benzene (E1=9.2 eV) but not
isopropanol (E1= 10.2 eV). Electrons are collected by an electrode linked to an
electrometer.
This detector can function at more than 4000C and is not destructive since the
ionization is reversible and affects only a small fraction of the molecules of each
compound passing through. ECD and PID are examples of class-specific detectors
often used in trace environmental analysis.

Fig: Photo- ionization detector

37
 The PID contains a UV source from which the photons are emitted, having a pre-
selected energy, using a filter which prevents undesired carrier gas ionization. On
contact with the electrodes the molecules return to uncharged state, ionization being
therefore reversible.

6. Sulphur chemiluminescence detector (SCD):

7. Flame photometric detector (FPD):

38
39
GC instruments:

40
41
 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
High performance liquid chromatography is basically a highly improved form of column
chromatography. Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressure of up to 400 atmospheres, which makes
it much faster.

It also allows using a very smaller particle size for the column packing material which
gives a much greater surface area for interactions between the stationary phase and the
molecules flowing past it. This allows a much better separation of the components of the
mixture.

The other major improvement over column chromatography concerns the detection
methods which can be used. These methods are highly automated and extremely
sensitive.

Types of HPLC

42
Depending upon the polarity of stationary phase and mobile phase there are two types of
HPLC i.e.
1. Normal phase HPLC
2. Reversed phase HPLC

1. NP-HPLC
The column is filled with tiny silica particles (polar), and the solvent is non-polar (for
example- hexane). A typical column has an internal diameter of 4.6 mm (and or may be
less than that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will stick longer to the
polar silica than non-polar compounds will. The non-polar ones will therefore pass more
quickly through the column.

2. RP-HPLC
In this case, the column size is the same, but the silica is modified to make it non-polar by
attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon
atoms in them. A polar solvent is used - for example, a mixture of water and an alcohol
such as methanol.
In this case, there will be a strong attraction between the polar solvent and polar
molecules in the mixture being passed through the column. There won't be as much
attraction between the hydrocarbon chains attached to the silica (the stationary phase) and
the polar molecules in the solution. Polar molecules in the mixture will therefore spend
most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon
groups because of Van Der Waal‟s dispersion forces. They will also be less soluble in the
solvent because of the need to break hydrogen bonds as they squeeze in between the
water or methanol molecules, for example. They therefore spend less time in solution in
the solvent and this will slow them down on their way through the column. This means
that now it is the polar molecules that will travel through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.

PRINCIPLE: A liquid mobile phase is pumped under pressure through a stainless steel
column containing particles of stationary phase with a diameter of 3-10µm. The analyte
is loaded onto the head of the column via a loop valve and separation of a mixture occurs
according to the relative lengths of time spent by its components in the stationary phase.
It should be noted that all components in a mixture spend more or less the same time in

43
the mobile phase in order to exit the column. Monitoring of the column effluent can be
carried out with a variety of detector.

APPLICATIONS
 The combination of high-pressure liquid chromatography (HPLC) with monitoring
by UV/visible detection provides an accurate, precise and robust method for
quantitative analysis of pharmaceutical products and is the industry standard
method for this purpose.
 Monitoring of the stability of pure drug substances and in drugs in formulations
with quantification of any degradation products.
 Measurement of drugs and their metabolites in biological fluids.
 Determination of partition coefficients and pKa values of drugs and of drug
protein binding.

LIMITATIONS
 There is still a requirement for reliable and inexpensive detectors which can
monitor compounds that lack a chromophore.
 Drugs have to be extracted from their formulations prior to analysis.
 Large amounts of organic solvent waste are generated, which is expensive to
dispose.

INSTRUMENTATIONS
Modern HPLC essentially comprises of the following main components namely:
a. Solvent reservoir and degassing system
b. Pressure, flow and temperature
c. Pumps and sample injection system
d. Columns
e. Detectors
f. Strip-chart recorder and
g. Data handling device and microprocessor control

44
Fig: Schematic Modular Structure of HPLC

45
 Fig: Flow Diagram of HPLC

a. Solvent reservoir and degassing system:

