BP701T Instrumental Method of Analysis

Unit III:
(Dr. S. K. Patro, Asst. Professor, IPT Salipur)

A. Chromatography
1. Introduction to Chromatography
2. Column Chromatography
3. Thin layer Chromatography
4. Paper Chromatography
B. Electrophoresis
1. Paper Electrophoresis
2. Gel electrophoresis
3. Capillary electrophoresis

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 A. Chromatography
Introduction:
Chromatography was invented by the Russian botanist Mikhail Tswet in the
year 1903. He employed the technique to separate various plant pigments (i.e.
Chlorophylls and Xanthophylls) by passing solutions of these substances (in
petroleum-ether extract) through a glass column packed with finely powdered CaCO 3.
The separated species appeared as separate bands having colored bands; the various
pigments migrating through the column at different rates (because of differences in
their distribution ratios). The separated species appearedasseparate bands having
colored bands: the various pigments migrating through the column at different rates
(because of differences in their distribution of ratios). The various solutes were
isolated by cutting and sectioning of the chalk packing. Twswett choose to designate
the name of such a process of separation as chromatography (Chroma-color,
graphein-writing). Tswett’s original experiments remained unnoticed in the literature
for several decades. It was not until 1931 when kuhan and Lederer investigated
polyene pigments that interest in such a technique was renewed.
But later on a diversify group of techniques which allow the separation of
closely related components of the complex mixtures. In this technique, the sample is
moved in a mobile phase, may be a gas, a liquid or a supercritical fluid. Such a
mobile phase is then allowed to flow through an immiscible stationary phase.
Chromatography is a physical method of separation in which the
components to be separated or distributed between two phases, one of which is
stationary (stationary phase), while the other, the mobile phase moves in a definite
direction.

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 Classification of Chromatographic methods:

Type of
General Type of Stationary Mobile
equilibration Name of the Technique
Classification method Phase Phase
Process
Paper Chromatography
(PC)
Liquid- Liquid Partition Thin layer
Liquid
Liquid Supported between the Chromatography
Chromatography Liquid
Or on a solid immiscible (TLC)
(LC) surface liquids
Partition High Performance thin
layer Chromatography
(HPTLC)
Liquid-Solid,
Adsorption Column
Or Solid Liquid Adsorption
Chromatography (ACC)
adsorption
Adsorption
(using very
Very finely
much higher
Liquid-Solid divided
pressures for the HighPerformance liquid
Or solid Liquid
flow of mobile Chromatography (HPLC)
adsorption packed in a
phase)
column

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 Type of
General Types of Stationary Mobile Name of the
equilibration
Classification method Phase Phase Technique
Process
Solid (ion- Ion-exchange
Ion-
exchange Liquid Partition/Sieving chromatography
Exchange
resign) (IEC)
Group –
Affinity Specific liquid
bonded to a
usually uses Partition between
enzymes solid surface (an Liquid Affinity
antibody , surface liquid
Or chromatography.
immobilized on (immobilized)
Antigen- a stationary and mobile phase
Antibody phase by
highly covalently
specific binding to it –
interactions an affinity
ligand)
Gas-solid
Gas – Solid chromatography
Adsorption
(or Gas (GSC)
Solid
adsorption)
Gas
Gas-Liquid
Chromatography Liquid adsorbed Partition between
Gas – liquid Chromatography
on a solid Gas and liquid (GLC)
( or Gas
Partition)

Super-critical
fluid
Supercritical Bio specific Organic Species Partition between
Supercritical chromatography
fluid adsorption or bonded to a Super-critical fluid
fluid (SFC) or
chromatography bio affinity solid surface and bonded species
Bioaffinity
(SFC)
chromatography
(BC)

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 Column Chromatography
When a column of stationary phase is used, the technique is called as column chromatography.
Based on the nature of the stationary phase i.e. whether it is solid or liquid, it is called as column
adsorption chromatography or. Column partition chromatography is not widely used.
Principle:
1. This technique is based on the principle of differential adsorption where different molecules
in a mixture have different affinities with the adsorbent present in the stationary phase.
2. The molecules having higher affinity remain adsorbed for a longer time decreasing their
speed of movement through the column.
3. However, the molecules with lower affinity move with a faster movement, thus allowing the
molecules to be separated in different fractions.
4. Here, the stationary phase in the column chromatography also termed the adsorbent is a
solid (mostly silica) and the mobile phase is a liquid that allows the molecules to move
through the column smoothly. The type of interaction between the stationary phase
(adsorbent) & the solute is reversible in nature.
The rate of movement of a component (R) is given as follows
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑚𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
R = 𝑅𝑎𝑡𝑒 𝑜𝑓 𝑚𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝𝑕𝑎𝑠𝑒
The equation can be simplified as follows:

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒔𝒐𝒍𝒖𝒕𝒆

R=
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒕𝒉𝒆 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
When a liquid mobile phase is used, the equation is written as
𝑨𝒎
R=
𝑨𝒎+ 𝜶 𝑨𝒔
𝐶𝑜𝑛𝑐 .𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝𝑕𝑎𝑠𝑒
Where α is the Partition coefficient =
𝐶𝑜𝑛𝑐 .𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝𝑕𝑎𝑠𝑒
Am is the average cross section of mobile phase
As is the average cross section of stationary phase
Practical Requirement
1. Stationary Phase
2. Mobile Phase
3. Column characteristics
4. Preparation of the column
5. Introduction of sample
6. Development technique (elution)
7. Detection of components
8. Recovery of components

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Stationary Phase
Adsorbents are used in this technique may be organic and inorganic classes of compounds. The
ideal requirements of adsorbent are:
i. It should produce only adsorption of the analyte over it
ii. The particles should have uniform size distribution and have spherical shape. Particle
size: 60-200µ.
iii. It should have high mechanical stability
iv. It should be inert & should not react with the solute or other components.
v. Insoluble in the solvents or mobile phases used.
vi. It should be colorless to facilitate observations of zones and recovery of components.

Figure: 1 Column chromatography.

