Instrumental methods of Analysis(BP 701T)

CHROMATOGRAPHY

BASICS, INSTRUMENTATION
&
APPLICATION
By

Mr Sanjay Jain
Department of Medicinal Chemistry
B R Nahata College of Pharmacy, Mandsaur University, Mandsaur
Instrumental methods of Analysis(BP 701T)

CHROMATOGRAPY
Chromatography is a groups of method for separating
molecular mixture that depends on the differential affinities of
the solutes between two immiscible phases.
Stationary phase is a fixed bed of large surface area.
Stationary phase can be porous or finally divide solid, or a
liquid coated as thin layer on an inert supporting material.
Mobile Phase is a fluid that moves through or over the
surface of the fixed phase. Mobile phase can be liquid or
gas.
Eluent - Fluid entering column/ solvent that carries the
analyte.
Eluate - Mobile phase leaving the column.
Instrumental methods of Analysis(BP 701T)

CHROMATOGRAPY
Chromatographic methods can be classified on the basis of
nature of the stationary and mobile phases.
If stationary phase is a solid the process is called adsorption
chromatography, whereas if the stationary phase is a liquid
the process is called as partition chromatography.
Instrumental methods of Analysis(BP 701T)

PURPOSE OF CHROMATOGRAPHY
• Analytical
Determine Chemical composition of a sample

• Preparative
Used to purify sufficient quantities of a substance
Instrumental methods of Analysis(BP 701T)

CHROMATOGRAPHY TERMS
Eluent - Fluid entering column/ solvent that carries the
analyte.
Eluate - Mobile phase leaving the column.
Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner wall of
the column tubing.
Examples : Silica layer - Thin Layer Chromatography
Instrumental methods of Analysis(BP 701T)

CHROMATOGRAPHY TERMS
Mobile phase - Moves in a definite direction. Liquid (LC),
Gas (GC). The mobile phase moves through the
chromatography column (the stationary phase) where the
sample interacts with the stationary phase and is separated.
Retention time: Time takes for a particular analyte to
pass through the system (from the column inlet to the
detector) under set conditions.
Sample (Analyte) :Substance analyzed in
chromatography.
Solvent:Any substance capable of solubilizing another
substance.
Instrumental methods of Analysis(BP 701T)

ADSORPTION CHROMATOGRAPHY
In adsorption chromatography the mobile phase containing the dissolved
solutes passes over the surface of the stationary phase. Retention of the
components and their consequent separation depends on the ability of
the atoms on the surface to remove the solutes from the mobile phase
and adsorb them temporarily by means of electrostatic forces

Stationary phase
Solutes
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)

ADSORBENTS
“An adsorbent is a substance, usually porous in nature
and with a high surface area that can adsorb substances
onto its surface by intermolecular forces.”
The Ideal adsorbent must fulfill the following requirements:
Insoluble in mobile phase
Inert to solutes (adsorptive)
Colorless especially when work with colored
mixtures
Suitable particle size enough to give good
separation and reasonable flow rate
Instrumental methods of Analysis(BP 701T)

COMMON ADSORBENTS
 Hydrated silica gel
 Silica gel G
 Silica gel S
Silica gel GF254
 Silica gel H
 Silica gel N
 Silica gel HF254
Silica gel PF254
Instrumental methods of Analysis(BP 701T)

PARTITION CHROMATOGRAPHY
“This form of
chromatography is based
on a thin film formed on
the surface of a solid
support( silica gel,
diatomaceous earth) by a
liquid stationary phase.
As the mobile phase containing the solutes passes in close
proximity to this liquid phase retention and separation occur
due to relative solubility of the analyte in two fluid
determined by their partition coefficient.
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)

THEORY OF CHROMATOGRAPHY
Plate theory
Theoretical plate is single
equilibration or transfer of
the solute between
stationary and mobile phase
and the length of the column
required for one
equilibration is called the
height equivalent to a
theoretical plate(HETP)
Instrumental methods of Analysis(BP 701T)

Plate theory

The plate theory based on the work of Martin and Synge

considers the chromatographic system as a series of
discrete layers of theoretical plates. At each of these
equilibration of the solute between the mobile and
stationary phase occurs. The movement of the solute is
considered as a series of stepwise transfers from plate to
plate.
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)
Instrumental methods of Analysis(BP 701T)

The Rate Theory considers the dynamic of solute particles as it

passes through the void spaces between the stationary phase
particles in the system as well as its kinetics as it is transferred to and
from the stationary phase.
chromatographic separation is achieved by selective retardation of the
passage of some compounds through the stationary phase while
permitting others to move more freely. This selective retardation is
evaluated by Retardation factor(Rf) used in complete chromatography
such as thin layer and paper chromatography
Retardation factor(Rf) is a measure of the fraction of its total elution
time that any compound spends in the mobile phase.

