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Presented By
Zohaib Aslam
Usama Sattar
M Talha
Imran Majeed
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CONTENTS:-
Paper chromatography:-
1. Introduction of chromatography
2. Classification of chromatography
3. Types of chromatography
4. Paper chromatography
5. Principle, Procedure, Types, Application,
Advantages &disadvantages
6. Thin layer chromatography
7. Principle, Procedure, application, Advantages &
disadvantages
8. Comparison of paper chromatography & thin layer
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chromatography
Gas chromatography:-
1. Introduction
2. Types of gas chromatography
3. Principle
4. Working
5. Applications
6. Reference
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Introductionofchromatography:-
Definition:-Chromatography is a technique employed
for separation of the components of mixture by continuous
distribution of the components between two phases. (mobile
phaseand stationary phase).
Chromatography is relatively a new technique which was
first invented by Michael Tswett, a Russianbotanist in
1906.
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Types of chromatography:-
1. Thin layer chromatography
2. Column chromatography
3. Paper chromatography
4. Gas chromatography
5. High performance liquid chromatography
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Paper chromatography
Paper chromatography (PC) was first introduced by German
scientist Christian friedrich schonbein(1865)
Principle:-this technique is a types of partition
chromatography in which the substances are distributed
between two liquids.
i.e., one is the stationary liquid (usually water) which is held
in the fibers of the paper and called stationary phase; the other
is the moving liquid or developing and called the moving phase.
The Components of the mixture to be separated migrate at
different rates and appear as spots at different points on the
paper.
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Procedure:-
1. In this technique, a drop of the test solution is
Applied as a small spot on a filter paper and the
spot is dried.
2. The paper is kept in a close chamber and the
Edge of the filter paper is dipped into a solvent
called developing solvent.
3. As soon as filter paper gets the liquid through its
capillary axis and when it reaches the spot of
the test solution.
4. The various substances are moved by solvent
system at various speeds. when solvent has
moved these substances to a suitable
height(15-18cm),the paper is dried and the
various spots are visualized by suitable
reagents called visualizing reagents.
5. The movement of substances relative to the
solvent is expressed in terms of Rf values.
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the
distance traveled by the solvent.
places.
If Rf value of a solution is zero, the solute remains in the
If Rf value=1 then the solute has no affinity for the
Rf=D1/D2
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1. Descending chromatography: - in this type,
development of the chromatography is done by
allowing the solvent to travel down the paper.
2. Ascending chromatography: - Here the solvent travel
upward direction of the chromatography paper.Both the
descending and ascending chromatography paper are used
for separation of organic and inorganic substances.
3. Ascending-descending chromatography paper:-
It is the hybrid of the above techniques. The upper partof
the ascending chromatography can be folded over arod
and allowing the paper to become descending after
crossing the rod
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4. Radial chromatographypaper:
It is also called a circular chromatography. Here a circular
filter paper is taken and the sample is given at the centerof
the paper.
After drying the spot the filter paper is tied horizontally on
a Petridis containing solvent.so that wick and the paper is
dipped inside the solvent. The solvent rises through the
wick and the component get separated in form of
concentrate circular zone.
5. Two-dimensional chromatography paper:-
In this technique a square or rectangular paper is
used. Here the sample is applied to one of the corners
and developments are performed at right angle to the
direction of first run.
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Application of paper chromatography:-
Paper chromatography is more useful for the analysis
of polar compounds like amino acid,natural products etc.
the different types of applications are listed below.
Separation of mixture of drugs of chemical or
biological origin, plant extracts etc.
Separation of carbohydrates (sugars),vitamins,
Antibiotics,Proteins,Alkaloids,Glycosides,Amino acid
etc.
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Advantages & disadvantages of paper
chromatography:-
Advantages:-
1. Paper chromatography requires very less quantitative
material.
2. Paper chromatography is cheaper compared to other
chromatography methods.
3. Both unknown inorganic as well as organic compounds can
beidentified by paper chromatography method.