Table : 1 Adsorbents and Solvents

Adsorbents Solvent
Sucrose Petroleum ether
Starch Carbon tetrachloride
Weak
Inulin Cyclohexane
talc Carbon di-sulphide
Sodium carbonate Ether (ethanol free)

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 Calcium carbonate Acetone
Medium
Calcium phosphate Benzene
Magnesium carbonate Toluene
Magnesium oxide Esters
Calcium hydroxide Chloroform
Activated magnesium silicate Acetonitrile
Activated alumina Alcohols
Activated charcoal Water
Strong
Activated magnesia Pyridine
Activated silica Organic acids
Mixtures of acids or bases
Fuller's earth with ethanol or pyridine

The most commonly used adsorbent is Silica gel of 80-100 mesh or 100 – 200 mesh size
which has a particle size of 60-200µ.
Selection of Stationary Phase
The selection of stationary phase in column chromatography depends on the following:
1. Removal of impurities: When a small quantity of impurity is present and there is
difference in affinity when compared to the major component, a weak adsorbent is
sufficient.
2. No. of components to be separated: When few components are to be separated, weak
adsorbent is used. When more components are to be separated, a strong adsorbent is
used.
3. Affinity differences between different components: When components have similar
affinities, a strong adsorbent will be effective. When there is more differences in
affinities, a weak adsorbent is selected.
4. Length of the column used: When a shorter column is used, strong adsorbent has to be
used. When a longer column is used, a weak adsorbent can be used.
5. Quantity of adsorbent used: 20 or 30 times the weight of the adsorbent is used for
effective separation.
Adsorbate: Adsorbent = 1: 20 or 1: 30.
Mobile Phase:Mobile Phase is the very important and they are several functions. Mobile is acting
as solvent, developer, and as eluent. The functions of a mobile phase are:
To introduce the mixture into the column – As solvent
To develop the zones for separation – As developing agent
To remove pure component out of the column – As eluent

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Different mobile phases used: It is used in increasing order of polarity or elution strength. The
solvents are given in the above Table 1. These solvents can be used in either pure form or as a
mixture of solvents of varying compositions
ColumnCharacteristics
Column is mostly best quality of neutral glass since it should not be affected by solvents, acids or
alkalies. An ordinary burette can be used as column for separation.
Length/diameter ratio is 10-15:1.
For more efficiency, the length/diameter ratio is 100:1.
Column length
a. Multi-component system long column
b. Components with similar affinities for adsorbent long column
c. Components with different affinities for adsorbent short column
Preparation of the Column
 The column mostly consists of a glass tube packed with a suitable stationary phase.
 Glass wool/cotton wool or an asbestos pad is placed at the botton of the column before
packing the stationary phase.
 After packing, a paper disc kept on the top, so that the stationary layer is not disturbed
during the introduction of sample or mobile phase.
There are two types of preparing the column, they are:
1. Dry packing / dry filling
In this the required quantity of adsorbent is poured as fine dry powder in the column and the solvent
is allowed to flow through the column till equilibrium is reached.
2. Wet packing / wet filling
In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It is
considered as the ideal technique for packing.
 Before using column, it should be washed properly and dried.
Introduction of the Sample
 The sample which is usually a mixture of components is dissolved in minimum quantity of
the mobile phase or a solvent of minimum polarity
 The entire sample is introduced into the column at once and gets adsorbed on the top portion
of the column.
 From this zone, individual sample can be separated by a process of elution.
C. Elution (Development technique)
 By elution technique, the individual components are separated out from the column.
 It can be achieved by two techniques:
 Isocratic elution technique: Same solvent composition or solvent of same polarity is used
throughout the process of separation.

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Eg. Use of chloroform alone or [Link]: Benzene = 1:1 only, etc.
 Gradient elution technique: Solvents of gradually ↑ (increasing) polarity or ↑ (increasing)
elution strength are used during the process of separation.
E.g. initially benzene, then chloroform, then ethyl acetate then chloroform
Other techniques like Frontal analysis and Displacement analysis where a graph of concentration of
eluate Vs. volume of eluate will give an idea of how compounds are eluted out from the column.
D. Detection of Components
1. If the compounds separated in a column chromatography procedure are colored, the progress
of the separation can simply be monitored visually.
2. If the compounds to be isolated from column chromatography are colorless. Then the
technique depends upon the properties of the components. Different properties which can be
used are
3. Absorption of light (UV/Vis) – Using UV-Visible Spectropotometer
4. Flourescence or light emission characteristics – Using fluorescence detector
5. By using flame ionization flame detector
6. Refractive index detector- based on the refractive index difference between the mobile
phase and mobile phase + component
7. Evaporation of the solvent and weighing the residue
8. Small fractions of the eluent are collected sequentially in labeled tubes and the composition
of each fraction is analyzed by TLC (thin layer chromatography).
Recovery of components: Earlier, recoveries of the components were done by cutting the column
into several distinct zones. Later, extrusions of the column into zones were done by using plunger.
The best technique is to recover the components by a process called aselution. 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. The different elution techniques like isocratic elution technique and
gradient elution [Link] is done by collecting different fractions of mobile phase of
equal volume like 10ml, 20ml etc or unequal volume. They can also be collected time wise i.e. a
fraction every 10 or 20 minutes etc. The recovered fractions are detected by using the techniques
discussed earlier. Similar fractions are mixed so that the bulk of the compound of each type is
obtained in a pure form. If a fraction still contains several components, it can be resolved by using
another column.
Applications:
1. Separation of mixture of compounds: Separation of glycosides, amino acids, plant extracts
2. Removal of impurities Isolation of the active constituents from the plant extract or from
formulations
3. Isolation of metabolites from the biological fluids: 17-ketosteroids from urine, cortisol
4. Estimation of drugs in formulations or crude extracts
i. Determination of % w/w of strychnine in syrup of ferrous phosphate
with quinine and strychnine
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 ii. Separation of diastereomers.
iii. Separation of tautomers and racemates
Factor affecting Column efficiency

1. Dimensions of the column

2. Particle size of the adsorbent
3. Nature of the solvent
4. Temperature of the column
5. Pressure

Advantages:
1. Any type of mixture can be separated by column chromatography.
2. Any quantity of the mixture can also be separated (µg to mg of substance).
3. Wider choice of mobile phase.
4. In preparative type, the sample can be separated and reused.
5. Automation is possible.
Limitation or Disadvantages of Column chromatography
1. Time consuming method.
2. More amounts of solvents are required which may be expensive.
3. Automation makes the technique more complicated and costly.
________________________________

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 Thin layer Chromatography (TLC)
Introduction:The history of thin layer chromatography dates back to 1938 when Izmailov and
Shraiber separated plant extracts using 2mm thick and firm layer of alumina set on glass plate. In
1944, Consden, Goden and Martin used filter papers for separating amino acids. In 1950, Kirchner
identified terpens on filter paper and later glass fibre paper coated with alumina. Only in 1958,
Stahl developed standard equipment for analyzing by Thin layer chromatography.
Principle:
Thin Layer Chromatography can be defined as a method of separation or identification of a mixture
of components into individual components by using finely divided adsorbent coated or spread over
a chromatographic plate. The mobile phase solvent flows 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 thin layer chromatographic plate based on the affinity of the components towards the
stationary phase.

