You are on page 1of 24

Chromatography

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

Solvent mixture:-n-butanol 75ml.


Acetic acid 25ml.
Distilled water 6ml.
Aniline-diphenylamine locating reagent:-
(A)Solution 1:-
Aniline 1ml.
Diphenylamine 1g.
Acetone 99ml.
(B)Solution 2:-
85%Phosphoric acid
For use , 10 volumes of solution1 are mixed with
1 volume of solution2.

Standards:-Prepare standard solutions of


glucose , lactose , fructose , galactose and xylose
in 10% aqueous isopropanol. The concentration
Silica gel:-Use20X10 Cm plates with 250m
thickness.
Method:-The optimum amount of urine sample
used will vary with the concentration of the
sugars present.
The reagent strips can be used to roughly
estimate the amount of sugars in the urine.
(1)Determine the volume of the sample required
in such a way that 5l will contain5-10g of sugar.
(2)Apply the sample and the standards 1Cm apart
along the line of application.
(3)Place the plate in the chromatography tank.
(4)Let the solvent rise to 12 to15 Cm for about 2
to 3hours.
(5)Remove the plate from the tank. Dry in under
hot air in a fume cupboard.
(6)Spray with the locating agent and heat for 5
minutes in hot air oven at 120C.
...Loaing
Aproximate Rf values of urinary sugars
Aprrox.Rf Characteristic Sugar
colour
0.14 Grey Lactose
0.34 grey Galactose
0.39 pink Fructose
0.42 Grey Glucose
>0.5 Grey brown pentoses
Amino acid chromatography
Requirements:-(1)Air tight tank.
(2)TLC sheet or plate.
(3)Solvent:-(V/V)n-Butanol 35 ml, Acetone 35 ml ,
Acetic acid (glacial) 10 ml , Water (de- ionized) 20
ml.
(4)Amino acid standard solution:-Solution A
5mmol/l each of leucine , phenylalanine ,
tryptophan , valine , proline , hydroxyproline ,
Threonine , glycine , aspartic acid and lysine in
0.1molar HCl.
Solution B 5mmol/l each of isoleuocine ,
methionine , tyrosine ,alanine ,glutamic acid ,
(5)Locating agents:-(i)Ninhydrin reagent:-General
reagent for detection of amino acid.
(ii)Iodoplatinate:-Detect sulphur containing amino
acids.
(iii)Pauly reagent:-To detect histidine or citrulline.
(iv)Ehrlichs reagent:-Used for tryptophan.
(6)Specimen:-plasma or uirne.
Urine:-no pre-treatment.
Plasma:-1ml plasma +4ml ml ethanol.
Mix and stand for 30min.
Centrifuge and use supernatant.
Precautions for amino acid
chromatography
(1)Antibiotics interfere with amino acid
chromatography.
(2)Protein rich milk interfere with amino acids
chromatography.
(3)Early morning of urine is preferable.
Ion exchange
General principle
chromatorgraphy
Ion exchange resins are usually crosslinked
polymers containing ionic groups as part of their
structure.
They have negligible solubility , but are porous
enough for ions to difuse through the resin.
Ion exchange resins are either anion exchange
resin(R-NH3)+OH- which are bases , or cation
exchange resins (R-SO3)H+ which are acids.
R represents a polystyrene resin.
Anion exchangers are usually supplied in the Cl-
form because they are more stable , then they
Cation exchangers are supplied in the H+ form.
If water containing sodium chloride is passed
through a column of cation exchange resin , the
Na+ cations replaced the H+ cations of the resin:-
(RSO3)-H++Na+(RSO3)-Na++H+.
The water now contains H+ ions (obtained from
the resin) together with the original Cl- anions.
If the water is passed through an anion
exchange resin , the Cl- replaces the OH- anion of
the resin:-(RNH3)+OH-+Cl-(RNH3)+Cl-+OH-.
The water now contain H+ and OH- ions which
combine to form H2O.
In this way the water is prepared ion-free.
Immunochromatography
test (ICT)
Immunochromatography technique is used for
qualitative detction of HCG , ILH , malaria ,HBs Ag
, HIV , Leishmania ,etc.
ICT is an qualitative sandwich immunoassay.
Components of ICT
The membrane or strip is pre-coated with:-
(1)Primary antibody at the test band there is
primary antibody.
(2)At the control band there is second
antibody(anti-Ig).
Procedure
The mixture move chromatographically by
capillary action.
ICT test is positive usually there is two bands.
If test is negative there is only one band at the
control region.