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The following points highlight the top ten techniques of chromatography. The techniques are: 1. Adsorption Chromatography 2. Paper Chromatography 3. Two Dimensional Chromatography 4. Circular Paper Chromatography 5. Ion Exchange Chromatography 6. Thin Layer Chromatography 7. Gas Liquid Chromatography 8. Column Chromatography 9. Affinity Chromatography 10. High Performance Liquid Chromatography.
Technique # 1. Adsorption Chromatography:
This was the original chromatographic technique employed by Tswett (1903). An adsorbent is a solid which has the property of holding molecules at its surface, especially when it is porous and finely divided. This differs from ion exchange resin in that the adsorption of molecules here, to the surface of solids, does not involve electrostatic forces.
Adsorption can be selective in that one solute alone and can be adsorbed selectively from a mixture. Separation of components by this method depends on differences in their degree of adsorption by the adsorbent and its solubility in the solvent used for separation. All these, however, are governed by the molecular structure of the compound.
Separation of Leaf Pigments:
Principle:
The separation of plant pigments is based on the solid-liquid partitioning in column chromatography. The partitioning takes place between the static phase of calcium carbonate and the mobile liquid phase.
Requirements:
1. Chromatography column (20 X 1 cm2).
2. Alumina-100 g.
3. Calcium carbonate-200 g.
4. Sucrose-finely sieved icing sugar 200 g.
5. Anhydrous sodium sulphate 100 g.
6. Petroleum ether (B.P. 60°-80°C) 1 litre.
7. Spinach leaves.
8. Methanol 500 ml.
9. Benzene (toxic) 500 ml.
10. Waring blender.
11. Filter paper.
12. Funnel.
13. Conical flasks.
14. Fume chamber.
15. Water bath.
16. Filter paper discs.
17. Test tubes.
18. Spectrophotometer.
Procedure:
a. Extraction:
1. Cut Spinach leaves and homogenize them in a waring blender.
2. Shake it with a mixture of petroleum ether : methanol: benzene (45 : 15 : 5).
3. Remove residue by filtration and wash filtrate four times with distilled water to remove methanol (avoid vigorous shaking to avoid formation of emulsion).
4. Add anhydrous sodium sulphate to remove last traces of water, filter and remove solid.
5. Concentrate filtrate to a few millilitres by careful evaporation in a fume chamber.
b. Preparation, of coloumn
6. Make slurries of the column material in petroleum ether and pack the column with alumina (5 cm) CaC03 (7 cm) and sucrose (7 cm) inserting filter paper discs in between each adsorbent (gentle suction can be applied to the bottom of the column to assist packing).
7. Wash the column several times with eluting solvent [Benzene : petroleum ether (1:4)].
c. Separation and elution of pigments
8. When column top is dry, add spinach extract and elute with solvent (if flow rate is slow, apply little pressure on top of the coloumn).
9. Collect fractions in separate tubes and plot absorption spectrum of each colour peak.
Technique # 2. Paper Chromatography:
Paper chromatography was introduced by Consden et al. (1944). This technique afford a simple and quick method for analyzing compounds of amino acids, sugars, organic acids etc. There are different types of paper chromatography.
i. Unidimensional Ascending Chromatography:
Cellulose in the form of paper which is the stationary phase is saturated with water in between the cellulose fibers. This acts as a stationary hydrophilic phase. The solute (e.g. amino acids) is spotted at one end of the filter paper. When this is placed in the solvent mixture, the solvent moves across the spot of solute by capillary action towards the top of the paper.
After drying the chromatogram it is sprayed with ninhydrin which reacts with amino acids; ninhydrin is reduced and amino acids are decarboxylated and deaminated. One molecule of reduced ninhydrin with one molecule of unreduced ninhydrin and ammonia react to give rise to a purple coloured complex. Proline and hydroxyproline give yellow colour.
Principle:
In paper chromatography, separation of solutes is based on liquid-liquid partitioning of solutes. Partitioning takes place between the static water molecule adsorbed to paper and the organic mobile phase.