Fig 2: Silica extended structure and surface.

Silica (SiO2) is a solid with an extended structure of tetrahedral silica atoms bridged together by
bent oxygen atoms. On the surface of the silica particles, the solid terminates in very polar silanol
(Si-O-H) groups. The silica is the stationary phase because it remains adhered to the glass plate and
does not move during the chromatography process.
The Silica extended structure and surface is shown in the Fig 2. The developed TLC plate is shown
in the Fig 3.

Fig 3: Developed TLC Plate

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Advantages of TLC
1. It is a simple process with a short development time.
2. It helps with the visualization of separated compound spots easily.
3. It helps in isolating of most of the compounds.
4. The separation process is faster and the selectivity for compounds is higher (even small
differences in chemistry is enough for clear separation).
5. The purity standards of the given sample can be assessed easily.
6. It is a cheaper chromatographic technique.
7. TLC offers a faster and more efficient separation than paper chromatography and the
majority of paper chromatographic separations have now been superseded by the TLC
Procedures.
Practical Requirement:
1. Stationary Phase: There a several adsorbents which can be used as stationary phases. Some
of the stationary phases, their composition and the ratio in which they have to be mixed with
water or other solvents to form a slurry for preparing thin layer chromatographic plates are
given in the below Table 2:
Name Composition Adsorbent: Water ratio
Silicagel H Silicagel without binder 1:1.5
Silicagel GF Silicagel + Binder + Fluorescent indicator 1:2
Silicagel G Silicagel + CaSO4(gypsum) 1:2
Alumina 1:1.1
Neutral Al2O3 without binder
Basic
Acidic
Al2O3 G Al2O3 + binder 1:2
Cellulose Cellulose without binder 1:5
powder
Cellulose Cellulose with binder 1:6
powder
Kieselguhr Diatomaceous earth + binder 1:2
G
Polyamide Polyamide 1:9 (CHCl3 : CH3OH=
powder 2:3)

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Fluoroscent indicator Zinc silicate
Silica gel and alumina are available with different specific surface areas and these grades are
identified by a number, e.g., silica gel 60 (or 40 or 150) which indicates the mean pore size in
Angstroms (10 -10m).The particle size of silica gel for TLC is 10-40 µm (average 15 µm).
2. Preparation of the Glass Plates: The sizes of the glass plates for use with commercially
available spreaders are usually 20 X 20, 20 X 10 or 20 X 5 cm.
Microscopic slides can also be used for some applications like monitoring the progress of
chemical reaction.
In general, the glass plates should be of good quality and should be withstand temperatures
used for drying the plates.
General method:
Mix 30gm of the adsorbent in a mortar to a smooth consistence with the requisite amount of
water or solvent specified in the manufacturer’s instruction and transfer the slurry quickly to
the spreader. Spread the mixture over 4 to 5 plates (20 X 20cm) or a proportionately larger
number of smaller plates and allow the thin layers to set (about 4minutes when CaSO4 is
present). Transfer the plates carefully to a suitable holder and after a further 30minutes, dry
at 100-120 0C for 1 hour to activate the adsorbent. Cool and store the plates in a desiccator
over silica gel. The thickness of the moist thin layer should be about 0.25 mm.
Special methods:
a. Preparative thin layer. The layers are 0.5 – 2mm thick, prepared as described under the
general method, but using a smaller quantity of water and allowing a longer time for the
initial drying of the plate.
b. Microscopic slides are conveniently coated by a dipping technique in the following way:
prepare slurry of the adsorbent by shaking withchloroform or chloroform-methanol (2:1)
and insert two microscope slides (back to back) into the slurry. Withdraw the slides;
allow draining, separating the slides and drying.
c. The slurry, prepared in the normal way, is sprayed onto the surface of glass plates, using
a laboratory spray gun.
d. Theadsorbent, mixed with an organic solvent, e.g., chloroform or ethyl acetate, is
distributed evenly over a glass plate by careful tilting, and, after evaporation of solvent,
is dried in the normal way.
In all the methods the plates should be tidied before use by cleaning the edges and backs
(microscope slides).
3. Application of Sample: In order to get good spots, the concentration of the sample or
standard solution can be 2-5µl of a %1 solution of either standard or test sample is spotting
using a capillary tube or micropipette. The spots can be placed at random or equidistant
from each other by using a template, with markings. The spot should be kept at least 2cm
above the base of the plate and the spotting area should not be immersed in the mobile phase
in the development tank.

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4. Development Tank: For the purpose of development, a developing tank or chamber of
different sizes to hold TLC plates of standard dimensions are used. These require more
solvents for developing the chromatogram. When a new method is developed, it is better to
develop in glass beakers or specimen jars, etc, to avoid more wastage of solvents. When
developed method or standard method is used, it is better to use development tank. In the
new type of development tanks have hump in the middle, which require less solvent. The
development chamber or tank should be lined inside with filter paper moistened with the
mobile phase so as to saturate the atmosphere. If this kind of saturation of the atmosphere is
not done, “edge effect” occurs where the solvent front in the middle of the TLC plate moves
faster than that of the edge. The development tank examples are shown in the below Fig 3.

Fig 3: Development tank

5. Mobile Phase:Selection of the mobile phase depends upon the below factors
i. Nature of the substances to be separated
ii. Nature of the stationary phase used
iii. Mode of chromatography (Normal phase or reverse phase)
iv. Separation to be achieved – Analytical or preparative
Pure solvents or mixture of solvents are used. The following gives a list of solvents (of
increasing polarity).
Petroleum ether, Carbon tetrachloride, Cyclohexane, Carbon di-sulphide, Ether, Acetone,
Benzene, Toluene, Ethyl acetate, Chloroform, Alcohols like methanol or ethanol, Water,
pyridine. The solvent composition is done by trial and error method only but with a review of
literature and other logical considerations like solubility of the substance, polar or non-polar
character of the samples, etc.
6. Development technique: Different development techniques are used for efficient
separations. They are
i. Vertical development (One dimensional)
ii. Two dimensional development
iii. Horizontal development
iv. Multiple development