Mobile phase consisting of a mixture of organic solvents or an aqueous-organic mixture
or a buffer-solution may be employed depending upon the chromatographic method and
the detector to be used. Special grades of solvents are commercially available for HPLC
that have been adequately refined to eliminate completely the UV-absorbing impurities
and any particular matter. In case, other grades of solvent are employed, purification may
have to be done at all cost because impurities present would, if are strong UV-absorbing,
affect the detector or, if of higher polarity (e.g. traces of H2O or EtOH, commonly
included as a stabilizer, in CHCl3), influence the separation.
Solvent reservoir comprise of a 1 dm3 glass bottle having a lid and a 1/8 inch diameter
ptfe-tube to convey the mobile phase from the reservoir to the degassers and then to the
pump. As described above, any liquid entering the pump should be free from dust and
particulate matter; otherwise these foreign substances may invariably give rise to
irregular pumping action, damage seals and valves, irregular behavior of column owing
to its contamination, and ultimate blockade of column. Sometimes a stainless steel filter
element (filter size 2 m) that could be conveniently positioned either in the ptfe-tube in
the reservoir or an in-line filter may be employed.

46
Degassing: Many liquids dissolve appreciable amounts of atmospheric gases, e.g. air or
suspended air-bubbles that may be a major cause of practical problems in HPLC,
specifically affecting the operation of the pump and the detector. However, all such
problems may be avoided by degassing the mobile phase by subjecting the mobile-phase
under vacuum distillation, spurge with a fine spray of an inert gas of low solubility such
as Argon or Helium or by heating and ultrasonic stirring.

b. Pressure, flow and temperature:

Pressure: HPLC column are packed usually up to 700 times atmospheric pressure and
therefore, the operating inlet column pressure in HPLC may be to a maximum of 200
times atmospheric pressure.
Hence, 1N atmospheric pressure = 105 Pascal
Where,
1Pa = 1Nm-2 and 1bar= 105 Pascal =14.5 psi (pounds per square inch)
Flow: The flow can be measured periodically at the column outlet by collecting the
liquid for a known period, and thereafter, either measuring the volume or weighing it
physically.
Temperature: In reality, the maintenance of strict temperature control plays a vital role
in measuring the retention data correctly and precisely. It makes use of the refractometer
detectors specifically. In HPLC, difficult separations may be achieved by increasing the
temperature carefully, but this must be done initially on a hit and trial basis.

c. Pump and sample injection system:

Pumps: The two major functions of the pump in a modern HPLC are, namely:
a. To pass the mobile phase through the column at a high pressure and
b. To pass the mobile phase at a constant controlled flow rate.
The types of pumps used for HPLC can be divided into two categories: constant-pressure
pumps (e.g.: the inexpensive gas-displacement pump) and the constant-volume pumps
(e.g.: the reciprocating and syringe pumps). The most commonly used pumps in HPLC
are the single- or multi-head reciprocating type. The former delivers the flow as a series
of pulses which must be damped using a pulse dampener; dual-and triple-head
reciprocating pumps can be operated without a pulse dampener since they minimize
pulsation, but are more expensive than singe-head pumps.

47
 Fig: schematic diagram of a dual- headed reciprocating pump.

Sample injection system: In HPLC, the injection of a precise volume of sample onto the
head of the column must be made as fast as possible in order to cause the minimum
disturbance to the dynamic regime of the mobile phase whose flow must be stable from
column to detector. This is done by a special high pressure valve, either manual or
motorized, possessing several flow paths, which is situated just prior to the column (fig:
3.4). This must be a component of precision able to resist pressures greater than 30000
KPa. The valve functions in two positions:

48
In load position: In this position, only communication between the pump and the
column is assured. The sample, contained in a solution, is introduced at atmospheric
pressure with the aid of a syringe into a small tubular curved section named a loop. Each
loop has a small defined volume. They are either integrated into the rotor of the valve or
are connected to the outside of the valve‟s casing.

In the inject position: In this position, the sample which is in the loop is inserted into the
flow of the mobile phase by the 600 rotation of a part of the valve, thus connecting the
sample loop to the mobile phase circulation. Highly reproducible injections art attained
only if the loop has been completely filled with the sample.

49
 Fig: schematic (flow) diagram of sample injection system

d. Column used in HPLC:

The columns most commonly used are made from precision-bore polished stainless steel
tubing, typical dimension being 10-30cm long and 4 or 5 mm internal diameter. The
stationary phase or packing is retained in the column by thin stainless steel frits at each
end having a mesh of 2 m or less.