Requirements:
1. Rectangular chromatographic Jars.
2. Whatman No. 1 filter papers.
3. Solvent mixture n-butanol : acetic acid : water (4:1:5 V/V).
4. Known and unknown samples of amino acids/sugars.
5. Ninhydrin (0.2% in acetone).
6. Micropipette/capillaries.
7. Sprayer.
8. Oven at 100°C.
Procedure:
1. Cut Whatman No.l filter paper slightly smaller than the size of jar.
2. Add solvent mixture in the jar.
3. Mark a line 1 cm above the lower end of paper and spot known an unknown amino acids using a micropipette. Size of the spot should be as small as possible and concentrated (25 µl). To saturate the paper, keep it hanging overnight in the jar without touching the solvent.
4. Next morning lower the paper so that the lower edges of it touch the solvent evenly, taking care to see that the edges of the paper does not touch the sides of the jar and that the paper should not be slanted. Cover the jar and allow the solvent to run till it reaches one inch below the upper edge of the paper (4-5 hours).
5. Remove the chromatogram, mark solvent level with lead pencil and suspend it on the clamp of a burette stand. Dry it till it is free of the smell of the solvent mixture.
6. Spray the chromatogram with 0.2% Ninhydrin in acetone and develop by heating it in a hot air oven at 100°C for 10-45 minutes. The spots develop.
7. Outline the spots with lead pencil, measure the distance between the baseline (from the centre of spot) to the centre of the developed spot.
8. Calculate Rf (relative to front) values
RF = Distance travelled by solute / Distance travelled by solvent front
In a given set of temperature, pH etc. the Rf values in a particular solvent system’ will be constant and can be used (Table below) to identify amino acids.
Technique # 3. Two Dimensional Chromatography:
Principle:
Resolution of separation is best achieved by using two different solvents.
Requirements:
1. 8 cm square Whatman No.1 filter paper.
2. Chromatographic jars.
3. Known and unknown samples of amino acids/sugars.
4. Solvent n-butanol: acetic acid : water (4 : 1 : 5) and Phenol: water (1 : 1).
5. Micropipettes /capillaries.
6. Spraying agent—ninhydrin 0.2% in acetone.
Procedure:
1. Load the paper as in the above experiment with known and unknown samples and run the chromatogram in n-butanol: acetic acid : water.
2. Dry the paper well, turn it at right angles and allow it to run in a second jar with phenol : water (1:1).
3. After running, dry the paper and spray it with the reagent (0.2% ninhydrin in acetone).
4. Heat the paper, mark spots that develop, find out their Rf values and identify them based on known samples.
Decending Chromatography:
In decending chromatography, the solvent system is held in a tray above, from where the spotted paper is hung.
Technique # 4. Circular Paper Chromatography:
Principle:
Here the compound to be analysed is spotted on circular filter paper and the solvent is allowed to run radially. Multiple running helps in the separation of overlapping amino acids/sugars which have closer Rf values.
Requirements:
1. Whatman Nol. circular (27 cm diameter) filter paper.
2. Known and unknown samples (amino acids, sugars etc.).
3. Circular tray: A large flat bottomed circular basin with a glass cover and a glass rod ring to support the edges of the paper.
4. A small Petri dish in the center to hold the solvent mixture.
5. Wicks made of chromatographic paper/ cotton.
6. Solvent mixture-n-butanol : acetic acid : water or phenol water.
7. Micropipette/capillaries.
8. Spraying agent (ninhydrin/aniline phthallate).
9. Lead pencil.
10. Hair drier.
Procedure:
1. Mark the centre of the paper, make 4-8 equidistant spots about 2.5 cm away from the center with lead pencil.
2. Load known and unknown samples with micropipette and dry with hair drier.
3. Make a small hole in the centre, and introduce a wick made of 1″ filter paper rolled. Keep the upper level of the wick the same as that of the filter paper.
4. Before inserting the wick, keep the loaded paper in the jar, with solvent in the small Petri dish kept in the centre. Allow to remain overnight for saturation.
5.Next day insert the wick, dip it in the solvent with edges of paper resting on the glass ring (with stand).
6. Allow the solvent to run a distance of 12- 12.5 cm from the center (5-6 hours).
7. Remove the chromatogram holding it by the dry edge with a clean forcep and mark the solvent boundary with a lead pencil and hang it.
8. Allow it to dry overnight (till there is no smell of the solvent).
9. Insert a new ‘wick’ and rerun the chromatogram in the same solvent system till the level of the solvent is a little above the boundary.
10. Repeat this a third and fourth time (multiple running).
11. Spray with 0.2% ninhydrin in acetone for aminoacids and 0.2% solution of aniline phthallate in acetone and dry at 100°C in an oven.
