Slide 1: CHROMATOGRAPHY BY
PHARMACEUTICS CHROMATOGRAPHY : CHROMATOGRAPHY Definition:
The term chromatography (In Greek, Kromatous-Colour and Graphos-Written) meaning colour writing.
It is the method in which the components of a mixture are separated on an adsorbent.
It is a method used primarily for the separation of the components of a sample, in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be solid or a liquid supported on a solid or a gel, and may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be gaseous or liquid. CLASSIFICATION : CLASSIFICATION Partition Chromatography:
This involves liquid or gas as a mobile phase and another liquid or solid as a stationary phase.
This involves liquid or gas as mobile phase and adsorbent solid as stationary phase. Slide 4: CHROMATOGRAPHY Adsorption
solid and Partition
liquid and Ion exchange
Exchange of ions Moleular
(GSC) Liquid Liquid Ion Exchange
Chromatography Thin Layer
Chromatography Thin Layer
(GSC) HPTLC HPLC PAPER CHROMATOGRAPHY : PAPER CHROMATOGRAPHY PRINCIPLE:
The principle involved in separation
by paper chromatography is largely
by partition coefficient phenomenon.
Separation of components depends
on both their solubility in the mobile
phase and their differential affinity
to the mobile phase and stationary phase. OPERATION TECHNIQUE : OPERATION TECHNIQUE Choice of filter paper:
Whatman filter papers are used as chromatography
paper. In general this paper contains 98-99% of
There are various grade and types of paper
available for separation of a sample.
Factors That governs the choice of paper:
Nature of Sample and solvents used.
Based on Quantitative or Qualitative analysis.
Based on thickness of the paper. PREPARATION OF PAPER: : PREPARATION OF PAPER: Cut the paper into desired shape and size depending upon work to be carried out.
The starting line is marked on the paper with an ordinary pencil 5cm from the bottom edge.
On the staring line marks are made 2cm apart from each other. APPLICATION OF SAMPLE: : APPLICATION OF SAMPLE: The sample mixture to be separated is applied as a small spot on the origin line.
The spot is dried on the filter paper and is placed in developing chamber.
Micropipette or glass
capillary is used for
sample application. SOLVENTS: : SOLVENTS: A number of solvents can be used in the paper chromatography. The solvent selection depends upon nature of substance to be separated.
Some Eg of solvents,
Ethyl alcohol n-Butanol these solvents are used in
N-Hexane water different ratio with different
Benzene methanol mixture…
Toluene chloroform CHROMATOGRAPHIC CHAMBER: : CHROMATOGRAPHIC CHAMBER: The chromatographic chamber are made up of many materials like glass, plastic or stainless steel.
Glass tanks are preferred most. They are available in various dimensional size depending upon paper length and development type.
The chamber atmosphere should be saturated with solvent vapour. DEVELOPMENT OF CHROMATOGRAM: : DEVELOPMENT OF CHROMATOGRAM: The paper is dipped in solvent in such a manner that the spots will not dip completely into the solvent.
The solvent will rise up by capillary action. It is allowed to run 2/3rd of paper height for better and efficient result.
After development is complete paper is taken out of the chamber carefully. Slide 12: DRYING OF CHROMATOGRAM:
The chromatogram is dried after its development.
They are dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms.
LOCATION OF SPOT:
If the substance are coloured they are visually detected easily.
But for colourless substance.
Physical and chemical methods are used to detect the spot.
Physical method: In this method observation are done under UV light,
detection of fluorescence and radioisotope measurements.
Chemical method: In this method chemical reagent is used to develop
>Amino Acids-Ninhydrin Reagent
>Alkaloids-Dragendroff’s Reagent Rf VALUE: : Rf VALUE: In paper chromatography the results are represented by Rf value which represent the movement or migration of solute relative to the solvent front. The Rf value is calculated as : Distance travelled by the solute Distance travelled by the solvent front DEVELOPMENT TECHNIQUE: : DEVELOPMENT TECHNIQUE: There are various methods of development of paper chromatography…
In this method solvent moves from top to bottom so it is called descending chromatography.
In this case the solvent migrates upward by capillary action. Slide 15: ASCENDING-DESCENDING CHROMATOGRAPHY:
A hybrid of above two technique is called
Initial ascending chromatography is
performed, often crossing the glass rod
changes to descending.
This is rarely used method, in this case a
circular piece of paper is taken which has
a wick cut parallel to the radius from the
edge to centre. The sample is applied at
the centre of the paper. The paper is then
laid on the edge of circular disk with wick
dipped into the solvent at the bottom of
the dish. Slide 16: TWO DIMENSIONAL CHROMATOGRAPHY:
In this method a square paper is taken the sample is applied to the one
of the corner. Using solvent system the first development is carried as
ascending method. The paper is taken out dried and second
development is performed at right angels to the first dimensional
APPLICATION OF PAPER CROMATOGRAPHY:
Used in the separation of various organic mixture.
Used in almost all area to solve complicated problems in chemistry, biology, biochemistry.
Used for both qualitative and quantitative analysis. Slide 17: REFERENCE:
Dr. A.V Kasture
Dr. K.R Mahadik
Dr. S.G Wadodkar
Dr. H.N More