The following points highlight the four main types of chromatography for biochemical investigation. The types are: 1. Ion-Exchange Chromatography 2. Partition Chromatography 3. Gel Chromatography 4. Gas Liquid Chromatography (GLC).
Type # 1. Ion-Exchange Chromatography:
(i) Ion-exchange resins are nothing but cross-linked polymers. The polymers must have negligible solubility but be porous enough for the ions to diffuse freely through it.
(ii) Ion-exchange resins are of cation and anion exchangers. Strong cation-exchange resins contain sulphuric acid groups (-SO3−), weak ones carboxylic acid groups (-COO−), whereas strong anion-exchange resins have -N (R1 R2 R3) and weak ones —N(R1 R2).
(iii) The most important resins are polystyrene resins formed by condensation of styrene (vinyl benzene) and divinyl benzene. Acidic or basic groups are introduced before or after polymerizing.
(iv) Strong alkaline cellulose treated with chloroacetic acid introduces the carboxy methyl group to give the weak cation-exchange resin carboxymethyl-cellulose (CM-cellulose) while condensation with 2-chlorotriethylamine gives the weak anion-exchange diethylaminoethyl-cellulose (DEAE-cellulose). Cellulose ion-ex-change materials are specially suitable for protein separations.
(v) The resins can be looked on as insoluble acids or bases which form insoluble salts shown below.
H+ – Resin− + Na+ → Na+ – Resin− + H+ (Cation-exchanger)
Resin+ – OH− + CI− → Resin+ – CI− + OH− (Anion-exchanger)
The more strongly acidic the ion-change resin, the greater is the ionisation of the acidic group and the lower the pH at which it will exchange.
(vi) Ion-exchange resins have been widely used for the separation of amino acids and peptides. These have also been used to separate organic weak cations or anions from inorganic salts – ion-exchange resulting.
(vii) Mixed beds of anion and cation exchange resins have the property of replacing cations and anions of any salt in water by equivalent amounts of H+ and OH− respectively. This process is used in the preparation of “deionised” water in the laboratory. Non-ionic contaminants are not removed.
Type # 2. Partition Chromatography:
This term covers liquid-liquid partition chromatography (Paper chromatography and thin layer chromatography). Gel chromatography, Gas liquid chromatography.
Paper Chromatography of Amino Acid Mixture:
Introduction:
In biology and chemistry, it is most often necessary to separate components of a mixture which are very similar and are difficult to separate by chemical or physical methods. Chromatography and electrophoresis arc two powerful modern methods utilised for such purpose.
Tswett (1906) the Russian biologist, first appreciated the possibilities of chromatography and he put the term “Chromatography”. Consden. Gorden, and Martin (1944) described paper chromatography in which separations were done mainly by partition.
Principle:
The separation of components of a mixture by a chromatographic system depends on multiple partition process. Small differences in partitioning of each component of a mixture are multiplied many fold. The greater such differences the greater is the ease of separation.
A small drop of solution containing the mixture of compounds is put on a strip of filter paper and allowed to dry. A suitable solvent (mixture of two solvents) is allowed to flow along the filter paper over this spot.
The substances in the initial spot are extracted by the flowing solvent and carried forward along the filter paper to a distance which appears related to their partition coefficient between the free and bound solvent phases of the filter paper. After the solvent has run for a suitable distance along the paper, the paper is removed, dried and subjected to suitable tests to locate the various compounds.
Rf value is defined as the ratio of the distance travelled by the component to the distance covered by the solvent. Rf value depends on the nature of the solvent, the temperature, and the presence of other substances.
Procedure:
Whatmann filter paper is cut into 35×15 cm sheet.
A pencil line is drawn about 3 cm above the shorter edge of the paper and 5 points are marked at equal spacing leaving 2.5 cm from the two edges. On the middle point, a mixture of four amino acids is applied with the help of a fine capillary. It is dried with the help of hot air blown by a hair dryer.
Again, another small quantity of the mixture is spotted at the same place and dried. This process is repeated 2 to 3 times more. On the other points, the individual amino acids of the mixture are similarly applied. The positions of these amino acids are marked with a pencil.
The solvent for developing the chromatogram is a mixture of n-butanol: acetic acid: water (12:3: 5) respectively. This mixture is freshly prepared. If a chromatographic tank is not available, specimen jar (about 40 x 15 cm) with fitting lid can very well serve the purpose.
In such ajar, put about 100 to 150 ml of the solvent mixture and replace the lid so that the lid is airtight. In case of doubts, apply Vaseline to the lid to avoid leakage. In an hour, the inside atmosphere will be saturated with the solvent vapour.
Now fold the paper in which sample has been applied in the shape of a cylinder and tie the opposing ends of the paper with staples or thread. Open the lid and place this folded paper in upright position in the jar, the pencil line lower most and about a centimeter above the solvent.
Replace the lid. The paper should stand absolutely vertically. Leave the chromatogram to develop for 10 to 15 hours or earlier if the solvent has ascended quite near the upper margin of the paper.
Take out the paper at the desired time, cut the stitches and let it dry completely in the air. After the paper has dried thoroughly, the location reagent (0.2 per cent ninhydrin in acetone) is sprayed uniformly on the paper with the help of an all glass sprayer.
