CHROMATOGRAPHY

Prashant Pandey Pharmaceutical Analysis

 CHROMATOGRAPHY

CHROMATOGRAPHY

 Chromatography is usually introduced as a technique for separating and/or identifying the

components in a mixture.
 The basic principle is that components in a mixture have different tendencies to adsorb onto
a surface or dissolve in a solvent.
 It is a powerful method in industry, where it is used on a large scale to separate and purify
the intermediates and products in various syntheses.
 There are several different types of chromatography currently in
use.
 Examples
 Paper chromatography
 Thin layer chromatography (TLC)
 Gas chromatography (GC)
 Liquid chromatography (LC)
 High performance liquid chromatography (HPLC)
 Ion exchange chromatography.
 Gel permeation or gel filtration chromatography
 Affinity chromatography etc.

Principle:
All chromatographic methods require one static part (the stationary phase) and one moving part
(the mobile phase). The techniques rely on one of the following phenomena: adsorption;
partition; ion exchange; size/molecular exclusion or affinity.
Adsorption:
 The first true chromatography is usually attributed to the Russian-Italian botanist Mikhail
Tsvet; He used a liquid-adsorption column containing calcium carbonate to separate yellow,
orange, and green plant pigments (currently they are known as xanthophyll’s, carotenes, and
chlorophylls respectively) in petroleum ether extract by using liquid hydrocarbon mobile
phase.
 It has a solid stationary phase and a liquid or gaseous mobile phase.
 The different solutes travelled different distances through the solid, carried along by the
solvent.
 Each solute has its own equilibrium between adsorption onto the surface of the solid
and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly.
 The result is a separation into bands containing different solutes.
 Liquid chromatography using a column containing silica gel or alumina is an example
of adsorption chromatography.
 The solvent that is put into a column is called the eluent, and the liquid that flows out of
the end of the column is called the elute.

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 CHROMATOGRAPHY

Figure: Adsorption chromatography using a column

Partition:

 In partition chromatography the stationary phase is a non-volatile liquid which is held as

a thin layer (or film) on the surface of an inert solid.
 The mixture to be separated is carried by a gas or a liquid as the mobile phase.
 The solutes distribute themselves between the moving and the stationary phases, with
the more soluble component in the mobile phase reaching the end of the chromatography
column first.
 Paper chromatography is an example of partition chromatography.

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 CHROMATOGRAPHY

Figure: Partition chromatography

Ion exchange:

 Ion exchange chromatography is similar to partition chromatography in that it has a coated

solid as the stationary phase.
 The coating is referred to as a resin, and has ions known as fixed ions (either cations
or anions, depending on the resin) covalently bonded to it and ions of the opposite charge
are electro-statically bound to the surface and known as counter ions.
 When the mobile phase (always a liquid) is eluted through the resin the electrostatically
bound ions are released as other ions are bonded preferentially.
 Domestic water softeners work on this principle.

Counter ions
Fixed ions

Figure: Ion exchange chromatography

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 CHROMATOGRAPHY

Size exclusion/Molecular exclusion

 Molecular exclusion differs from other types of chromatography in that no equilibrium state
is established between the solute and the stationary phase.
 The pore size is designed to allow the large solute particles to pass through uninhibited.
 The small particles, however, permeate the gel and are slowed down so the smaller the
particles, the longer it takes for them to get through the column. Thus separation is according
to particle size.
 When soft gels (Sephadex/Dextran, Sepharose/Agarose, Biogel/Polyacrylamide) are used
the method is known as gel filtration. Aqueous mobile phase is used.
 When semisolid or rigid gels (Cross linked polystyrene, controlled porosity glass beads,
alkylated dextran) are used the method is known as gel permeation. Non-aqueous mobile
phase is used. Eg. Chloroform, Pyridine, Acetone etc.

Figure: Gel permeation chromatography.

