specialist pharmacist
Lecture ( 11 A )
Ghada Ali AL-Hussiany
Chromatographic technique
is a physical method of separation in which the components to be
separated are distributed between two phases ,one of which is
stationary phase while the other the mobile phase moves in a definite
direction .
Chromatography represents a group of methods for separating
molecular mixtures depend on the differential affinities of the solute
between two immiscible phases .
Stationary phase is fixed ,large surface area may be a porous or finely
divided solid or liquid as a thin layer on an inert support material , while
the mobile phase is a pure liquid or mixture of solution or gas or
mixture of gases which moves through or over the surface.
Type of chromatography
1. Thin layer chromatography (TLC)
This method based on
adsorption chromatography ,the adsorbent silica gel G or C is coated
to a thickness of 0.3 mm on clean TLC plate using spreader, the
plates are activated at 1050C for 30min & used .The selection of
Hmobile depend upon type of constituents to be analyzed .After the
development of chromatogram (is the visual output of the
chromatograph.) by ascending technique ,the resolved spots are
revealed by spraying with suitable detecting agent such as
concentrated sulphuric acid.
This technique is useful in analysis of alkaloid ,glycosides , isoprenoids
,lipid component sugars ….etc
Rf = distance travelled by component /solvent front
Rf may varying depend on purity of solvent ,nature of substance to be
resolved ,composition of solvent ,% of impurity , adsorbent used
,polarity of solvent ,…..etc. This method is important analytical tool
for qualitative &quantitative analysis of natural products.
2.High performance thin layer chromatography (HPTLC)
It's useful in qualitative & quantitive analysis of pharmaceuticals .
The principle is shorter time &better resolution ,the difference
between TLC &HPTLC is only particle &pore size of the adsorbent.The
plates are similar to TLC ,layers of HPTLC are in the form of pre coats
.Silica gel of very fine particle size is widely used as adsorbent in
HPTLC, this fine particle helps in greater resolution &sensitive ,3-6cm
solvent front migration is sufficient to effect proper separation
.HPTLC plates are 4-5um silica gel with an inert binder to form a
200um layer. About 7cm development distance is achieved in about
4minute.Sample preparation in HPTLC needs a high concentrated
solution ,the size of the sample spot must not exceed 1mm in
diameter.
3.Gas liquid chromatography.
Separation volatile substance by percolating a gas steam over a
stationary phase .The bases of separation in GLC is the partition of
the sample in &out of the film of liquid ,spreading over an inert solid
,it’s the most selective &versatile form of gas chromatograph. The
choice of carrier gas shall determine the efficiency &inertness ;N2,He
the most common carrier gas .The important application of GLC
include examination of many volatile oil ,plant acids ,alkaloid of
opium, tobacco ,conium & belladonna ,the resins of cannabis
,steroidal compound ,cardio active glycoside &aglycones ,
sugars&amino acid.
4.High performance liquid chromatography(HPLC).
This method takes place with a packed column .The packing material
is inert material .A liquid mobile phase is used as the eluent.In HPLC
,the mobile phase is forced through the column under high pressure
either autocratic elution or gradient elution .HPLC has become the
most versatile ,safest, dependable ,fastest & sensitive
chromatography technique for the quality control of drug
components .The drugs like morphine ,papaverine , codeine ,emetine
antibiotics ,steroids ,ergot alkaloids ,cardiac glycosides, sennosides
,capsaicin ,vitamins ,rhubarb….etc are analyzed by HPLC.
5.Column chromatography.
It is a liquid chromatography in which the mobile phase in the form of
liquid passes over the stationary phase packed in a column .The
column is either a glass or metallic .The adsorbent agent are used like
starch ,calcium carbonate, magnesium , lime ,silica gel ,alumina
,charcoal &to optimum resolution ,various mobile phases are used
either single or combination, petroleum ether cyclohexane
chloroform ,acetone water , pyridine &organic acid CO2 used for
extraction purposes.