Introduction to Chromatography:
The name of this technique comes from the Greek word Khroma meaning ‘color’. So ‘chromatography‘ means, ‘color writing’. This technique was discovered by Tswett in 1903.
Definition:
A technique for the preparation of components of a become of the distribution of the components between two phases, is called chromatography. This separation is due to the difference in solubilities of the component between these phases.
Stationary phase:
The phase over which mobile phase flows in chromatography is called the stationary phase.
Important points:
- It may be solid or liquid supported on solid or a gel.
- It may be packed in a column.
- It may be spread as a layer or distributed as a film.
- Chromatographic had is a term used to denote any of the different forms in which the stationary phase is used.
- Examples: Silica gel, alumina, filter paper, etc. are some important stationary phases.
Mobile phase:
The solvent or mixture of solvents used for separating components in chromatography is called the mobile phase.
Important points:
- It is also called eluant.
- It competes with the stationary phase for components of the mixture.
- Examples: Water, ethanol, acetic acid, acetone or gas, etc, are some important mobile phases.
Principle of chromatography:
The principle involved in chromatography depends upon the relative solubilities of the components between the two phases. It is governed by the distribution coefficient (KD). The partition coefficient is the ratio of the concentration of components in the mobile phase to the concentration of the component in the stationary phase.
There are possibilities for the value of KD.
- A component having a smaller value of KD mostly remains in the stationary phase as the mobile phase flows over it.
- A component having a higher value of KD remains largely dissolved in the mobile phase and it quickly passes over the stationary phase.
Classification of chromatography
It can be classified in three ways:
- On the basis of shape
- On the basis of phase
- On the basis of the mechanism
On the basis of shape:
Chromatography is classified into various types depending upon the way in which it is carried out.
Examples: Column chromatography, thin layer chromatography (T.L.C), paper chromatography, ascending, and descending paper chromatography.
On the basis of phase:
It is also classified on the basis of phases. As described before the mobile phase can be gas or liquid while the stationary phase can be liquid or solid. Thus we have four combinations:
- Liquid-liquid chromatography
- Liquid – solid chromatography
- Gas-liquid chromatography
- Gas – solid chromatography
On the basis of mechanism:
Two types of machines are found in chromatography. In some cases absorption takes place and sometimes separation involves simple partitioning. From this point of view, chromatography is classified into two types:
Adsorption chromatography:
When physical forces are involved in retentive ability (ability to hold water) of the stationary phase, the chromatography is an adsorption chromatography. Simply we can say that chromatography in which solid substance is used at the stationary phase is called adsorption chromatography.
Partition chromatography:
If separation involves the distribution of the components between liquids then it called partition chromatography. Chromatography in which liquid is used as the stationary phase is called partition chromatography.
Paper Chromatography
in 1944, Consider Gondon and Martin introduced paper chromatography. They employed shoes of filter paper as stationary support. The phase is a house that passes within the porous structure of the paper.
Introduction:
in paper chromatography, the piece of Whatman No. I grade filter paper contains water which is loosely combined or absorbed with the of the paper. The thin film of adsorbed water acts as a stationary phase. The phase usually an organic liquid flows over the paper. It is a kind of partition chromatography. In partition chromatography solutes are distributed between two liquids.
Different ways of paper chromatography:
There are 3 common ways of carrying out this technique.
- Ascending paper chromatography
- Descending paper chromatography
- Radial/circular paper chromatography
Ascending paper chromatography:
In this method, the solvent is in a pool at the bottom of a vessel in which paper is supported. This vessel is known as the chromatographic tank. The mobile phase mines upwards due to capillary action.
A solvent mixture (mobile phase) prepared in accordance with the sample to be separated Thus mixture is put into the chromatographic tank. A 20 cm strip of Whatman No. I grade filter paper is
taken. A thin pencil line is drawn on filter paper about 2.5 cm from one end.
Then spot a point with a narrow capillary tube on the line with the sample mixture solution. To make identification of components easy, spot the points of known compounds on the same line. All spots should be pointed using separate capillary tubes Fig (2.6) B and C are two pure components. A is a mixture of components B and C.
Let the spots dry for some time and then suspend the paper with clips in such a way that the sound on which points are spotted, dips into the solvent up to 5-6 mm. As the solvent rises through the paper, a competition starts. The paper can absorb the solute particles and solvent can dissolve them When the solvent front has risen to about
3/4th of the length of the paper, remove the strip. Mark the solvent front with a pencil and allow the strip to dry. If mixture contain colored components their bands can
be visually identified. If the components are colorless, they can be located by spraying certain reagents. These reagents are called locating agents. The important locating agents are ninhydrin, rubeanic acid, etc.
After locating the bands of components, Rt (retardation factor) values of the components are calculated as:
Each component has its specific R, the value from which it is identified. The chromatography indicates that sample A has both components B and C. Their Rr, values are given by:
Uses of chromatography:
Although it is a simple technique yet it has many important applications:
- It is very useful in the separation and identification of colored pigments.
- Medicines and proteins, which are complicated molecules can be identified.
- Amino acids travel different distances with solvents and hence can be identified.
- Substances produced by a body like urine can also be analysed by chromatography.
- The purity of the compounds was checked by chromatography. Pure substance gives one spot only.
- It separates substances from complete mixtures of closely related substances which cannot be separated by ordinary chemical methods
- It separates a mixture of very small quantities at very low concentrations.
- It is equally important in qualitative and quantitative analysis.
- It is very useful in organic synthesis for separation, purification, and identification of products.
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