14
 i. Vertical development (One dimensional): In this technique, the plates are
kept vertical and the solvent flows against gravity, because of capillary
action.
ii. Two dimensional techniques: For complex mixtures this technique is used.
First, the plates are developed in one axis and the plates after drying are
developed in the other axis. When large number compounds cannot be
separated by using one dimensional technique.
7. Detecting or Visualizing Agents
After the development of TLC plates, the spots should be visualized. Detecting colored
spots can be done visually. But for detecting colorless spots, any one of the following
techniques can be used.
a. Specific methods: In this method particular detecting agents are used to find out the
nature of compounds or for identification purposes. Examples are
i. Ferric chloride- for phenolic compounds and tannins.
ii. Ninhydrin in acetone- for amino acids
iii. Dragendroff’s reagent – for alkaloids
iv. 2,4 – Dinitrophenyl hydrazine – for aldehydes and ketones
b. Nonspecific methods: Where the number of spots can be detected, but not the exact
nature or type of compound.
Examples
i. 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.
ii. Sulphuric acid spray reagent: 70-80% v/v of sulphuric acid with few mg of either
potassium dichromate or potassium permanganate or few ml of nitric acid as
oxidizing agent is used. This reagent after spraying on TLC plates is heated in an
oven . Black spots are seen due to charring of compounds.
iii. Using fluorescent stationary phase: When the compound s are not flourscent, a
fluorescent stationary phase is used. When the plates are viewed under UV
chamber, dark spots are seen on a fluorescent background. Examples of such
stationary phase is Silica gel GF
The detecting techniques can be categorized as
i. Destructive technique: Specific spray reagents, Sulphuric acid spray reagent, etc where
the samples are destroyed for detection.
ii. Non-Destructive technique: like UV chamber method, Iodine chamber method,
densitometric method, etc where the sample is not destroyed even after detection. These
detecting techniques are used in TLC method development and in preparative TLC.
In densitometric method, Densitometer is used which measures quantitatively the density of the
spots. When the optical density of the spots for the standards and test solution are measured, the
quantity of the substance can be calculated. The plates are neither destroyed nor eluted with the
solvents to get the compounds. This method is also called as in-situmethod.

15
8. Qualitative analysis
The Rf value is calculated for identifying the spots in qualitative analysis. Rf value is the
ratio of distance travelled by the solute to the distance travelled by the solvent front.
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
𝐑𝐟 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡𝑕𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The 𝑅𝑓 value ranges from 0 to 1. The ideal value is 0.3 to 0.8.
The 𝑅𝑓 value is constant for every compound in a particular combination of stationary and
mobile phase. When the Rf value of a sample and reference compound is same, the
compound is identified by its standard. When the 𝑅𝑓 value differs, the compound may be
different from its reference standard.
𝑹𝒙 value is the ratio of distance travelled by the sample and the distance travelled by the
standard. Rx value is always closer to 1.
𝑹𝒎 value is used to find out whether the compounds belong to a homologous series. If they
belong to a homologous series, the ΔRm values are constant. The ΔRm values for a pair of
adjacent member of a homologous series are determined by using the formula:
1
𝑅𝑚 = 𝑙𝑜𝑔 ( − 1)
𝑅𝑓
9. Quantitative Analysis
Indirect method: Quantitative analysis can be done after eluting the individual spotswith
solvent and filtering off the stationary phase. The solution can be concentrated and the exact
quantities of the compound determined by the methods like UV-Visible spectrophotometry,
fluorescence method, flame photometric method, electrochemical methods of analysis.
Direct method: It can be done after eluting the individual spots with solvent and filtering
off the stationary phase. The solution can be the concentrated and the exact amount of the
compound deterimined by the various methods like UV-visible spectrophotometry,
flourscence method, flame photometric method, electrochemical methods of analysis etc.

10. Application of TLC

i. Separation of mixtures of drugs of chemicals or biological origin, plant extracts etc
ii. Separation of carbohydrate, vitamins, antibiotics, proteins, alkaloids, glycosides etc
iii. Identification of drugs
Drug Stationary Phase Mobile Phase Detecting agent
Amoxycillin trihydrate Silica Gel G.F-254 Buffer pH 6: NaOH+
acetone (4:1) Starch+glacial acetic
acid+Iodinein
potassium iodide
Ampicillin for oral Cellulose M.N-300 Citric acid : Butyl Starch iodide reagent
suspension alcohol(5:1)

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 Test for impurities, decomposition & related substances in pharmaceutical products (as per
British Pharmacopoeia substances and preparations)
Substance Tested for Mobile Phase Detection
Chlorpropamide p-Chlorobenzene Chloroform : Sodium
sulphonamide and methanol:cyclohexane:13.5M hypochlorite
NN’-diropylurea ammonia followed by
(0.33%) ([Link].5) potassium iodide
in starch
mucilage
Nitrazepam Tablets Decomposition and Nitromethane : ethyl 254nm radiation
related substances acetate(85:15)
0.5%
Desipramine Iminodibenzyl Toluene: ethyl acetate: Potassium
Hydrochloride (0.2%) ethanol: diethylamine dichromate
([Link]) (0.5%) in
sulphuric
acid:water(4:4)

17
 Paper Chromatography
Paper chromatography (PC) is a type of a planar chromatography whereby chromatography
procedures are run on a specialized [Link] is considered to be the simplest and most widely used
of the chromatographic techniques because of its applicability to isolation, identification and
quantitative determination of organic and inorganic [Link] was first introduced by German
scientist Christian Friedrich Schonbein (1865).
Types of Paper chromatography:
(i) Paper Adsorption Chromatography: Paper impregnated with silica or alumina acts as
adsorbent (stationary phase) and solvent as mobile phase.
(ii) Paper Partition Chromatography : Moisture / Water present in the pores of cellulose fibers
present in filter paper acts as stationary phase & another mobile phase is used as solvent In
general paper chromatography mostly refers to paper partition chromatography.
Principle of Separation
The principle of separation is mainly partition rather than adsorption. Substances are distributed
between a stationary phase and mobile phase. Cellulose layers in filter paper contain moisture
which acts as stationary phase. Organic solvents/buffers are used as mobile phase. The developing
solution travels up the stationary phase carrying the sample with it. Components of the sample will
separate readily according to how strongly they adsorb onto the stationary phase versus how readily
they dissolve in the mobile phase.
Instrumentation of Paper chromatography
2. Stationary phase & papers used
3. Mobile phase
4. Application of sample
5. Developing Chamber
6. Detecting or Visualizing agents
1. STATIONARY PHASE AND PAPERS: Whatmann filter papers of different grades like No.1,
No.2, No.3, No.4, No.17, No.20 etc are used.
In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose. These papers differ in
sizes, shapes, porosities and thickness.
Other modified papers like Acid or base washed filter paper, glass fiber type paper.
Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.
Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used
for reverse phase chromatography. Silicon pretreatment and organic non-polar polymers can also be
impregnated to give reverse phase chromatographic mode.
Impregnation of silica, alumna, or ion exchange resins can also be made.
Size of the paper used: Paper of any size can be used. Paper should be kept in a chamber of suitable
size.