50
 Columns are expensive and easily degraded by
dust or particles in the sample or solvent and by
irreversible adsorption of impurities from the
sample or solvent. Therefore, the entrance to the
main column is protected by a short guard
column containing the same stationary phase as
the main column.

Fig: HPLC column with replaceable guard column

Types of packing: Modern HPLC makes use of packing which essentially consist of
small and rigid particles with a very narrow particle size distribution. Broadly speaking
three types of packing are invariably used in HPLC column, namely:
i. Porous, polymeric beads based on styrene-divinylbenzene copolymers: These are
used for ion exchange and size exclusion chromatography but have been replaced for
many analytical applications by silica-based packing which are more efficient and
mechanically stable.
ii. Porous-layer beads: (diameter 30-55 m) consisting of a thin shell (1-3 ) of silica,
or modified silica or other material, on an inert spherical core (e.g. glass beads). These
pellicular-type packing are still used for some ion exchange applications, but their
general use in HPLC has declined with the development of totally porous
microparitculate packing.

51
iii. Totally porous silica particles: (diameter less than 10 m, with narrow particle size
range) are now the basis of the most commercially important column packings for
analytical HPLC. Compared with the porous-layer beads, totally porous silica
particles give considerable improvements in column efficiency, sample capacity, and
speed of analysis.

Silica gel: Silica gel is a very polar material corresponding to a three-dimentsional

network. Though it does not possess the well-ordered structure of crystalline silica, it
does maintain the tetrahedral arragement of four bonds around the silicon atom. This is a
reticulated inorganic polymer as shown in fig below. It contains a variable number of
silanol groups.

52
The preparation of silica gel in the form of irregular grains; used in preparative
chromatography, proceeds in a different fashion. First, orthosilicic acid is formed which
comprises the unstable [Si(OH)4], by acidification using sodium silicate [Na2SiO3]
obtained from definite purified sands by alkaline fusion. This ustable orthosilicic acid
initially dimerizes then poly-condenses progressively to colloidal particles having a
hydroxylated surface. Through aggregation a hydrogel of gelatinous silica is obtained, the
calcinations of which yields dense grains of silica gel (xerogel).

Bonded silica:
Monomeric phases (10-15 m thickness): They are obtained by reaction of an alkyl-
monochlorosilane in the presence of an alkaline agent with surface silanol groups. The
RP-8 (dimethyloctylsilane) or RP-18 ( dimethyloctadecylsilane groups, ODS) are
prepared in this way. However a part of the Si-OH groups remain intact and can be the
origin of interfering polar interactions. A more complete reaction is obtained with other
silyl reactants such as chlorotrimetylsilane (ClSiMe3) or hexamethyldisilazane
(Me3SiNHSiMe3). The sits that remain non-transformed are usually as inaccessible to
the reactants as they are to the analytes.

53
Polymeric phases (25 m or greater thikness): Here a di- or trichlorosilane is used in
the presence of water vapour which provokes a polymerization of the reactiant in solution
prior to deposit and bonding with the silica. A reticulated polymer layer is obtained. At
the molecular scale the final framework or the coating is difficult to imagine: it is mono-
or multilayer.

e. Detectors used in HPLC:

The function of the detector in HPLC is to monitor the mobile phase as it emerges from
the column. The detection process in liquid chromatography has presented more
problems than in gas chromatography; there is, for example no equivalent to the universal
flame ionization detector of gas chromatography for use in liquid chromatography.
Suitable detectors can be broadly divided into the following two classes:
i. Bulk property detectors: They measure the difference in some physical property
of the solute in the mobile phase compared to the mobile phase alone, e.g.
refractive index and conductivity detectors. They are generally universal in
application but tend to have poor sensitivity and limited range. Such detectors are
usually affected by even small changes in the mobile-phase composition which
precludes the use of techniques such as gradient elution.
ii. Solute property detectors: e.g. specytrophotometric, fluorescence and
electrochemical detectors. These respond to a particular physical or chemical
property of the solute, being ideally independent of the mobile phase. In practice,
54
 however, complete independence of the mobile phase is rarely achieved, but the
signal discrimination is usually sufficient to permit operation with solvent
changes, e.g. gradient elution. They generally provide high sensitivity (about 1 in
109 being attainable with UV and fluorescence detectors) and a wide linear
response range but, as a consequence of their more selective natures, more than
one detector may be required to meet the demands of analytical problem. Some
commercially available detectors have a number of different detection modes built
into a single unit, e.g. the Perkin-Elmer‟3D‟ system which combines UV
absorption, fluorescence and conductimetric detection.