12. Calculate Rf values as given in the first experiment. For quantitative estimation, each spot can be cut and eluted in different solvents.
Technique # 5. Ion Exchange Chromatography:
This is the separation of compounds on an insoluble and chemically inert matrix containing labile ions capable of exchanging with ions into the surrounding media.
Ion Exchange Matrix:
The first synthetic ion exchange material was produced by Adams & Holmes (1935) by condensing phenol, sulphonic acid and formaldehyde to form an insoluble resin. Following this, several resins have been produced, mostly from aromatic compounds.
One such matrix is the one obtained by polymerizing divenyl benzene and styrene.
Copolymerisation of Styrene and Divenyl benzene (Dowex):
Sulphonation (SO3) of the cross linked polystyrene results in a strong cation exchanger, like Dowex 50. However, cross linking styrene with chloromethyl ethyl groups [CH2N+(CH3)] makes a strong anion exchanger such as Dowex 1.
The relative amounts of divenyl benzene and styrene controls the degree of cross linking between polystyrene chains and this in turn effects the water regain. The cross linking can be carefully controlled to produce a sieving effect of the molecules to be separated.
Ion exchange resins are good for separating small molecules like amino acids and not large ones like proteins which cannot penetrate the closely linked structure of this resin.
Separation of Amino Acids:
Principle:
The affinity with which a particular polyelectrolyte binds to a given ion exchanger, depends on the identities and concentrations of the other ions in solutions, because of the competition among these various ions for the binding sites on the ion exchanger. The binding affinities of polyelectrolytes, bearing acid- base groups are also highly pH dependent because of the variation of their net charges with pH.
Requirements:
1. Chromatographic columns (20 X 1.5 cm)
2. Strongly acidic resin 30.0 g
3. HCl (4 M) 1.0 L
4. HCl (0.1 M) 4.0 L
5. Amino acid mixture—aspartic acid— 10 mg histidine and lysine each dissolved in 1 M soln. of HCl.
6. Glass wool.
7. Tris HCl buffer (0.2 M, pH 8.5) 3.0L
8. NaOH (0.1 M) 2.0L
9. 500 ml separating funnels 10 No.
10. Acetate buffer (4 M. pH 5.5) 250 ml
11. Ninhydrin dissolve 20 g in 1000 ml and 3.0 g of hydrindantin in 750 ml of methyl cello-solve and add 250 ml of acetate buffer. Prepare fresh and store in brown bottle.
12. Methyl cellosolve (Ethylene glycol monomethyl ether)—1L.
13. Ethanol (50% V/V) 1L.
14. Ninhydrin 0.2% in acetone-100 ml.
15. 105°C Oven.
16. Water bath.
Procedure:
Preparation of column:
1. Slowly stir the resin with 4 M HCl until fully swollen (15-30 ml/g dry resin) in the tube (column).
2. When resin settles, decant the acid and repeat washing with 0.1 (M) HCl and resuspend this in this solution.
3. Prepare the column containing 5.0 g of this strongly acidic resin and wash with distilled water.
4. When dry, carefully add 0.2 ml of the amino acid mixture to the top of the column and open the tap of the column allowing sample to flow into the resin.
5. Add 0.2 ml of 0.1 (M) HCl, open the tap and allow it to flow into the resin. Repeat this twice.
6. Apply 2 ml of 0.1 (M) HCl to the top of the resin and connect the column to a reservoir containing 500 ml of 0.1 (M) HCl.
7. Adjust the height of the reservoir to give a flow rate of about 1 ml/minute. Collect a total of forty 2 ml fractions in tubes.
8. Test five of the tubes at the same time by spotting samples from each tube on to a filter paper, spray it (dip it) with ninhydrin solution in acetone, dry in an oven at 105°C for 5-6 minutes and test till first amino acid is eluted from the coloumn. Detection of Amino acids
9. Add a few drops of acid/alkali and adjust pH of tubes to 5.
10. Add 2 ml of buffered oinhydrin reagent, heat in a boiling water bath for 15 minutes.
11. Cool tubes to room temperature and add 3 ml of 50% (V/V) ethanol and allow tubes to stand for 10 minutes.
12. Read extinction at 570 nm.
13. With appropriate standards and blanks plot the amount of amino acid in each fraction against the volume eluted.
Technique # 6. Thin Layer Chromatography (TLC):
Izmailov and Schraiber (1938) described separation of plant extracts on a thin layer of alumina. This is in many ways similar to paper chromatography with added advantage in that a variety of supporting media and even mixtures can be used; fluorescent dyes can be incorporated into the medium to identify spots and that separation can be by adsorption, ion exchange, partition chromatography or gel Alteration, depending on the nature of the medium employed.