Where VM= volume of mobile phase

VS= effective volume of stationary phase
CM and CS are the concentration of solute I

in mobile and stationary phase

Instrumental methods of Analysis(BP 701T)

Where K is partition coefficient (CS/CM) is the

ratio of the solute concentration in stationary
and mobile phase.
Therefore a compound with large partition
coefficient attracted strongly to stationary
phase will have small Rf and long elution time
By dividing each term by VM equation become

Where K’= CAPACITY FACTOR

Instrumental methods of Analysis(BP 701T)

Retention time (RT, tR) is a measure of the time taken for

a solute to pass through a chromatography column. It is
calculated as the time from injection to detection.
tR is the function of the length of the column and the rate
of travel of the solute
The rate of travel is determined by

Where Is the linear velocity of the mobile phase(cm/sec) therefore

Retention volume, VR which is equal to the volume of mobile phase required

to elute a compound from the system
Instrumental methods of Analysis(BP 701T)

Thin Layer Chromatography

Thin Layer Chromatography is a
technique used to isolate non-volatile
mixtures. Thin Layer Chromatography
is conducted on a sheet of aluminium
foil, plastic, or glass which is coated
with a thin layer of adsorbent material.
The material usually used is aluminium
oxide, cellulose, or silica gel.
It is useful in
Identification of components of a
mixture (using appropriate standards)
Analyzing fractions collected during
purification.
Analyzing the purity of a compound.
Instrumental methods of Analysis(BP 701T)

Thin Layer Chromatography

TLC consisting of:
A mobile phase (developing solvent).

A stationary phase (a plate or strip coated with a form of

silica gel).

Analysis is performed on a flat surface under atmospheric

pressure and room temperature.
Instrumental methods of Analysis(BP 701T)

Principle of Thin Layer Chromatography

In Thin layer chromatography(TLC)
separation is based on the principle
of adsorption.
In adsorption chromatography the
mobile phase containing the
dissolved solutes passes over the
surface of the stationary phase.
Retention of the components and
their consequent separation
depends on the ability of the atoms
on the surface to remove the solutes
from the mobile phase and adsorb
them temporarily by means of
electrostatic forces.
Instrumental methods of Analysis(BP 701T)

Principle of Thin Layer Chromatography

The mobile phase solvent flows through
the stationary phase because of
capillary action.

The compound with greater affinity for

stationary phase moves slower rate and
compounds with lesser affinity moves
fast.
Identification of components is done by
calculating the Rf value for each
compound.
distance travelled by solute
Rf =
distance travelled by solvent
 Instrumental methods of Analysis(BP 701T)

Thin Layer Chromatography

The two most common classes of TLC are:
Normal phase Chromatography is used in which the
stationary phase is polar; for example silica gel, and the
mobile phase is an organic solvent or a mixture of organic
solvents which is less polar than the stationary phase.

Reversed phase Chromatography is used when the

stationary phase is a silica bonded with an organic
substrate such as a long chain aliphatic acid like C-18 and
the mobile phase is a mixture of water and organic solvent
which is more polar than the stationary phase.
Instrumental methods of Analysis(BP 701T)

STEPS IN TLC ANALYSIS

The following are the important components of a typical
TLC system:
 Apparatus (developing chamber)
Glass plate
Stationary phase layer and mobile phase
Application of sample
Development of the plate
Detection of analyte
Instrumental methods of Analysis(BP 701T)

1)STATIONARY PHASE
Adsorbents mixed with water or other solvents→ slurry
Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF (Silica gel + binder + fluorescent indicator)
Alumina, Cellulose powder
Kieselghur(Diatomaceous earth +binder)
Instrumental methods of Analysis(BP 701T)

2) GLASS PLATES
Glass plates which are specific dimensions like 20 cm X 20 cm
(Full plate), 20 X 10 cm(Half plate), 20 cm X 5 cm (Quarter
plate) can be used. These dimensions are used since
the width of the commercially available TLC spreader is 20 cm.

