Paper Chromatography

Paper chromatography has proved to be very successful in the analysis of chemical compound and lipid sample in particular.

Nature of the paper:

The paper commonly used consists of highly purified cellulose. Cellulose, a homopolysaccharide of glucose. Contains several thousand anhydro-glucose units-linked through oxygen atoms. The paper exhibits weak ion exchange and adsorptive properties. Modified forms of paper have been produced in which the paper has been impregnated with alumina, silica gel, and ion-exchange resin etc.

The chemical composition of whatmann filter paper no: 1 is: a-cellulose (98 to 99%), b-cellulose (0.3 to 1%), Pentosans (0.4 to 0.8%), Ash (0.07 to 0.1%) & ether soluble matter (0.015 to 0.1%).

Apparatus:

The apparatus required for paper chromatography are

1) Support for paper

2) Solvent trough

3) Airtight chamber

4) Whattmann filter paper number 1

5) Capillary tubes

6) Samples – Amino acids (or) Pigments

7) Solvents

8) Platinum loop

Paper development

There are two main techniques, which may be employed for the development of paper Chromatograms.

1) Ascending techniques

2) Descending techniques

3) Radial development

4) Two-dimensional chromatography

1) Ascending techniques:

The filter paper is then dried and equilibrated by putting it into on airtight cylindrical jar, which contains an aqueous solution of a solvent. The most widely applicable solvent mixture is n-butanol: acetic acid: Water (4:1:5), which is abbreviated as BAW.

The sheet of paper is supported on a frame with the button edge in contact with a trough with solvent. The arrangement is contained in an airtight tank lined with paper saturated with the solvent to prove a constant atmosphere and separations are carried out in a constant temperature room. Thus, the solvent will ascend into the paper this process is, therefore, termed “Ascending Chromatography”

2) Descending techniques:

The end of the filter paper may be put into the solvent mixture contained in a narrow trough mounted near the top of the container. In this chromatography, the solvent will descend into the paper and this process is then termed “Descending Chromatography”.

This method is convenient for compounds, which have similar Rf values since the solvent drips off the bottom of the paper, thus giving a wider separation.

3) Two dimensional chromatography (3D):

The mixture is separated then the first solvent, which should be volatile: then after drying, the paper is turned through 90⁰ and separation is carried out in the second solvent.

After locating the migrated unknown sample along with standard known sample, a map is obtained and comparing their position with a map of known compounds can identify compounds.

Locating the compounds:

Strip is removed when the solvent has migrated over most of the available space. The distance to which the solvent has run is marked. In most cases, the completed Chromatogram is colorless with no indication of the presence of any compounds. Such a chromatogram is said as “Undeveloped” for locating the various compounds. The filter paper strip is first dried, then sprayed with 0.5% Ninhydrin in acetone and at least heated for a few minutes at 80 to 100⁰ C. the reaction occurs and the colored spots appear at the sites of the amino acids, such as Chromatogram is now called “Developed”.

In paper chromatography, the stationary cellulose phase is more polar than the mobile organic phase.

Identifying the compounds:

The ratio of the distance travelled by a component (i.e. amino acid) to that travelled by the solvent front, both measured from the marked point of the application of the mixture, is called the “Resolution front (Rf)” value for that component.

   Distance from origin run by the compound

Rf = -----------------------------------------------------------------------------

Distance from origin run by the solvent

 

Detection:

The filter paper strip may be sprayed with ninhydrin and heated so that the colored spots indicating the location of amino acids may develop. The color densities of these spots may be measured with a recording transmittance (or) reflectance photometer device.

Ninhydrin test:

• Amines (including α-amino acids) react with ninhydrin to give a coloured product.
• It can be used qualitatively (e.g. for chromatographic visualisation) or quantitatively (e.g. for peptide sequencing).
• The α-amino acids typically give a blue-purple product.
• Proline, a secondary amine, gives a yellow-orange product.
• The test is sensitive enough that ninhydrin can be used for the visualisation of fingerprints.

Applications:

By using this technique

1) To check the control of purity of pharmaceuticals,

2) To the detection of adulterants,

3) To detect the contaminants in foods and drinks,

4) To the study of ripening and fermentation,

5) To the detection of drugs and dopes in animals & humans

6) To the analysis of cosmetics

7) To the analysis of the reaction mixtures in biochemical labs.