The process is rapid and the spots are compact, so that it is possible to detect compounds at lower concentration than on paper. Detection of separated compounds by corrosive sprays and elevated temperatures with some thin layer material is possible unlike that with paper.
Preparation of thin layer:
While preparing this layers of silica gel the Rf values will be affected if the thickness of the layer is below 200 µm. A depth of 250 µm is most suitable for most of the separations.
CaSO4 (10-15%) is sometimes incorporated into the adsorbent to bind the layer to the plate. Therefore, once the adsorbent is mixed with water, work should be rapid.
The adsorbent is applied to the surface of the glass plate by a glass rod or pipette or a TLC applicator. Air dry the plates for 15-20 minutes and dry in an oven at 100°C for 10- 15 minutes for activation and store the plates in a desiccator.
Development:
The atmosphere of the jar should be fully saturated; otherwise Rf values will vary. This can be done by using a small tank and lining its walls with filter paper soaked in solvent mixture. Plates should be developed by ascending method for 5 hours.
Experiment :
Identification of sugars in fruit juices using TLC
Materials:
1. Silica gel plates – 10 Nos.
Prepare a slurry of silica gel G (E. Merek) in 0.02 mol/l sodium acetate (chloroform). Dip in the slurry the cleaned glass plates (20 X 5 cm) or spread it to an even thickness of 250 µm using a spreader. Dry at room temperature, remove excess silica gel from edges and activate, before use, in an oven by heating at 105°-120°C for an hour. Cool at room temperature before spotting.
2. Separation chambers with lid – 5 Nos.
3. Solvent system (Ethyl acetate : isopropanol water: pyridine 26 :14 :7:2). Five times the tank volume.
4. Fresh fruit juice — 5 ml. (orange, pineapple etc.)
5. Absolute ethanol – 100 ml
6. Location Reagent. Aniline diphenyl amine (prepare fresh by mixing 5 vol of 10 g/l aniline and 5 volumes of 10 g/l diphenylamine in acetone with 1 volume of 85% phosphoric acid).
7. Desiccator.
8. Oven at 105°C.
9. Hair drier.
10. Sprayers.
11. Standard sugar solution – 10 ml
(10 g/l in 10% V/V isopropanol of rhamnose lactose, ribose, glucuronic acid xylose, glucosamine, fructose, glucose).
Procedure:
1. Add 3 ml of ethanol to 1 ml of fruit juice and centrifuge to remove denatured protein.
2. Carefully spot the supernatant on to TLC plates along with standard sugar solutions. The spotting should be done with micropipettes equidistantly 2 cm above the lower line of silica gel.
3. After the spots dry, place the plates in the chamber saturated with solvent using filter papers dipped in solvent and kept on the -sides of the jar. This saturation is known as equilibration. Solvent should be about 1.5 cm.
4. Allow it to run till one inch below the top and mark the line of solvent level.
5. Dry the plates at room temperature (28°-30°C).
6. Spray with aniline—diphenylamine in a fume chamber and heat them to 100°C.
7. Measure the distance moved by the sugars from their origin and determine Rf values.
Rf = Distance in cm travelled by the compd. from origin / Distance in cm moved by solvent from origin
Preparative TLC:
When TLC is used for the isolation of separated compounds, it is called preparative TLC. Here instead of a thin layer, a thick layer (upto 5 mm) of adsorbent is coated , and a greater quantity of sample is loaded as a streak rather than a spot since it helps in loading more quantity. The compound also separates as streaks which may be scraped, eluted and recovered in a comparatively pure state.
Solvent systems (TLC):
For Lipids:
Petroleum ether (B.P. 60°-70°C) : diethyl ether : glacial acetic acid (80:20:1).
For Alkaloids:
Unidimensional chromatography:
(i) Chloroform: ethyl acetate (1:1)
(ii) Benzene : ethyl acetate (1:1)
(iii) Ether : Ethyl acetate (1:1)
(iv) Benzene : Ether (1:1)
These solvent mixtures (fresh) are shaken with equal volume of liquor ammonia and used.