Some of the important characteristics required of a detector are the following:

a. Sensitivity: It is often expressed as the noise equivalent concentration, i.e. the
solute concentration, Cn, which produces a signal equal to the detector noise level.
The lower the value of Cn for a particular solute, the more sensitive is the detector
for that solute.
b. A linear response: The linear range of a detector is the concentration range over
which its response is directly proportional to the concentration of solute.
Quantitative analysis is more difficult outside the linear range of concentration.
c. Type of response, i.e. whether the detector is universal or selective. A universal
detector will sense all the constituents of the sample, whereas a selective one will
only respond to certain components. Although the response of the detector will not
be independent of the operating conditions, e.g. column temperature or flow rate,
it is advantageous if the response does not change too much when there are small
changes of these conditions.
A summary of these characteristics for different types of detectors is given in table
below:

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Some detectors used in HPLC:
1. UV-detectors 2. Fluorescence detectors
3. Refractive index detector 4. Multipurpose detector

UV- detectors: The UV absorption detector is the most widely used in HPLC, being
based on the principle of absorption of UV visible light as the effluent form the column is
passed through a small flow cell held in the radiation beam. It is characterized by high
sensitivity (detection limit of about 1x10-9 gml-1 for highly absorbing compounds) and,
since it is a solute property detector, it is relatively insensitive to changes of temperature
and flow rate. The detector is generally suitable for gradient elution work since many of
the solvents used in HPLC do not absorb to any significant extent at the wavelength used
for monitoring the column effluent. The presence of air bubbles in the mobile phase can
greatly impair the detector signal, causing spikes on the chromatogram; this effect can be
minimized by degassing the mobile phase prior to use.

Fig: Schematic diagram of double beam of UV-Detector

Fluorescence detectors: These devices enable fluorescent compounds present in the

mobile phase to be detected by passing the column effluent through a cell irradiated with
ultraviolet light and measuring any resultant fluorescent radiation. Although only a small
proportion of inorganic and organic compounds are naturally fluorescent, many
biologically active compounds and environmental contaminants ( e.g. polycyclic
aromatic hydrocarbons) are fluorescent and this , together with the high sensitivity of
these detectors, explains their widespread use. Because both the excitation wavelength
and the detected wavelength can be varied, the detector can be made selective. The
application of fluorescence detectors has been extended by means of pre- and post-
column derivatisaiton of non-fluorescent or weakly fluorescing compounds.

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 Fig: Fluorescence Detector

Refractive Index detector:

Fig: block diagram of RI-Detector

These bulk property detectors are based on the change of refractive index of the effluent
from the column with respect to pure mobile phase. Although they are widely used, the
refractive index detectors suffer from several disadvantages-lack of high sensitivity, lack
of suitability for gradient elution, and the need for strict temperature control ( 0.0010C)
to operate at their highest sensitivity. The two main types of RI detector are as follows:

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a. Deflection refractometer (as shown above): it measures the deflection of a beam
of monochromatic light by a double prism in which the reference and sample cells
are separated by a diagonal glass divide. When both cells contain solvent of the
same composition, no deflection of the light beam occurs; if, however, the
composition of the column mobile phase is changed because of the presence of a
solute, then the altered refractive index causes the beam to be deflected. The
magnitude of this deflection is dependent on the concentration of the solute in the
mobile phase.
b. The Fresnel refractometer which measures the change in the fractions of
reflected and transmitted light at a glass-liquid interface as the refractive index of
the liquid changes. In this detector both the column mobile phase and a reference
flow of solvent are passed through small cells on the back surface of a prism.
When the two liquids are identical there is no difference between the two beams
reaching the photocell, but when the mobile phase containing solute passes
through the cell there is a change in the amount of light transmitted to the
photocell, and a signal is produced. The smaller cell volume (about 3 L) in this
detector makes it more suitable for high-efficiency columns but for sensitive
operation, the cell windows must be kept scrupulously clean.

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