THIN LAYER CHROMATOGRAPHY (TLC)

TLC may be either carried out by the adsorption principle (if the thin layer is prepared by an adsorbent such as “Keiselguhr” (or) “Alumina” (or) by the partition principle (if the layer is prepared by a substance such as “Silica gel” which hold water like the paper).

Preparation of the layer:

The glass plate should be washed thoroughly & dried before layer application. The material to be used for layer preparation is a follows:

The selected material is usually mixed with water, it form thick suspension, known as ”Slurry”. This slurry is applied to a plate surface uniformly with 0.25mm thickness. To this layer mix the binder “Calcium sulphate” for better adhesion of the stationary phase. The plates are dried after application of the slurry. If adsorption chromatography is to be performed, the thin layer is activated by heating at 110⁰ C for several hours.

Compounds	Adsorbents	Solvent systems
Mono and Disaccharides	Kieselguhr.G (Sod.acetate) Kieselguhr.G (Sod.Phosphate, pH-5.0)	Ethylacetate/Propanol (65/35) Butanol/Acetone/Phosphate buffer (pH-5.0) (40-50-10)
Amino acids	Silica Gel.G	96% ethanol/water (70/30)
Plant pigments	Kieselguhr.G	Petroleum ether/Propanol (99/1)

Procedure:

Chromatographic plates (20X20cm) of 200m thicknesses are prepared by using a suspension of 30 grams of silica gel G in 63ml of 0.1M Na₂CO₃ solutions by shaking vigorously for 90 seconds. The silica gel slurry is applied on to the glass plate in the form of a uniform layer. These plated are activated at 110⁰C for 30 minutes immediately prior to use.

Then the samples (5 to 100mL) are applied on the silica plate in the form of small drops at regular intervals. In this plate these samples are applied as a spot of less than 5 minutes diameter on the lower right corner of the plates under a stream of warm air.

This plate is introduced into the saturated standard Brinkman developing chamber with the vapor of the solvent mixture with Chloroform: Methanol: Acetic acid: Water (250:74:19:3 v/v) to dip 4.5 cm of its bottom.

When solvent migrates about 15cm, plates are dried in air for 15 minutes and develop in the second dimension (90⁰ rotation clockwise) with CHCl₃: CH₃OH: 7M NH₄OH (230: 90: 50 v/v). The solvent front is again allowed to stand (or) more about 15cm. Then the plate is dried in air for 5 minutes and exposed to iodine vapor (or) UV light the sample molecules can be visualized. When a permanent record of developed plates is desired, plates are sprayed lightly with 10N H₂SO₄ and then heated at 110⁰ for 15 minutes.

By calculating the Rf values are can easily identified the molecules present in the mixture.

Detection: Several detection methods are available. They are,

1) Spraying the plate with 25 to 50% H₂SO₄ in ethanol and heating. This results in charring of most of the compounds, which show up as Brown spots.

2) Iodine vapours are used extensively as a universal reagent for organic compounds. This iodine spot disappears rapidly but can be made more permanent by spraying with 0.5% benzidine solution in absolute ethanol.

Applications:

1) The constituents of the mixture of amino acids, and the constituents of natural lipids and phospholipids are separated and estimated in a short time.

2) Enzymes, nucleic acids, pigments, sugars can also be separated by using this technique.

3) TLC has often been used to identify drugs, contaminants & Adulterants.

Advanced TLC:

TLC can be automated using forced solvent flow, running the plate in an vacuum-capable chamber to dry the plate, and recording the finished chromatogram by absorption or fluorescence spectroscopy with a light source. The ability to program the solvent delivery makes it convenient to do multiple developments in which the solvent flows for a short period of time, the TLC plate is dried, and the process is repeated. This method refocuses the spots to acheive higher resolution than in a single run. See for example: Poole, C. F.; Poole, S. K. "Instrumental Thin-Layer Chromatography," Anal. Chem. 1994, 66, 27A.

Two-dimensional TLC uses the TLC method twice to separate spots that are unresolved by only one solvent. After running a sample in one solvent, the TLC plate is removed, dried, rotated 90^o, and run in another solvent. Any of the spots from the first run that contain mixtures can now be separated. The finished chromatogram is a two-dimensional array of spots.