For Amino Acids:
One phase system
Methyl ethyl ketone: Pridine : water.
Two phase system
1.5% ethanol is used to stabilise chloroform.
Chloroform: methanol (90: 10)
Chloroform: formic acid (100: 5)
Heptane system
M.heptane : ethylene chloride : formic acid (90 : 30 : 21 : 18) use 100 ml of upper phase.
Reagents for Alkaloids:
Mayer’s reagent: (Potassium mercuric iodide) 1.36g HgCl2 in 60ml distilled water plus 5.0g of KI in 10 ml distilled water and make up volume to 100 ml with distilled water.
Wagner’s reagent I2-KI solution:
1.27 g of I2 plus 2.0 g of KI dissolved in 5 ml distilled water and made upto 100 ml with distilled water.
Identification (TLC):
1. If commercially available adsorbent contains a fluorescent dye, the separated compounds, when viewed under U.V. light will be seen as green, blue or black spots. Such areas can be scraped, eluted with suitable solvents and quantitative estimation of the separated compounds can be carried out.
2. If such a dye is not used in the adsorbent, one of the following methods can be used:
(a) Spray with 50% H2S04 and heat. The charred spots appear as brown.
(b) Observe plates under U.V. light which will show spots of U.V. absorbing or fluorescent compounds
(c) For unsaturated compounds, expose to I2 vapour.
(d) For amino acids spray with ninhydrin.
(e) For sugars spray with aniline- diphenyi amine in water saturated with n-butanol or in butanol: methanol (1:1) containing 5% trichloro acetic acid, heat at 100°C for 10-15 minutes,
(f) For radioactive compounds, subject the plates to auto radiography and then dark spots can be detected on X-ray film or the plate may be scanned by a radio chromatogram scanner.
Technique # 7. Gas Liquid Chromatography (GLC):
This technique is applicable to compounds that exert significant vapour pressure at temperatures below those of excessive pyrolysis. Such compounds can be converted to stable volatile derivatives that can be separated by vapour-phase chromatography.
GLC is based on the distribution of compounds between a liquid and a gaseous phase. This is applicable for the qualitative and quantitative analysis of a wide range of compounds because of its rapidity and high sensitivity.
A stationary phase of liquid material such as silicon grease is supported on an inert granular solid. This material is packed into a narrow glass or steel column. Through this, the inert gas (mobile phase) such as nitrogen or argon passes. The column is maintained in an oven at an elevated temperature which volatalises the compound to be analysed.
The basis of separation of the compounds being analysed is the difference in the partition coefficients of the volatalised compounds between the liquid and gaseous phases as -they are carried through the column by the gas. As the compounds pass through the column, they pass through a detector. This is linked via an amplifier to a chart recorder which records a peak as a compound passes through the detector.
Method has been described under ‘Nitrogenase estimation’,
Technique # 8. Column Chromatography:
In column chromatography, the stationary phase is packed in a cylindrical column made of glass or plastic chromatographic columns, have a number of adjacent zones in each of which there is sufficient space for the solute in order to achieve complete equilibrium between the mobile and the stationary phases. Each zone is called a theoretical plate and its length in the column is known as plate height. If the number of theoretical plates are more, the column will be more efficient.
Principle:
The solute competes with the solvent for surface sites of the adsorbent and depending on the distribution coefficients, the compounds are distributed on the surface of the adsorbent.
Requirements:
1. Adsorption columns:
Long cylindrical tube, like burette, constricted at one end and connected to a suction flask. Size of the tube may be much longer depending on the material to be separated.
2. Filling material:
Sephadex G 100-several materials are available and selection should be made based on the material to be separated. Permutit is one such material.
3. Elution buffer.
4. Fraction collector.
5. Peristalitic pump.
6. Material to be resolved along with known sample.
Procedure:
a. Preparation of material:
1. Suspend sephadex G 100 in elution buffer overnight or until it is fully swollen and make a good slurry.
Large number of materials are used in chromatography. They Have to be equilibrated with solvent before packing the column. In addition, some type of pretreatment is required e.g. some gel filtration material should be ‘activated’. This is done by heating or by acid treatment and ion exchange resins have to be obtained in the required ionized form by washing. During equilibration with solvent, allow the material to settle down and suspended fine particles should be decanted in order that the flow rate may not get reduced.
