Methodology for Separation Procedures

Thin layer chromatography

The separation and identification of organic compounds is a routine work in many service laboratories. Thin layer chromatography (tlc) is an easy technique to adopt for the said purpose. It is highly useful in research laboratories to separate, identify and characterize unknown compounds. A variety of small molecules like amino acids, sugars, organic acids, lipids etc. are separated by tic technique. The greater advantage of tic is the speed at which separation is achieved. When volatile solvents are used the time required to effect separation is only about 30 min and with nonvolatile solvents it is seldom longer than 90 min.

Principle

The general Principle involved in tlc is similar to that of column chromatography i.e. adsorption chromatography. In the adsorption process, the solute competes with the solvent for the surface sites of the adsorbent. Depending on the distribution coefficients, the compounds are distributed on the surface of the adsorbent. Of course, in tic the partition effect in the separation is also not ruled out. The adsorbent normally used contains a binding agent such as calcium sulphate which facilitates the holding of the adsorbent to the glass plate.

Materials

» Glass Plate (20 x 20 cm or 20 x 10 cm)
» Glass Tank with Lid
» Spreader
» Developing Solvents
» Adsorbent Silica GelG/Alumina
» Sample (should be extracted following the procedures indicated for each group of compounds. For e.g., extraction with 80% alcohol for amino acids and sugars)
» Standards
» Spraying Agent (This also differs as for the group of compounds of interest).

Procedure

Preparation of Plates
Place dry, clean glass plates (5 nos, 20 x 20cm) on the plastic base plate over a plane surface.

1.	Prepare a slurry of the adsorbent in water (sometimes buffer) in the ratio 1:2 (w/v).
2.	Stir the slurry thoroughly for 1-2 min and pour into the applicator positioned on the head glass plate.
3.	Coat the slurry over the glass plates at a thickness of 0.25mm for qualitative analysis by moving the applicator at a uniform speed from one end to the other. (One has to gain some experience by practicing to prepare uniformly coated plates.) Leave the plates to dry at room temperature for 15-30 min.
4.	Heat the plates in an oven at 100-120°C for l-2h to remove the moisture and to activate the adsorbent on the plate. The dried plates in a rack can be stored in a desiccator over silica gel to prevent moisture absorption.

Sample Application

1.	Leave 2.5cm from one end of the glass plate and at least an equal distance from the edges.
2.	Apply the sample and standards by means of a micropipette or syringe as small spots. All spots should be placed at equal distance from one end of the plate. See that the adsorbent does not flake off at the sample application point. (Measured volumes are applied for quantitative analysis.)
3.	Allow the sample to dry so that spotting can be done repeatedly for a more concentrated sample spot.

Developing Chromatogram

1.	Pour the developing solvent into the tank to a depth of 1.5cm. Allow it to stand for at least an hour with a cover plate over the top of the tank to ensure that the atmosphere within the tank becomes saturated with solvent vapor. This is called equilibration.
2.	After equilibration, remove the cover plate, and place the thin layer plate (sample applied) vertically in the tank so that it stands in the solvent with the spotted end dipping in the solvent.
3.	Replace the cover plate. The separation of the compounds occurs as the solvent moves upward. Develop the chromatogram at constant temperature in order to avoid anomalous solvent-running effects.
4.	Once the solvent reaches the top of the plate, remove it from the tank, dry and proceed for the identification of the separated compounds.

Two-dimensional Chromatography
In order to improve the resolution of a particular separation, the two- dimensional chromatography may be used. Here the separation of the compounds is done in two different solvent systems.

1.	Apply the sample as a single spot at one corner of the plate.
2.	Develop in the first solvent system, remove from the tank and dry.
3.	Again develop in the second solvent system in the direction at right angles to the first development.

Identification

If the commercially available adsorbent contains a fluorescent dye, the separated compounds will show up as blue, green or black spots when viewed under UV light. These areas can be scrapped, eluted with a suitable solvent, and quantitative estimations of the separated compounds can be carried out.

When such a dye is not used in the adsorbent, one of the following methods of identification can be followed.
a. Spray with 50% sulphuric acid and heat. This will result in most compounds get charred and show up as brown spots.
b. Examine the plates under U V light which will show the locations of UV absorbing or fluorescent compounds.
c. Expose to iodine vapor for unsaturated compounds.
d. Spray with ninhydrin for amino acids.
e. If the compounds are radioactive, the plates may be subjected to autoradiography and can be detected as dark spots on X-ray film. Alternatively the plate may be scanned by a radiochromatogram scanner.
f. See the table given at the end of this exercise for the detection of specific compounds.

Preparative tlc
The tlc technique can also be used for the isolation of separated compound and in that case it is called as preparative tic. Instead of a thin layer, with a thick layer (up to 5mm) of adsorbent coated, a greater quantity of the sample can be applied onto the plate. After separation, the area of the separated compound is scraped off, eluted with a suitable solvent and recovered in a relatively pure form.
In preparative tic usually, the sample is applied as a streak rather than a spot, for a large quantity can be applied. The compounds are also separated as series of bands which may be scraped off and eluted.