The paper is then allowed to dry first in the air and then in a hot air oven at 105°C for 3 minutes. Purple coloured amino acid spots are seen on the chromatogram. Identify the amino acids in the mixture with the help of spots produced by known amino acids.
The Rf values of the amino acids can be calculated as:
Rf = Distance travelled by particular amino acid/Distance covered by the solvent from the point of origin
Reagents:
a. N-butanol (Chromatographic grade).
b. Glacial acetic acid.
c. Solvent mixture of butanol, acetic acid, and distilled water in the proportion of (12 : 3 : 5)
d. Ninhydrin : 0.2 per cent solution of ninhydrin in acetone. It is prepared just before use.
Thin Layer Chromatography (TLC):
Introduction:
In recent years, thin layer chromatography has been developed. This technique consists of a thin layer of absorbents (silica gel, alumina. Kiselguhr or cellulose) on a glass plate or plastic sheet. Since absorbent is used, it is also termed as absorption chromatography.
This technique also provides superior results than that of paper chromatography. The spots are more compact with better resolution and the run is comparatively of shorter duration. Therefore, quicker run is possible.
Preparation of Plates:
Chromatographic plates (20 x 20 cm) of 200 µ thickness are prepared by using a suspension of 30 grams of silica gel G in 63 ml of 0.1 M Na2CO3 solution by shaking vigorously for 90 seconds. Only these plates are used which appear to be uniform in both transmitted and reflected light. These plates are activated at 110°C for 30 minutes immediately prior to use.
Procedure:
Samples (5-100 (JL) are applied as a spot of less than 5 mm diameter on the lower right corner of the plates under a stream of warm air. Plates are first developed in a standard Brinkmann developing chamber previously staturated with the vapour of the solvent mixture with chloroform: methanol: acetic acid: water (250 : 74 : 19 : 3 ; V/V).
When the solvent front migrates about 15 cm, plates are dried in air for 15 minutes and develop in the second dimension (90° rotation clockwise) with chloroform: methanol: 7 M. ammonium hydroxide (230 : 90 : 15 ; V/V). The solvent front is again allowed to move about 15 cm.
Developed plates are then dried in air for 5 minutes and exposed to iodine vapour in a sealed chamber for 30 to 60 seconds. The pale yellow areas are quickly outlined using a dental probe and the plates are exposed to air until the iodine has evaporated from the spots. When a permanent record of developed plates is desired, plates are sprayed lightly with 10 N. H2SO4 and then heated at 110°G for 15 minutes.
The silica gel in each spot is scraped with the aid of a sharp edged polyethylene blade on paper. The weighing papers are then transferred to a 12 ml conical centrifuge tube and eluted by different solvents for estimation by photoelectric colorimeter.
Discussion:
The constituents of the mixture of amino acids, and the constituents of neutral lipids and phospholipids are separated and estimated in a short time.
Type # 3. Gel Chromatography:
(i) This type of Chromatography uses a porous gel. The dry gel particles are first allowed to take up the chosen solvent. This is accompanied by swelling; the liquid taken up constitutes the stationary phase.
(ii) These swollen particles are when made into a column with the same solvent, the spaces between them are filled by solvents. The mobile phase is the “void volume” (Vo); the gel particles are sponge like and the channels within them are of similar diameter (pore size) for a particular grade of gel.
(iii) Molecules can enter the stationary phase only within the gel if their diameter is less than this pore-size. Thus small molecules have the whole fluid volume (bed volume), but molecules above this limit are confined to the void volume and are rapidly washed through the column as mobile phase.
(a) This type of separation is usually performed using the pressure of a small head of the liquid phase to force this through the column. By per-fusing the mobile phase at much higher pressures the separation can be carried out quickly with high resolution.
(b) Dense column packing gives high resolution and high pressure metering systems maintain extremely reproducible solvent flows.
(c) This equipment consists of a column, a solvent delivery system, a sample injecter and a detection. The column is of stainless steel of 10-50 cm long and 2-5 mm internal diameter. The solvent delivery is effected by solvent pumps which generate pressure at several thousand Psi and direct a pulse-free delivery of the solvent.
Constant pressure and constant volume pumps are used for the purpose. The sample injection is done with a syringe. The detectors may be ultraviolet with fixed wavelength Hg lamp 254 nm, refractive index detectors of deflection type, fluorescence detectors.
(d) This method is highly applicable for the separation of carbohydrates, proteins, peptides, amino acids, vitamins, steroids, neuropeptides, hormones and drugs. Separations are achieved very quickly. Hence this method is indispensable in laboratories where advance research and sophisticated analyses are performed.
Type # 4. Gas Liquid Chromatography (GLC):
(i) The substances to be separated are carried as vapours in an inert gas like nitrogen, argon or helium over liquids when there is partitioning of the substances between the gas and the liquid. The liquids used are silicone, oils, lubricating greases, etc. held in inert solids like di-aomaceous earth or ground firebrick.
(ii) Glass or metal tube 1-2 metres long and of 0.2 – 2 cm diameter can be used.
(iii) If fatty acids are to be separated, they are first converted to methyl esters which are easily evaporated. The vapours are swept constantly by nitrogen through the tube containing the liquid phase at a temperature of 170-225°C, so that the vapours of the esters may remain as vapours.
Separation takes places owing to the partitioning of the esters between the gas and the liquid. The ingredients can be identified by physical, chemical means or flame ionisation.