18
[Link] of sample: The sample to be applied is dissolved in the mobile phase and applied
using capillary tube or using micropipette. Very low concentration is used to avoid larger zone
3. PAPER CHROMATOGRAPHY MOBILE PHASE
Pure solvents, buffer solutions or mixture of solvents can be used. Some of the Examples of
Hydrophilic mobile phases
Isopropanol: ammonia:water [Link]
Methanol: water 4:1 or 3:1
n-Butanol: glacial acetic acid: water [Link]
Hydrophobic mobile phases
kerosene: 70% isopropanol
Dimethyl ether: cyclohexane
The commonly employed solvents are the polar solvents, but the choice depends on the nature of
the substance to be separated.
If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be
applied.
[Link] CHAMBER: The chromatographic chambers are made up of many
materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in
various dimensional sizes depending upon paper length and development type. The chamber
atmosphere should be saturated with solvent vapor.
Development technique:
Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and
waited until the solvent front reaches near the edge of the paper.
Different types of development techniques can be used:
a. ASCENDING DEVELOPMENT
Like conventional type, the solvent flows against gravity. The spots are kept at the bottom portion
of paper and kept in a chamber with mobile phase solvent at the bottom. (Same as in TLC) (Fig 4)

Fig 4

19
 b. DESCENDING TYPE: This is carried out in a special chamber where the solvent
holder is at the [Link] spot is kept at the top and the solvent flows down the
[Link] this method solvent moves from top to bottom so it is called descending
chromatography (Fig: 5).

Fig 5: Descending technique and sprayer to spray the visualizing agent

c. ASCENDING – DESCENDING DEVELOPMENT: A hybrid of above two
techniques is called ascending-descending [Link] length of
separation increased, first ascending takes place followed by descending.
d. CIRCULAR / RADIAL DEVELOPMENT

Fig 6: Circular/Development technique

Spot is kept at the centre of a circular [Link] solvent flows through a wick at the centre &
spreads in all directions uniformly. Hence the individual spots after development look like
concentric circles. By making perforations radially, number of quadrants can be created allowing
more number of samples to be spotted (Fig 6).
e. Two dimensional developments: This technique is very similar to 2-Dimensional
TLC. Here the chromatogram development occurs in two directions at right angles
(Fig 7). In this mode, the samples are spotted to one corner of rectangular paper and
20
 allowed for first development. Then the paper is again immersed in the mobile phase
at a right angle to the previous development for the second chromatogram. In the
second direction, either the same solvent system or different solvent system can be
used for development.

Fig 7: Two dimensional techniques

f. Drying of Chromatogram: After the development, the solvent front is marked and
the left to dry in a dry cabinet or oven.
g. Detection: After the development of chromatogram, the spots should be visualized.
Detecting colored spots can be done visually. But for detecting colorless spots, any one
of the following technique can be used.
a. Nonspecific methods: where brown or amber of spots can be detected, but not the
exact nature or type of the compound.
Examples
(i) Iodine chamber method where brown or amber spots are observed when
the developed papers are kept in atank with few iodine crystals at the
bottom.
(ii) UV chamber for fluorescent compounds: When compounds are viewed
under UV chamber, at 254nm (short λ) or at 365nm (long λ), fluorescent
compounds can be detected. Bright spots can are seen against a dark
background.
b. Specific methods: Specific spray reagents or detecting or visualizing agents are used
to find out the nature of compounds or identification purposes.
a. Ferric chloride- For phenolic compounds and tannins
b. Ninhydrin in acetone- for amino acids
c. Dragendroff’s reagent- for alkaloids
d. 3,5-Dinitro benzoic acid- for cardiac glycosides
e. 2,4- Di-nitrophenyl hydrazine- for aldehydes and ketones

21
The detecting techniques can also be categorized as
a. Destructive technique: Specific spray reagents etc where the samples are
destroyed before detection e.g. Ninhydrin reagent.
b. Non-destructive technique: UV chamber method, Iodine chamber method,
densitometric method, e.t.c, where the sample is not destroyed even after
detection.
For radioactive materials, detection is by using autoradiographyor Geiger muller
counter.
For antibiotics, the chromatogram is layed on nutrient agar inoculated with
appropriate strain and the zone of inhibition is compared.
Quantitative Analysis
Direct technique: Densitometer is an instrument whichmeasures quantitatively the density of the
spots. When the optical densities of the spots for the standard and test solution are determined, the
quantity of the substance can be calculated. The papers are neither destroyed nor eluted with
solvents to get the compounds. The method is also known as in-situ method.
Indirect techniques: In this technique, the spots are cut into portions and eluted with solvents. The
solution can be analyzedby any conventional techniques of analysis like spectrophotometry,
electrochemical methods, etc.
Qualitative Analysis: a. Rf value
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The Rf value ranges from 0 to 1. But the ideal values are from 0.3 to 0.8.
b. Rx value: It is always closure to 1.
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑥 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
c. Rm value: It is mainly used to find out whether the compounds belong to a homologous series. If
they belong to a homologous series, the 𝚫Rm values are constant. The 𝚫Rm values for a pair of
adjacent member of a homologous series are determined by using the below formula:
1
𝑅𝑚 = log( − 1)
𝑅𝑓
Application:
(i) To check the control of purity of pharmaceuticals,
(ii) For detection of impurities
Drug Mobile phase Detecting agent
Hydroxocobalamin Butyl alcohol: acetic Elution and measurement of
acid:potassium cyanide absorbance at 361nm.