Notes

1.	Cellulose powder is also used as adsorbent in tic in certain cases. This combines adsorption and partition effects. Here, the plates have to be air dried.
2.	Argentation tic: Some compounds may not get separated with the normal adsorbents. In such cases, silver nitrate dissolved in water is used to make the adsorbent slurry and therefore the adsorbent is impregnated with silver nitrate when the plates are dried. This method of preparing the plates is suitable for separating compounds differing in the number and position of the double bonds (e.g. lipids).
3.	The adsorbent, solvent system and the method of identification for certain group of compounds are listed below.

Compound	Adsorbent	Solvent system	Mode of identification	Color
1. Amino acids	Silica gel G	(a) 96% Ethanol/water (70/30) (b) Butan-1-ol/acetic acid/water (80/20/20)	Spray with 0.1% ninhydrin in acetone and heat the plates for 5min at 100 to 110°C	Pink or purple
2. Mono and disaccharides	KieselghurG (sodium acetate)	Ethyl acetate/propan-1-ol (65/35)	(a) Spray with 2% solution of diphenylamine in water saturated with n-butanol or in butanol and methanol (1:1) containing 5% trichloro- acetic acid. Heat at 100°C for 10-15 min.	Aldohexose give brown spots Aldopentoses give purple spots
	Kieselghur G (sodium phosphate)	Butan-1-ol/acetone/phosphate buffer pH 5 (40/50/10)	(b) Spray with 0.5% solution of 3, 5 dinitrosalicylic acid in 4% NaOH. Heat 100°C for 5 min.	Reducing sugars give brown spot on a yellow background
	Silica gel G impregnated with 0.2M sodium borate buffer pH 8.0 or 0.02M sodium acetate or boric acid	Benzene/glacial acetic acid/Methanol (20/20/60)	(c) Spray with 0.2% naphthoresorcinol in ethanol and 2% aq. TCA (1:1) (Mix just before use). Heat at 100°C for 5 to 10 min.	Fructose, sucrose, sorbose and Raffinose give red color. Pentoses and uronic acids give blue color on standing.
3. Neutral lipids (alkanes, triglycrides, diglycerides and monoglycerides	Silica gel G	Petroleum ether/diethyl ether/acetone (90/10/1) (or) Isopropyl ether/acetic acid (24/1)	(a) Spray with ethanolic 0.2% 2,7 dichlorofluorescein. View under UV light (b) Spray with 0.5% ethanolic rhodamine B or 6 G	Light green florescent spots Yellow or blue violet spots on a pink background
4. Plant acids	Silica gel G	Ethanol (96%)/water/25% Ammonium hydroxide (78/9.5/12.5)	Spray 0.1% solution of 2,6 dichlorophenol indophenols in 96% ethanol (Brief heating may aid in color development)	Pink spots on sky blue background. (Oxalic acid gives rocket-like spot)
5. Phenol and phenolic acids (Gallic, protocatechui c, Genetisic, P-hydroxybenzoic, syringic, vanillic and salicylic)	Silica gel G	Acetic acid/chloroform (1/9)	(a) Spray with dilute Folin-Ciocalteau reagent (1:1 with water) followed by spraying with 20% Na₂CO₃ solution; (b) Spray with diazotized sulphanilic acid reagent (Dissolve 50mg diazotized sulphanilic acid in 200mL of 20% Na₂CO₃ solution; Diazotization is done by dissolving 25g sulphanilic acid in 125mL 10% sodium nitrite solution. Add this mixture drop by drop to 60mL 8M HCl in ice-cold conditions. After 10min filter in the cold. Wash extensively with the cold water and then with ethanol, followed by ether. Air dry the crystals and store at 4°C. - Diazotized salt is occasionally explosive at room temp.)	Blue spots at room temperature Yellow to orange red spots
6 Phenols	Silica gel G	Acetic acid/chloroform (1/9) 9or) Ethyl acetate/Benzene (9/11)	Spray with vanillin HCl	*Orcinol and 2-methyl resorcinol give blueish-pink color, 4 methyl resorcinol and resorcinol give red color.
7 Purine and pyrimidine bases	Silica gel G	Ethanol/acetic acid/water (81:5:14)	Spray with 0.25% mercuric acetate and 0.05% diphenyl carbazone	White spots on violet background.

*For identification of other phenols visualize under UV light, elute the spots with ethanol, take A_max and characterize as follows.
Catechol - 279; Hydroquinone - 295; Pryogallol - 266; Phoroglucinol - 269, 273nm
Phloroglucinol shows absorption maximum at 350nm when eluted in ethanolic NaOH solution.

Extraction
1. Amino acids, sugars, phenols, phenolic acids and plant acids.
For extracting these compounds follow the procedure given for amino acid extraction in paper chromatography section.
2. Neutral Lipids: Macerate the fresh leaf tissue in 20 volumes of cold isopropyl alcohol, then re-extract with chloroform-methanol (2:1). Seed tissue can be extracted directly with chloroform-methanol (2/1) mixture. The lipids tightly bound as in cereals can be extracted with chloroform, ethanol, water (200/95/5) mixture. Store the extracts at 5°C in the
presence of antioxidants (Butylated hydroxytoluene BHT 0.005%).

References
1. Williams, B L and Wilson, K (1974) In: Principles and Techniques of Practical Biochemistry, p 52, Edward Arnold London.
2. Horborne, J B (1976) In: Phytochemical Methods Chapman and Hall London p 33.

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