basic concepts related to chromatography

1. Pharmaceutical analysis-III
Basic concepts related to chromatography
By:- Drx Jayesh.M.Rajput
Points:-
1. introduction to chromatography
2.history of chromatography
3.chromatographic selection of methods
4.classification
1. Introduction :-
Chromatography may be defined as method of separating a mixture of components into
individual component through equilibrium distribution between two phases. chromatography
is based on the differences in the rate at which components of mixture moves through a porous
medium ( called stationary phase ) under the influence of some solvent or gas ( called moving
phase ). Graph showing detector response as a function of a time is called Chromatogram. The
true separation of two consecutive peak on a chromatogram is measured by resolution.
Chromatography is non destructive procedure for resolving a multi-component mixture of
trace, minor, or major constituents into its individual fraction. While chromatography may be
applied both quantitatively, it is primarily a separation tool.
However final identification usually requires confirmation by some other analytical procedure
such as infrared spectroscopy, nuclear magnetic resonance or mass spectrometry.
Chromatrography can be used for the quantitative and qualitative analysis.
2. history :-

2. Chromatography is relatively new technique which was first invented by M. Tswett, a botanist
in 1906 in Warsaw.
Few methods of chemical analysis are truly specific to a particular analyte. It is often found that
the analyte of interest must be separated from the myriad of individual compounds that may
be present in a sample. As well as providing the analytical scientist with methods of separation,
chromatographic techniques can also provide methods of analysis
Chromatography may be defined as method of separating a mixture of components into
individual component through equilibrium distribution between two phases.
Essentially, the technique of chromatography is based on the differences in the rate at which
components of mixture moves through a porous medium ( called stationary phase ) under the
influence of some solvent or gas ( called moving phase ).
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which
may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an
immobile, immiscible stationary phase. The phases are chosen such that components of the
sample have differing solubilities in each phase. A component which is quite soluble in the
stationary phase will take longer to travel through it than a component which is not very
soluble in the stationary phase but very soluble in the mobile phase. As a result of these
differences in mobilities, sample components will become separated from each other as they
travel through the stationary phase.
Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas
Chromatography) use columns - narrow tubes packed with stationary phase, through which the
mobile phase is forced. The sample is transported through the column by continuous addition
of mobile phase. This process is called elution. The average rate at which an analyte moves
through the column is determined by the time it spends in the mobile phase.7,6
Basic chromatographic terminology :-
* Chromatograph: Instrument employed for a chromatography.
* Stationary phase: Phase that stays in place inside the column. Can be a particular solid or gel-
based packing (LC) or a highly viscous liquid coated on the inside of the column (GC).
* Mobile phase: Solvent moving through the column, either a liquid in LC or gas in GC.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
* Elution: The process of passing the mobile phase through the column.

3. * Chromatogram: Graph showing detector response as a function of a time.
* Flow rate: How much mobile phase passed / minute (ml/min).
* Linear velocity: Distance passed by mobile phase per 1 min in the column.3
The chromatographic method of separation, in general, involves the following steps:
1. Adsorption or retention of a substance or substance on the stationary phase.
2. Separation or the retention of a substance by the mobile phase.
3. Recovery of the adsorbed substances by a continuous flow of the mobile phase; the
method being called elution.
4. Qualitative and quantitative analysis of the eluted substances.

4. 3. classification:-
In all types of chromatography, separation of components of a mixture
results either by adsorption or partition for the column material. Binding of
compound to the surface of solid phase take place in adsorption while in
case of partition, a compound gets distributed into two liquid phases. The
chromatographic methods are classified as;
4.1 Partition chromatography:6
This involves liquid or gas as mobile phase and another liquid or solid as
stationary phase.
The operation includes:
(a) Partition column chromatography.
(b) Paper chromatography.
(c) Thin layer chromatography.
(d) Gas-liquid chromatography.
(e) High performance liquid chromatography.
4.2 Adsorption chromatography:
This involves liquid or gas as mobile phase and adsorbent solid as stationary
phase. The type under this category includes:
(a) Adsorption column chromatography.
(b) Thin layer chromatography.
(c) Solid-liquid chromatogrraphy
(d) Gas-solid chromatography.