Microscopic slides can also be used for some applications like

monitoring the progress of a chemical reaction. The
development time is much shorter like 5 minutes.

Glass plates of different dimensions can also be used when the

TLC plates are prepared without the use of TLC spreader. In
general, the glass plates should be of good quality and should
withstand temperatures used for drying the plates.
Instrumental methods of Analysis(BP 701T)

3) PREPARATION AND ACTIVATION OF TLC PLATES

• The slurry, which is a mixture of stationary phase and water is
prepared by using the ratio mentioned earlier. After preparing the
slurry, the TLC plates can be prepared by using any one of the
following techniques; pouring, dipping, spraying and spreading
can not be ensured.
• Pouring technique: the slurry is
prepared and poured on to a
glass plate which is maintained
on a leveled surface. The slurry
is spread uniformly on the
surface of the glass plate. After
setting, the plates are dried in an
oven is used for spotting. The
disadvantage is that uniformity in
thickness
Instrumental methods of Analysis(BP 701T)

Dipping technique: two plates (either of standard dimensions or

microscopic slides) are dipped in to slurry and are separated after
removing from slurry and later dried. The disadvantage is that a larger
quantity of slurry is required even for preparing fewer plates.

Spraying technique: resembles that of using a perfume spray on a

cloth. The suspension of adsorbent or slurry is sprayed on a glass plate
using a sprayer. The disadvantage is that the layer thickness cannot be
maintained uniformly all over the plate.
Instrumental methods of Analysis(BP 701T)

Spreading: is the best technique where a TLC spreader is used.

The glass plates of specific dimensions (20cm X 20cm/10 cm/5cm)
are taken. The prepared slurry is poured inside the reservoir of TLC
spreader. The thickness is adjusted by using a knob in the spreader.

Normal thickness of 0.25cm is used for analytical purpose and 2 mm

thickness for preparative purpose. Then the spreader is rolled only
once on the plate. The plates are allowed for setting(air drying). This
is done to avoid cracks,
The plates are activated by keeping in an oven at 110 oC to for 1 hour
Instrumental methods of Analysis(BP 701T)

4)Activation of TLC plates is nothing but removing

water/moisture and other adsorbed substances from the
surface of any adsorbent, by heating at high temperature
so that adsorbent activity is retained. The activated
plates can be stored in thermostatically controlled oven
or in desiccator and can be used whenever required.
Instrumental methods of Analysis(BP 701T)

5.Mobile phase:
It is a developing liquid which travels up the stationary phase,
carrying the samples with it. It depends on:
Nature of the substance to be separated i.e polar or non polar
Nature of stationary phase used
Mode of chromatography
Solvent used should be of high purity.
Solvents used:-
petroleum ether
benzene
carbon tetrachloride
chloroform
Instrumental methods of Analysis(BP 701T)

6.Spotting:
1% solution of sample or standard is spotted using a capillary
tube or micropipette. The spots should be kept at least 2cm
above the base of plate and the spotting area should not be
immersed in mobile phase in a developing chamber.
The sample is applied on the narrow-band.
The width of the band must be as narrow as possible.
Instrumental methods of Analysis(BP 701T)

7.Developing chamber:
It is used for the purpose of “TLC plate run
in mobile phase.”
After the mobile phase is poured into the
chamber it is kept closed with lid .
This is done to equilibrate the atmosphere
of empty space in chamber with the mobile
solvent.
This is also known as saturation of TLC
chamber.
Edge effect occurs when the solvent front
in the middle of TLC plate moves faster
than that of edge edge of plate.
Instrumental methods of Analysis(BP 701T)

8.Development of TLC plate:

Different development techniques are used for efficient
separations. They are
1. One dimensional development (ascending or descending
technique).
2. Two dimensional development
3. Horizontal development
4. Multiple development
Instrumental methods of Analysis(BP 701T)

One dimensional development (vertical)