Affinity Chromatography:
 A specific ligand, such as an antibody is bound to the inert stationary phase to achieve
a highly selective separation.
 When a mixture of solutes containing a molecule that preferentially binds to the ligand,
such as an antigen, is passed through the system, the antigen binds strongly to the ligand
(anti-body) and is retained, while the other solutes elute.
 The antigen then can be displaced and eluted in a purified state.

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CHROMATOGRAPHY

Classification of Chromatography
1. According to the principle
a. Adsorption
b. Partition
c. Ion exchange
d. Size/molecular exclusion
e. Affinity

2. According to the Polarity of the Phases

a. Normal Phase

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CHROMATOGRAPHY

b. Reversed Phase
3. According to the Phases
a. Liquid Chromatography
i. Liquid Liquid Chromatography
Eg.: Paper Chromatography, Ion Exchange Chromatography, Droplet
Counter Current Chromatography (DCCC)
ii. Liquid Solid Chromatography
Eg: Column Chromatography, TLC, HPLC, HPTLC etc.
b. Gas Chromatography
i. Gas Solid Chromatography (GSC)
ii. Gas Liquid Chromatography (GLC)
c. Supercritical Fluid Chromatography
4. According to Geometry
a. Planner
i. Paper Chromatography
ii. Thin Layer Chromatography
iii. High Performance Thin Layer Chromatography
b. Column
i. Gas Chromatography
ii. Column Chromatography
iii. Size Exclusion Chromatography
iv. HPLC
v. Flash Chromatography
vi. Ion Exchange Chromatography
vii. Droplet Counter Current Chromatography
viii. Affinity Chromatography

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 CHROMATOGRAPHY

Theories:
1. Plate Theory
Each single equilibration between the phases is termed a theoretical plate and the length of
the column required for one equilibration is called the height equivalent a theoretical plate
(HETP). The nomenclature has been adopted by chromatographers to describe the equivalent
transfer of solute between the mobile and stationary phases.
HETP= L/N
Where,
N is number of theoretical plates in a column
L= Length of the column

S= Stationary Phase, M= Mobile Phase.

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 CHROMATOGRAPHY

2. Rate Theory
A more realistic description of the processes at work inside a column takes account of the time taken
for the solute to equilibrate between model, which assumes that equilibration is infinitely fast). The
resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also
affected by the different paths available to solute molecules as they travel between particles of
stationary phase. If we consider the various mechanisms which contribute to band broadening, we
arrive at the Van Deemter equation for plate height;

HETP = A + B/µ + Cµ
Where
 µ is the average velocity of the mobile phase
 A, B and C are factors which contribute to band broadening.

A: Eddy diffusion
The mobile phase moves through the column which is packed with stationary phase. Solute molecules
will take different paths through the stationary phase at random. This will cause broadening of the
solute band, because different paths are of different lengths.

Eddy diffusion in chromatographic column

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 CHROMATOGRAPHY

B: Longitudinal diffusion
The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses
out from the center to the edges. This causes band broadening. If the velocity of the mobile phase
is high then the analyte spends less time on the column, which decreases the effects of longitudinal
diffusion.

C: Resistance to mass transfer

The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase.
If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary
phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase.
The band of analyte is broadened. The higher the velocity of mobile phase, the worse the
broadening becomes.

Retardation Factor (Rf)

 The Rf factor is used for the qualitative evaluation of a TLC separation. It is the quotient
of the distance of the substance zone from the sample origin (Zs) to the front of the mobile
phase (Zf).
 Historically, Schonbein and Goppelsroeder used this R f value (relation to front expression) to
characterize planar separations:

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CHROMATOGRAPHY

Rf = zs / (zf-z0)
Where,
Zs distance of the substance zone from the sample origin (mm)
Zf solvent front migration distance (mm)
Z0 distance between immersion line and sample origin (mm)