22
 (iii) Detect the contaminants in foods and drinks,
(iv) For the detection of drugs
Drug Mobile phase Detecting agent
Gentamycin Chloroform : Methanol : Ninhydrin in pyridine-
Ammonia : Water acetone mixture
([Link])
Vancomycin t-Amyl alcohol : Nutrient agar containing
Acetone: water ([Link]) Bacilus subtilis

(v) In analysis of cosmetics

(vi) Analysis of the reaction mixtures in biochemical labs.
(vii) Identification of decomposition products
(viii) Analysis of metabolites of drugs in blood, urine etc.
(ix) In the study of ripening and fermentation
Advantages of Paper Chromatography:
1. Simple and Rapid
2. Paper Chromatography requires very less quantitative material.
3. Paper Chromatography is cheaper compared to other chromatography methods.
4. Both unknown inorganic as well as organic compounds can be identified by paper
chromatography method.
5. Paper chromatography does not occupy much space compared to other analytical methods
or equipment’s.
Limitations of Paper Chromatography
1. Large quantity of sample cannot be applied on paper chromatography.
2. In quantitative analysis paper chromatography is not effective.
3. Complex mixture cannot be separated by paper chromatography.
4. Less Accurate compared to HPLC or HPTLC

23
 B. ELECTROPHORESIS
Electrophoresis is a physical method of analysis based on themigration of electrically charged
proteins, colloids, moleculesor other particles dissolved or dispersed in an electrolytesolution in the
direction of the electrode bearing the oppositepolarity when an electric current is passed through it.
The electrophoretic mobility is the rate of movement in metre per second of the charged particles
under the action of an electric field of I volt per metre and is expressed in square metres per volt
second. For practical reasons it is given in square centimetres per volt second, cm 2V- 1S-1. The
mobility is specific for a given electrolyte under precisely determined operational conditions.
𝑄
µ=
6𝜋𝑟ƞ
Where µ = Electrophoretic mobility
Q – Net charge on the ion
r – Ionic radius of the solute
ƞ – viscosity of the medium
The electrophoretic mobility is directly proportional to net charge and inversely proportional to
molecular size and viscosity of the electrophoresis medium.
The pH of the solution affects the mobility of the ion by
Depending on the method used, the electrophoretic mobility is either measured directly or
compared with that of a reference substance.
Based upon the type of apparatus used, electrophoretic methods may be divided into two categories,
one called free or moving boundary and the other called zone electrophoresis (using a supporting
medium). Zone electrophoresis include paper, gel such as agar, starch or poly acrylamide.

24
 Paper electrophoresis:
It is separation technique, where ions of different charges are separated on a medium of paper
(moistened with a buffer), by the application of a voltage between two electrodes, which are in
contact with the paper.
Principle of Separation: A mixture of ions or ionisable substances is applied on the centre of a
paper, previously immersed in a buffer of known ionic strength. This paper is placed across two
trays, filled with buffer, into which two electrodes are immersed. When a voltage is applied across
these electrodes, the ions or ionisable substances migrate towards anode or cathode, based on their
charge and other factors. Neutral or non-ionisable substances do not migrate. Anionic substances
move towards anode and cations move towards cathode. Ultimately, there is separation of anionic,
cationic and non-ionic / zwitterionic substances. The spots/bands which migrate can be detected bu
using appropriate spray reagents or visualizing agents as in paper chromatofraphy and can be
quantified by using densitometer. Both qualitative and quantitative analysis can be performed in
paper electrophoresis.
Components of paper electrophoresis
a. Normally Whatman® filter paper (Grade 3 MM or No. 1) of suitable dimension (2.5cm to
5cm) with a length so that ends of the strip of paper touch the buffer solution, kept in the
electrode vessels. The paper to be used is washed with double distilled water followed by
0.1 M HCl or 0.01 M EDTA to remove impurities.
b. Electrodes and voltage to be applied
The electrode in the form of a thin wire is made up of carbon or platinum. A DC voltage of
about 8-15 V/cm length of paper is normally applied. In low voltage electrophoresis, the
voltage across two electrodes is about 100-300V, with a current of 0.4mAmp per cm width
or 1.5mAmp/strip.
In high voltage electrophoresis, a potential of about 50-215V/cm(Total 10,000V/strip) is
applied across the electrodes.
c. Buffers used: The pH of buffer to be used depends upon the types of compounds to be
separated.
The following are some of the buffers used:
1. Barbitone buffer (Veronal buffer) (0.07mole/litre, pH 8.6). Ionic Strength – 0.05.
2. Tris-acetate buffer (0.07 mole/litre, pH 7.6)
3. Citrate buffer (0.07 mole/litr, pH 3.0 or pH 6.8)
Other buffers of different pH and ionic strength can also be for separation, based on the type
of compounds. Usually ionic strengths (IS) of 0.05-0.5 is used in most separations.
Types of paper electrophoresis (PE)
A. Horizontal/vertical/Continuous Electrophoresis
There are different types of electrophoresis instruments based on the design of the
instrument. The diagrams of these 3 types of instruments are shown in Fig 1 to 3.

25
Horizontal and vertical modes are used in analytical scale; whereas continuous
electrophoresis is used on a preparative scale (i.e. large amount of sample mixture is
used). The principles involved in all the modes are same, but the design of each
instrument varies.
In Horizontal type / Vertical (Fig 1 and 2), buffer solution of known pH and ionic
strength is filled into two troughs. Appropriate grade of Whatmann filter paper and
suitable width and length of filter paper are immersed in buffer solution. 10-20µl of
sample solution is applied at the centre of the paper and fixed in position.
The transparent lid is closed for safety as well as to prevent evaporation of
buffer/solvent. A suitable potential (100-300V) is applied across two electrodes dipped
in buffer solution.
Fig 1: Horizontal Paper electrophoresis

Fig: 2: Vertical Paper Electrophoresis

When such potential is applied across the electrodes, migration of cations and anions
take place towards cathode and anode respectively. Non-ionisable / neutral substances
do not move. Hence the separation of compound from the mixture takes place.

26
 In this vertical mode, the migrations of ions are assisted by gravity and hence a typical
separation takes place in about 6-8 hours. After sufficient migration, the paper is taken
out and dried, to fix the spots / bands. Then the compounds / bands /spots can be
visualized by using the visualizing agent. The quantitation of spots can be done by
densitometer.
The horizontal mode is similar to the vertical mode, in principle. However, the paper
is placed on a flat bed, as shown in Fig 2. The procedure to be followed is same as that
of vertical type. In horizontal mode, it takes about 12-14 hours for separation.
Continuous electrophoresis (Fig. 3) is meant for preparative samples, where a
predetermined sample volume through a valve device is applied continuously on the
centre of paper. The application of voltage causes migration of samples and hence
compounds are separated as bands. Thus each band is made to fall down and pure
compounds are collected in separate containers. The solvent is evaporated and pure
fractions are reused.
Various factors like charge of ions, size of the ions, viscosity of the medium, applied
voltage, pH of buffer and ionic strength affect the migration of ions in paper
electrophoresis

