Paper ChromatographyBCH 332 Lecture 10

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Paper chromatography

• Paper chromatography and TLC are examples of adsorption chromatography.

• Cellulose support is in the form of sheet of paper which has large amount of water bound to it.

• Partitioning occurs between the bound water and developing solvent.

• The solvent used is water.

• There is one effluent and substances are distinguished by their positions in the paper after the solvent has moved a given distance.

• Uses: To identify unknown samples

• Isolation of components of mixtures.

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Paper chromatograms can be developed by either ascending or descending solvent flow.

Ascending and descending Paper Chromatography

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Ascending Descending

Direction and mechanism of solvent movement

mobile phase separates the mixture by capillary action

mobile phase moves downwards by gravity

Speed Slower Faster

Convenience Easier More difficult to setup

Separation Mobile phase stops due to a balance formed between the capillary strength and downward gravitational pull

Calculation of Rf value is not possible in this process. So standards are run simultaneously

Rf values not suitable for slow moving solvents and low Rf values

For quantitative separations of substances with very small Rfvalues

Polarity Polar substances will be on the bottom

polar substances will be on the top with respect to the tank where as the least polar ones will be at the bottom.

Two Dimensional Paper Chromatography

• After chromatography has been carried out in a single direction the paper is dried and rechromatographed at right angles to the original direction of flow using a different solvent system.

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Detection of spots in the paper

• By color

• Fluorescence

• Chemical reaction after the paper is sprayed with various reagents

• Radioactivity.

• Identification is based on comparison with standards of known Rf or by elution.

• Elution is accomplished by cutting out the spot and soaking the paper in appropriate solvent.

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Thin Layer Chromatography (TLC)

• Originally used to separate lipids.

• Substances used include inorganic substances like silica gel, aluminium oxide, and magnesium silicate.

• Organic substances like cellulose, polyamide, polyethylene powder.

• Used for separation of small molecules.

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• Stationary phase is a layer (0.25-0.5 mm) of sorbent spread uniformly over the surface of a glass or plastic plate.

• A slurry of sorbent is made in a solvent for that particular sorbent.

• The plate is coated with the slurry and allowed to dry at 100-120 in an oven.

• This dries the slurry and activates the adsorbents.

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• The TLC plate is placed in a chamber containing the solvent and developed by ascending chromatography.

• After the solvent has reached the top the plate is removed from the chamber and dried.

• Spots are located as on paper by color, fluorescence or by spraying.

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Polarity in TLC

stationary phase

silica gel attached to the TLC plate.

mobile phase

TLC solution- usually mixture of a polar and a non-polar organic liquid, such as ethyl acetate (polar) and hexanes (non-polar).

Stationary phase (silica gel) is polar.

Polar compounds have an affinity for the silica gel

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Detection

• Commonly used sprays are

• Ninhydrin for amino acids

• Rhodamine B for lipids

• Sulfuric acid plus heating for organic substances

• Potassium permanganate in sulfuric acid for hydrocarbons

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Elution

By scraping off the sorbent and eluting the powder with a suitable solvent.

Advantages of TLC

• Greater resolving power because the spots are smaller.

• Greater speed of separation

• Wider choice of materials as sorbents.

• Easy detection of spots.

• Easy isolation of substances from chromatograam.

• Smaller amounts of materials (one nanomole can be detected).

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Various applications of thin layer chromatography (TLC)

• Purity of any sample: Purity of sample can be carried out with TLC. Direct comparison is done between the sample and the standard or authentic sample; if any impurity is detected, then it shows extra spots and this can be detected easily.

• Identification of compounds: Thin layer chromatography can be employed in purification, isolation and identification of natural products like volatile oil or essential oil, fixed oil, waxes, alkaloids, glycosides, steroids etc.

• Examination of reactions: Reaction mixture can be examined by Thin layer chromatography to access whether the reaction is complete or not. This method is also used in checking other separational processes and purification processes like distillation, molecular distillation etc.

• Biochemical analysis: TLC is extremely useful in isolation or separation of biochemical metabolites or constituent from its body fluids, blood plasma, serum, urine etc.

• In food and cosmetic industry, TLC method is used for separation and identification of colors, preservatives, sweetening agent, and various cosmetic products.

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