 The value of the retardation factor in a given separation system at constant temperature
depends entirely on the characteristic properties of the separated substances.
 It is important for identification purposes that R f values are accurate and reproducible, but
this is difficult to achieve, since it is almost impossible to adequately control all the
experimental conditions that influence the separation process.
 This problem is avoided by defining a retardation factor for a standard substance in the
experimental conditions.
Selection of Stationary Phase:
 The success of the separation of a complex mixture by thin layer chromatography
(TLC)
greatly depends on the choice of the stationary phase.
 Sorbents generally used in TLC including silica gel, alumina, kieselguhr, magnesia,
magnesium/Calcium silicate, cellulose, charcoal etc.
 High-performance thin-layer chromatography (HPTLC) is distinguished from
conventional TLC. HPTLC involves a layer prepared from a particle of approximately 5
µm with a narrow particle size distribution.
Silica gel:
 Silica gel (SiO2) is by far the most widely used adsorbent and remains the dominant
stationary phase for TLC.
 The great majority of TLC analyses are carried out using normal phase (NP) silica gel
layer.
 The method is complementary for achieving separation and confirming qualitative
and quantitative results.
 The surface of the silica gel was investigated with the best and most adequate methods
for analyses. The hydrated silica gel surface can contain three kinds of silanol
groups: free silanol, geminal and associated silanols.
 Surface of the silica gel is acidic in nature.

Alumina
 Next to silica gel, alumina (Al2O3) is the most widely used adsorbent in TLC.
 Alumina is prepared from aluminum hydroxide (Al[OH]3) by calcination at 500°C.

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CHROMATOGRAPHY

 Three types of alumina, according to the nature of their function groups: acidic, basic,
and neutral.
 The active groups on the surface of chromatography alumina are hydroxyl groups, oxide
(O 2–) ions, and an aluminium cation.

Diatomaceous earth (Kieselguhr):

 It is a naturally occurring, soft, siliceous sedimentary rock that is easily crumbled into a
fine white to off-white powder. It has a particle size ranging from 10-200 micrometres.
 The typical chemical composition of oven-dried diatomaceous earth is 80-90% silica, with
2-4% alumina and 0.5-2% iron oxide.
 It has neutral pH and available without binder, but less resolution capacity.

Magnesia:

 Magnesia (MgO) is too finely divided to allow filtration and can be mixed with filter aid .

Magnesium/Calcium Silicate (MgSiO3/ Ca2SiO4)

 These are used for separation of sugars.

Cellulose:
 Flow is faster than paper chromatography.
 Modified cellulose powders are used to obtain ion-exchange separations in TLC and can
be used with or without binder.
 Cellulose contains adsorbed water which brings separation by partition mechanism.
 These materials are commonly used for separating hydrophilic substances like amino
acids, sugars etc.
Charcoal (Activated):
 Charcoal has the specific property of adsorbing strongly aromatic substances.

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CHROMATOGRAPHY

TLC Visualizing Reagent

S. No. Reagent Details

1. Iodine Chamber  Iodine vapor reacts with spots and gives
dark
brown spots.
 Unsaturated & Aromatic Compounds
2. 10% Sulphuric acid in ethanol  Used as a charring reagent.
 Reacts with all spots and gives
black
coloration when heated in hot plate or oven.
 Used for all type of compounds.
3. p-Anisaldehyde  Universal Stain
 Good for nucleophiles and o xygenated
Compounds
Used for Acidic groups (pKa < 5)
4. Bromocresol Green  and

5. Cobalt Chloride (CoCl2)  Carboxylic Acids identification

Universal Stain
6. Dragendorff Reagent  Nitrogenous Compounds: Alkaloids, amines,
organics bases, etc.
 Spots shows Orange to red color.
7. Ferric Chloride (FeCl3)  Used to detect phenols.
 Spots are Red in color.
8. Ninhydrine  Used to detect Amino Acids, Amino Sugars,
Amines
9. Vanillin  Universal stain
 Very effective for same polarity products (Rf)

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