Disadvantages:-
1. Large quantity of sample cannot be applied on paper
chromatography.in quantitative analysis paper chromatography
isnot effective.
2. Complex mixture cannot be separated by paper
chromatography
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Gas chromatography
INTRODUCTION:-
Gas chromatography – “It is a process of
separating component(s) from the given crudedrug
by using a gaseous mobile phase.”
It involves a sample being vaporized and injected
onto the head of the chromatographic column. The
sample is transported through the column bythe flow of
inert, gaseous mobile phase. The column itself
contains a liquid stationary phase which is adsorbed
onto the surface of an inert solid.
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Carrier Gas:-
Hydrogen, helium, nitrogen and carbon dioxide are
commonly used.
Hydrogen has low density and better thermal conductivity.
However, it reacts with unsaturated compounds and is
inflammable and explosive in nature.
Nitrogen is inexpensive but it gives reduced sensitivity.
He is the most preferred gas.
Inlet pressure ranges from: 10-50 psi
-Flow rate : 25-150 mL/min for packed columns
-Flow rate: 2-25 mL/min for open tubular column
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Types of Gas chromatography
There are two major types:-
Gas-solid chromatography:-
Here, the mobile phase is a gas while the stationary
phase is a solid.
Used for separation of low molecular gases, e.g.,air
components, H2 S, CS2,CO2,rare gases, CO and
oxides of nitrogen.
Gas-liquid chromatography:-
The mobile phase is a gas while the stationary phase
is a liquid retained on the surface as an inert solids by
adsorption or chemical bonding.
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Principle of working
The mixture of component to be separated is
convertedto vapors and mixed with gaseous mobile
phase.
The component which is more soluble in stationary
phase travel slower and eluted later. The component
which is less soluble in stationary phase travels faster
and eluted out first.
No two components has same partition coefficient
conditions. So the components are separated according
to their partition coefficient.
Partition coefficient is “the ratio of solubility of a
substance distributed between two immiscible liquids ata
constant temperature.
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Working
Fill the syringe with sample.
Record the setting i.e., column temperature,
detector temperature and injection port
temperature.
Introduce sample into the injection port by
completely inserting the needle into the rubber
septum. Note down the injection time.
The sample gets vaporized due to higher temperature of
Injection port and is swept into column by carrier gas.
These sample components now get distributed
between the gas and stationary liquid phase depending
upon their solubilizing tendencies.
The components with minimal solubility move faster
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and those with maximum solubility travel slowly.
The components leaving the column activate detector
and recorder to give a plot.
Calibration curve- a graph is plotted by taking peak areas
on Y axis and concentration of standard compound on X
axis. Concentration ofunknown sample is then
determined by plottingits peak area on same graph.
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The components that slowly leave the column give a
bell shaped curve of shorter peak while the one which
travels faster gives a bluntly pointed curve of larger
peak.
In above graph, the component that first emerges out
of the column is component 4 followed by 2,5,3 and 1.
The area under the curve is determined in order to
obtain the percentage composition of the mixture.
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Applications
Qualitative Analysis – by comparing the retention time or
volume of the sample to the standard / by collecting the
individual components as they emerge from the
chromatograph and identifying these compounds by other
methods like UV, IR, NMR.
Quantitative Analysis- area under a single component elution
peak is proportional to the quantity of the detected
component/response factor of the detectors. It is done by:
Direct comparison method- A(sample) /
A(std) = α C(sample)/C(std)
Where, α is the response factor determined for every pure
compound under given conditions.
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References
Ravi Shankar, text book of pharmaceutical
analysis.
Skoog.D.A, Holler .F.J; principles of instrumental
analysis.
P.C.Kamboj; pharmaceutical analysis- II,
instrumental methods, pg.no: 281-322.
P.D.Sethi; quantitative analysis of drugs; 3rd
edition.
A.V.Kasture; pharmaceutical analysis- volume II.