27
 Fig 3: Continuous paper electrophoresis

B. There are two types of paper electrophoresis based on the voltage applied, i.e. Low
voltage or High voltage Paper electrophoresis
Voltage/cm Votage/strip
Low voltage PE 8-15 V/ cm 100-300
High voltage PE 50-215V/ cm 10,000v

High voltage paper Electrophoresis has the following advantages:

1. Separation is faster, hence less time is required for separation
2. Sharp bands are obtained, since there is less diffusion of bands.
3. As sharp bands are obtained, separation of closely related compounds can be
achieved.
4. More number of samples can be analysed simultaneously.
Disadvantages of High Voltage Paper Electrophoresis
1. It is dangerous to the operator, since high voltage is applied.
2. More heating effects are seen because of high voltage and the paper becomes
dry.
So in most of the laboratories, low voltage paper electrophoresis is used.
Advantages of Paper Electrophoresis:
1. The technique is easy to follow
2. Less expenditure
3. Number of samples can be separated on a sample paper, at a time.
4. Wide variety of ionisable substances such as amino acids, proteins and peptides,
antibiotics, alkaloids etc., can be separated.
Disadvantages
1. The time required for separation is more, i.e. 6-8 hours in vertical mode and 12-
14hours in horizontal mode.
2. Use of high voltage may be dangerous, unless precautions are taken.
Application of Paper Electrophoresis
Paper electrophoresis is used mainly for the separation of ionizable substances, by using
buffers of different pH and ionic strength. The following are some of the pharmaceutical
applications of paper electrophoresis.
1. Separation of amino acids into acidic or basic or zwitterionic type
2. Separation of proteins in serum (into albumin, α1, α2, β and gamma globulins). The
type of protein and the percentage of each component can be estimated using
densitometer.

28
 3. Separation of lipoproteins in serum (in case of hyperlipidemia)
4. Separations of enzymes in blood.
5. Separation of alkaloids and antibiotics in different samples can be carried out.

GEL ELECTROPHORESIS:
It is a separation technique. Gel is used as medium. The gel may be agar or agarose gel or
polyacrylamide gel.
The device consists essentially of a glass plate over the whole surface ofwhich is deposited a
firmly adhering layer of gel of uniform thickness. The connection between the gel and the
conducting solution is effected in various ways according to the type of apparatus used. Precautions
are to be takento avoid condensation of moisture or drying of the solid layer.
Vertical Gel Instrument- The schematic diagram of a vertical gel electrophoresis apparatus is
given in Figure 4. It has two buffer chambers, upper chamber and a lower chamber. Both chambers
are fitted with the platinum electrodes connected to the external power supply from a power pack
which supplies a direct current or DC voltage. The upper and lower tank filled with the running
buffer is connected by the electrophoresis gel casted in between two glass plates (rectangular and
notched). There are additional accessories needed for casting the polyacrylamide gel such as comb
(to prepare different well), spacer, gel caster etc.

Fig 4: Different components of the vertical gel electrophoresis apparatus

29
Casting of the gel: The acrylamide solution (a mixture of monomeric acrylamide and a bi-
functional cross linker bis-acrylamide) is mixed with the TEMED and APS and poured in between
the glass plate fitted into the gel caster. What is the mechanism of acrylamide polymerization?
Ammoinum persulfate in the presence of TEMED forms oxygen free radicals and induces the
polymerization of acrylamide monomer to form a linear polymer. These linear monomers are
interconnected by the cross linking with bis-acrylamide monomer to form a 3-D mesh with pores.
The size of pore is controlled by the concentration of acrylamide and amount of bis-acrylamide in
the gel. In a vertical gel electrophoresis system, we cast two types of gels, stacking gel and
resolving gel. First the resolving gel solution is prepared and poured into the gel cassette for
polymerization. A thin layer of organic solvent (such as butanol or isopropanol) is layered to stop
the entry of oxygen (oxygen neutralizes the free radical and slow down the polymerization) and make
the top layer smooth. After polymerization of the resolving gel, a stacking gel is poured and comb is
fitted intothe gel for construction of different lanes for the samples. Different steps involves the vertical
gel electrophoresis is shown in the below Fig 5.

Fig 5: Different steps in performance of vertical gel electrophoresis to resolve sample

Running of the gel: The sample is prepared in the loading dye containing SDS, β-mercaptoethanol
in glycerol to denature the sample and presence of glycerol facilitates the loading of sample in the
well. As the samples are filled vertically there is a distance drift between the molecules at the top

30
Vs at the bottom in a lane. This problem is taken care once the samples run through the stacking
gel. The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front whereas
Tris-HCl is moving fast. As a result, the sample gets sandwiched between glycine-Tris and get
stacked in the form of thin band. As the sample enters into the resolving gel with a pH 8.8, the
glycine is now charged, it moves fast and now sample runs as per their molecular weight (due to
SDS they have equal negative charge). After tracking dye reaches to the bottom of the gel, gel is
taken out from the glass plate with the help of a spatula. Gel is stained with coomassie brilliant blue
R250 dye. The dye stains protein present on the gel. A typical SDS-PAGE pattern is given in the
Fig. 6.

Potentials of discontinuous PAGE:

1. Number of disulfide bonds: Comparison of reducing and non-reducing denaturing gels can be used to
provide information related to the number of disulfide bonds present in the protein.
2. Seperating Proteins based on size alone: In the presence of SDS and reducing environment, PAGE
gel resolves two proteins of on the basis of molecular masses and the concentration of gel concentration.
In SDS-PAGE, the relative mobility and the log molecular weight as given by
𝐴−𝑙𝑜𝑔𝑀
𝒗′=𝐕𝐨
𝐴
Molecular weight of a protein can be determined by plotting relative migration Rf with the log
molecular weight of standard protein.
migration of protein from the lane
𝑹𝒇= migration of tracking dye

Fig 6: Determination of molecular weight using SDS-PAGE and Determination of Rf

31
Calculation of molecular weight of the unknown protein sample is a 5 step process:
1. Resolve the protein sample on the SDS-PAGE along with the molecular weight markers.
2. Calculate the relative mobility (Rf) using the following formula:
𝑹𝒇=𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧𝐨𝐟𝐩𝐫𝐨𝐭𝐞𝐢𝐧𝐟𝐫𝐨𝐦𝐭𝐡𝐞𝐥𝐚𝐧𝐞/𝐦𝐢𝐠𝐫𝐚𝐭𝐢𝐨𝐧𝐨𝐟𝐭𝐫𝐚𝐜𝐤𝐢𝐧𝐠𝐝𝐲𝐞