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
Two dimensional technique
This technique is similar to 2-
Dimensional TLC. The paper is
developed in one direction and
after development, the paper is
developed in the second
direction allowing more
compounds or complex mixtures
to be separated into individual
spots. In the second direction,
either the same solvent or
different solvent system can be
used for development.
Instrumental methods of Analysis(BP 701T)

9.Detecting agent:
After the development of chromatogram, the spots should
be visualised. Detecting coloured spots can be done
visually, But for detecting colourless spots, any one of the
following techniques can be used.
a. Non specific method: Where the number of spots can be
detected but not exact nature of compound
Example
i. Iodine Chamber Method: Where brown or amber spots
are observed when the paper is kept tank with few iodine
crystals at the bottom
ii. UV Chamber for fluorescent compounds: When
compounds are viewed under UV chamber at 245 nm or at
365 nm fluorescent compounds can be detected.
Instrumental methods of Analysis(BP 701T)

Specific methods: Specific spray reagents or detecting agents visualizing

agents are
used to find out the nature of compounds for identification purposes
Examples
i. Ferric chloride - For phenolic compounds and tannins.
ii. Ninhydrin in acetone - For amino acids
iii. Dragendroff’s reagent - For alkaloids
iv. 3,5-Dinitro benzoic acid - For cardiac glycosides
v. 2,4-Dinitrophenyl hydrazine - For aldehydes and ketones
Instrumental methods of Analysis(BP 701T)

How to Run Thin Layer Chromatography:

• Step 1: Prepare the developing container

• Step 2: Prepare the TLC plate
• Step 3: Spot the TLC plate
• Step 4: Develop the plate
• Step 5: Visualize the spots
Instrumental methods of Analysis(BP 701T)

Step 1: Prepare the developing container

• Take a beaker with a watch glass on the top.
• Required quantity of solvents are taken into the beaker.
• Cover the beaker with watch glass and mix the solvents.
• Keep them aside until the plate is prepared.
Instrumental methods of Analysis(BP 701T)

Step 2: Prepare the TLC plate

Take a TLC plate and cut it to required length and width.

Now Mark a line about 1 cm from the bottom.
On the line place two dots at equal space.
 Instrumental methods of Analysis(BP 701T)

Step 3: Spot the TLC plate

Take the capillary tube and by
the help of heat make it into two,
so that the end of the capillary
tube will be thin.

It helps to place a small amount

of sample.

Take the required solutions and

spot them at the marked points
Instrumental methods of Analysis(BP 701T)

Step 4: Develop the plate

Put the TLC plate carefully into the beaker.
The solution should not touch the marked line.
Close the beaker with watch glass.
Do not allow the solvent to run off the top of the plate.
Instrumental methods of Analysis(BP 701T)

Step 5: Visualize the spots

Take off the TLC plate from the beaker carefully.
Mark the solvent front level.
Let it dry.
Spray detection reagent solution
Observe the spot and round it with pencil
Instrumental methods of Analysis(BP 701T)

Quantitative analysis
Direct technique: Densitometer is an instrument which
measures quantitatively the density of the spots. When the
optical density of the spots for the standard and test solution
are determined, the quantity of the substance can be
calculated.

Indirect technique: In this technique, the spots are cut into

portions and eluted with solvents. This solution can be
analysed by any conventional techniques of analysis like
spectrophotometry, electrochemical methods etc.,
Instrumental methods of Analysis(BP 701T)

Qualitative analysis

The Rf value (Retardation factor) is calculated for identifying the spots

i.e. in Qualitative analysis. Rf value is the ratio of distance travelled by
the solute to the distance travelled by the solvent front.

The Rf value ranges from 0 to 1. But ideal values are fro 0.3 to 0.8. 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.

Rf =
distance travelled by solute
distance travelled by solvent
Instrumental methods of Analysis(BP 701T)

Applications:-
1.Separation of mixtures of drugs of chemical or biological origin, plant
extracts etc.,
2. Separation of carbohydrates (sugars), vitamins, antibiotics, proteins,
alkaloids, glycosides, aminoacids etc
3. Identification of drugs
4. Identification of related compounds in drugs
5. To detect the presence of foreign substances in drugs
6. To detect decomposition products in drugs