Professional Documents
Culture Documents
Introduction
IS the technique used for the separation of
number of similar components in a mixture.
These closely related compounds include
proteins , peptides , amino acids , lipids ,
carbohydrates , vitamins and drugs.
This technique is based on the principle of
adsorption , partition , ion-exchange and
exclusion properties.
The selection of a particular type of
chromatography to separate the components
depends on the material to be isolated.
General principle
Chromatography consists of a mobile and a
stationary phase.
The mobile phase refers to the mixture of
substance to be separated in a liquid or a gas.
The stationary phase is a porous or solid matrix
through which the sample contained in the
mobile phase enters.
The interaction between the stationary and the
mobile phase causes the separation of
compounds from the mixture.
These interactions include adsorption ,
Classification of
The type ofchromatography
interaction medium used for
stationary phase and mobile phase is the basis of
classification of chromatography.
(1)Column chromatography
In which the stationary phase is packed into
glass or metal columns.
There are several types of column
chromatography they are as follows:-
(A)Partition chromatography.
(B)Adsorption chromatography.
(C)Ion-exchange chromatography.
(D)Gel filtration chromatography.
(E)Affinity chromatography.
(F)High pressure liquid chromatography(HPLC).
(2)Paper chromatography
In which the stationary phase is supported by
the cellulose fibres of a paper sheet.
There are two types:-(A)Ascending.
(B)Descending.
(3)Thin layer
chromatography
The stationary phase is thinly coated onto
glass , plastic or foil plates.
(4)Gas-liquid
chromatography
The mobile phase is normally an inert gas , e.g.
argon , by which the various compounds is the
sample mixture are separated as they pass along
a special column containing a liquid stationary
phase coated on to a support matrix or on the
inside of a capillary column.
Applications
(1)The chromatographic technique is used for the
separation of amino acids , proteins ,
carbohydrates.
(2)It is also used for the analysis of drugs ,
hormones , vitamins and brain amines.
(3)Helpful for the qualitative and quantitative
analysis of complex mixtures.
(4)The technique is also useful for the
determination of molecular weight of proteins.
(5)The technique is used to separate and identify
the phytochemical constituents present in plant
extracts.
Chromatographic
identification of
Urinary sugars
Sugars in urine:-(1)Glucose(glycosuria).
(2)Lactose(lactosuria).
(3) Fructose(fructosuria).
(4)Galactose(galactosuria).
(5)Pentose(Pentosuria).
(6)Sucrose(sucrosuria).
galactos pentos Fructos Lactos Glucos
e e e e e
+ + + + + Benedicts
* - - - + Clinistix
- - - - + BM-test
* - + - - Seliwanof
* - - + - Methylamin
e
* + - - - Bial
Chromatography
Is considered a superior method for
identification of urine sugars.
Thin layer chromatography is quick and
sensitive ; and therefore it is the method of
choice.
Reagents