3. Plotlog molecular mass (y-axis) versus relative mobility (x-axis) of the standards.
4. Perform a linear regression using a calculator or using regression software such as
Microsoft Excel.
5. Use the linear regression equation (Y = mx + c) to estimate the mass of the unknown
protein.
Log Molecular Weight = (slope) (mobility of the unknown) + Y intercept

Buffer and reagent for electrophoresis- The different buffer and reagents with their purpose for
vertical gel electrophoresis is as follows-
1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide
polymerization.
2. Ammonium Persulfate (APS)-it is an initiator for the acrylamide polymerization.
3. Tris-HCl- it is the component of running and gel casting buffer.
4. Glycine-it is the component of running buffer
5. Bromophenol blue- it is the tracking dye to monitor the progress of gel electrophoresis.
6. Coomassie brilliant blue R250-it is used to stain the polyacrylamide gel.
7. Sodium dodecyl sulphate-it is used to denature and providenegative charge to the protein.
8. Acrylamide- monomeric unit used to prepare the gel.
9. Bis-acrylamide- cross linker for polymerization of acrylamide monomer to form gel.

32
 Capillary Electrophoresis
Capillary electrophoresis employing a narrow bore fused quartz silica capillary tube usually
50-75cm long with an i.d. of 25-100µm (and an o.d. of 400 µm) containing an appropriate
electrolyte using a direct current (DC) high voltage source, capable of producing a current of 250
µA at voltage ranging from 1000 to 30,000volts and on-line detector that similar to those HPLC are
involved (high voltage electrophoresis).
A cross – sectional view of such a capillary is shown in the Fig 7. The capillary is protected with an
outer layer of a polyimide (polymer of imide monomer)

Fig 7: A cross – sectional view of such a capillary is shown in the

Fig 8: Capillary electrophoresis system

A basic schematic of a capillary electrophoresis system is shown in fig. 8. The system's main
components are a sample vial, source and destination vials, a capillary, electrodes, a high voltage
power supply, a detector, and a data output and handling device. The source vial, destination vial
and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce the
sample, the capillary inlet is placed into a vial containing the sample. Sample is introduced into the
capillary via capillary action, pressure, siphoning, or electrokinetically, and the capillary is then

33
returned to the source vial. The migration of the analytes is initiated by an electric field that is
applied between the source and destination vials and is supplied to the electrodes by the high-
voltage power supply. In the most common mode of CE, all ions, positive or negative, are pulled
through the capillary in the same direction by electroosmotic flow (EOF) (Fig 9). The analytes
separate as they migrate due to their electrophoretic mobility, and are detected near the outlet end of
the capillary. The output of the detector is sent to a data output and handling device such as an
integrator or computer. The data is then displayed as an electropherogram, which reports detector
response as a function of time. Separated chemical compounds appear as peaks with different
retention times in an electropherogram.

Fig 9: Various events of migration of species and EOF in capillary electrophoresis

The efficiency, N (Number of theoretical plates) may be expressed by the equation:
µ𝐸𝑑
𝑁=
2𝐷
Where, D = the diffusion coefficients of migration species,
d = the distance travelled
µ = electrophoretic mobility of the species, and
E = the applied electric field
CE mechanism is entirely different from a chromatographic distribution mechanism, in that it is
readily applicable smaller as well as macromolecules. Thus it is used for the separation of large
biomolecules.
Detection in CE: The most commonly used detectors are a UV absorbance or a fluorescence
monitor or a diode array spectrometer producing absorbance data at multiple wavelengths, on
account of the very small volume (< 10-9L) of the separated analytes, the detection is carried out on
column (or on-line detection). For this a small outer part of the protective polyimide coating from
the capillary surface is removed either by burning, dissolving or scratching. This small opening of
the outer capillary surface then serves the purpose of a detector cell (optical window). The path
length for the focused beam to be passed through such an opening is very small (50-100µm) which
34
utilizes the small volumes. One example of representative Electropherogram which was obtained
from the seized heroin sample is showing in the Fig 10.

Fig. 10:Representative Electropherogram showing the separation of a seized heroin sample byusing
MEKC with short end injection (1) morphine, (2) heroin, (3) acetylcodeine, ISTD = internal
standard (N, N-dimethyl-5-methoxytryptamine). UV absorbance at 210 nm, uncoated fused silica
capillary 50 cm × 50 μm I.D. ×360μm O.D., effective separation length 8 cm, back- ground
electrolyte: 15 mM sodium borate, 25 mM sodium dodecylsulfate, 15% (v/v) acetonitrile, pH 9.5,
25 °C.–25 kV, hydrodynamicinjection.
Application:
1. Capillary electrophoresis (CE) is the primary methodology used for separating and
detecting short tandem repeat (STR) alleles in forensic DNA laboratories.
2. Capillary electrophoresis may be used for the simultaneous determination of the ions NH+4,
Na+, K+, Mg+2 and Ca+2 in saliva.
3. Illicit Drug Analysis: Applications of capillary electrophoresis to illicit drugs in seizures and
toxicology samples
Analytes Matrix Method
Coca alkaloids and sugars Illicit cocaine MEKC (Micellar electrokinetic
chromatography) with indirect UV
detection
Heroin, morphine, Heroin seizures MEKC with short-end
acetylcodeine,caffeine, injection,detection by UV
paracetamol absorbance
Methamphetamine, Urine from subjects CZE using cyclodextrins for
amphetamine,dimethylamph using separationof enantiomers with
etamine, methamphetamine MSdetection
and p- and
hydroxymethamphetamine dimethylamphetamine

35
 The analytical separations by capillary electrophoresis are done in many ways which are
called modes. There are four modes are: (i) capillary zone electrophoresis (CZE) (ii) capillary gel
electrophoresis (CGE) (iii) capillary isoelectric focusing(CIEF) and (iv) capillary
isotachophoresis(CITP).

36
References:
1. P. C. Kamboj Pharmaceutical Analysis – II (Instrumental methods). First edition,
Vallabh Publications; 2010.
2. A. H. Beckett, J. B. Stenlake, Practical Pharmaceutical Chemistry –Part Two Fourth
edition CBS Publishers and Distributors, New Delhi (India); 2001
3. F. Tagliaro, ... S.W. Lewis, in Encyclopedia of Forensic Sciences (Second Edition);
2013 (Science direct)
4. B. K. Sharma, Instrumental Methods of Chemical Analysis-Twenty Seventh Edition;
2011.
5. Indian Pharmacopoeia, Volume 1, The Pharmacopoeial commission Ghaziabad 2007.

37