Fine adsorbents like magnesium oxide is mixed with heat treated silicaceous earth since fine adsorbents show low filtration.
b. Packing the column:
2. Pack the neck of the burette with little glass wool (optional) and pour a little amount of buffer to avoid trapping of air bubbles. Immediately add the slurry. A metal rod fixed to a disc, having a diameter slightly smaller than that of the column, is used to press the gel and pack it. Apply little suction also.
Take care to see that the adsorbent is never sucked dry since it will shrink and the column will be useless. Every-time, before adding next portion of gel, the surface should be stirred with a glass rod. The level of the liquid should be just above the surface of the material.
3. Place a filter paper disc on top of the gel to avoid disturbing the surface during sample application after equilibrating the column thoroughly.
c. Application of sample
4. Dissolve the sample in solvent and apply it gently by a pipette and open the tap until top of the column is just above the miniscus (1/10 th –⅕ th of column).
5. Connect the solvent reservoier in which the liquid level is maintained and develop the chromatogram.
d. Elution
6. Using appropriate solvent, elute materials from the column. In displacement, development the solvent interacts more strongly with the chromatographic material than the compound on the column, thus displacing the bound compound molecules.
7. Collect the effluent into a series of tubes, preferably in a fraction collector (automatic).
8. Analyse each fraction.
9. The effluents can be routed through a suitable spectrophotometer to monitor absorbance (at either 280, 230 or 210 nm for proteins).
10. Record the data.
The volume of mobile phase required to elute a particular solute is known as the elution volume, while the corresponding time for elution of the solute at a given flow rate is known as the retention time. Elution is continued for 2-3 times the volume of buffer, until the absorbance monitored reaches base line value.
11. Repeat with reference compound, plot logarithms of molecular weight of marker proteins against their respective ratios of elution volume to column void volume. Compute elution volume of protein of interest and deduce its molecular weight from graph.
Technique # 9. Affinity Chromatography:
One of the important characteristics of many proteins is that they bind to specific molecules tightly, but noncovalently. This property helps in purifying such proteins by affinity chromatography. A molecule known as ligand specifically binds to the protein of interest. This is a covalent attachment to an inert and porous matrix.
Principle:
When an impure solution is passed through ligand, the desired protein binds to the immobilised ligand and other substances are washed through the column with buffer. Then the desired protein can be recovered in a highly purified form from the ligand by changing the elution conditions.
The chromatographic matrix in affinity chomatography must be chemically inert, should have high porosity and large number of functional groups capable of forming covalent linkages to ligands. For this, agarose which has several hydroxyl groups is the best.
If the ligand has a primary group that is not essential for its binding to the protein of interest, the ligand can be covalently linked to the agarose by the following steps.
1. Agarose is reacted with cyanogen bromide to form an “activated” but stable intermediate (commercially available).
2. Ligand reacts with activated agarose to form covalently bound product.
After a protein has bound to a column and washed of impurities, the protein should be released from the column by eluting with a solution or compound that has higher affinity for the protein binding site than the bound ligand.
Technique # 10. High Performance Liquid Chromatography (HPLC):
HPLC is increasingly used for analysis of substances like mycotoxins. The separations are improved to a great extent through the use of high-resolution columns and the column retention times are much reduced. By this technique, separation of various constituents of a sample, their detection and measurement is possible. There are two phases, a mobile liquid and a stationary liquid/solid phase.
The packing material is non-compressible matrix of fine glass or plastic beads coated with a thin layer of the stationary phase. Alternatively, the matrix may consist of silica whose available hydroxyl (OH–) groups can be derivatized with many of the commonly used functional groups of ion exchange chromatography or affinity chromatography. The mobile phase is a solvent system.
Separation is achieved by competitive distribution of the sample between the two phases. A column of 25 cm long and 4 mm internal diameter supports the stationary phase. Optimization of column parameters and in particular, particle size in column packing provides high separating efficiency. The mobile phase is pumped through the separating column at a pressure upto 5000 psi (pounds per square inch) leading to reduced analysis time. The elutants are detected as they leave the column according to their U.V. aborption, refractive index or flourescence.
When each component passes through the detector, there is a change in the electrical output which is recorded on a moving chart to give a chromatogram. The time taken for a substance like mycotoxin to pass through the column under fixed conditions is constant and is called the retention time. Comparison of this retention time with standards enables qualitative analysis. The area under each peak on the chromatogram is proportional to the concentration of the component and helps in the quantitative analysis.