The Separation Of Compounds Of Different Polarity

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Chromatography is a technique used to separate individual components in a mixture. The basic principle is, they all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas which carries the components of the mixture with it) The mobile phase flows through the stationary phase and carries Different components that travel at varied rates. Thin layer chromatography involves using a thin, uniform stratum of stationary phase coated onto a piece of metal or glass. The stationary phase contains a substance which fluoresces in UV light.

The mobile phase is a suitable liquid solvent or mixture of solvents.

PRINCIPLE AND MECHANISM INVOLVED

The stationary phase - silica gel

Silica gel is a supplement of silicon dioxide (silica). In a giant covalent structure the silicon atoms are joined via oxygen atoms. And, at the silica gel surface of the silicon atoms are attached to -OH groups. So, the surface contains Si-O-H bonds instead of Si-O-Si bonds.

The surface part is depicted by the following structureAs the surface of silica gel has the -OH groups and is very polar, it can form two types of bonds likeHydrogen bonds with suitable compounds around itVan der Waals dispersion forces and dipole-dipole attractions.

Development of chromatogram and separation of compounds

The solvent that soaks up the plate first dissolves the compounds in the dots put on the base line. They are carried up the plate with the solvent moving upwards.Carrying of compounds depends on two things:The solubility of compound in mobile phase. (It is attraction between the molecules of the compound and those of the 5% diethyl ether in methyl benzene)Sticking of compound to the stationary phase. (It is attraction between the molecules of the compound and the silica gel)If the spot contains two compounds - one forms hydrogen bonds, and the other take part in weaker van der Waals interactions.As the hydrogen bond having compound will stick to the surface of the silica gel more firmly than the other one, that one is more strongly adsorbed than the other.

Adsorption

The process by which molecules of a substance, such as a gas or a liquid, collect on the surface of another substance, such as a solid. The molecules are attracted to the surface but do not enter the solid's minute spaces as in absorption.There is a constant movement of a molecule between being adsorbed onto the silica gel surface and going back into solution in the solvent as adsorption isn't permanent. Obviously the compound can only travel up the plate during the time that it is dissolved in the solvent. While it is adsorbed on the silica gel, it is temporarily stopped - the solvent is moving on without it. That means that the more strongly a compound is adsorbed, the less distance it can travel up the plate.

It is very unlikely that both will hydrogen bond to exactly the same extent, and be soluble in the solvent to exactly the same extent. It isn't just the attraction of the compound for the silica gel which matters. Attractions between the compound and the solvent are also important - they will affect how easily the compound is pulled back into solution away from the surface of the silica.

The elution solvent passes the component during chromatography. If this reaches new adsorption places, it means that there will be equilibrium between the elution solvent and the adsorbent.

APPARATUS:

Chromatographic development tanks and paperPlastic-backed silica get TLC plates (with and without fluorescent indicator )Melting-point tubes

REAGENTS:

Phosphomolybdic acid, 12% in ethanolIodineChloroform solutions of squalene, Vitamin E (α-tocopherol), CholesterolChloroform, methyl stearate, methyl oleate and one or more mixturesEluant solution (5% diethyl ether in methyl benzene)

PROCEDURE:

Pencil line is drawn at 1cm from the bottom of the TLC plates without fluorescent indicators and small drops of given known and unknown samples are spotted on them and are labelled. The plates are dried and then eluted with 5% diethyl ether in methyl benzene in ascending manner in a covered lined beaker.After the solvent reached almost top of the plates, the chromatograms are again dried and then one plate Visualized by spraying with phosphomolybdic acid and heated and the other plate exposed to iodine in a covered tank.It is repeated using a plate with fluorescent indicator and observed under a UV lamp. Also repeated with varying sample sizes.Compared the effect of lining the tank with paper soaked in the eluting solvent with chromatograms developed in an unlined tank.

RESULTS:

The results obtained are

Plate 1

(Visualizing methods used UV & Phosphomolybdic acid with ethanol)Distance travelled by the solvent front is 7.5cm

KNOWN SAMPLES

Sl. No.SAMPLE NAMEDistance travelled(in cm )x/7.5(in cm)RF value1Methyl oleate4.44.4/7.50.582Squalene66/7.50.83Methyl stearate4.54.5/7.50.64Cholesterol0.60.6/7.50.085Vitamin -E3.43.4/7.50.45

UNKNOWN SAMPLES

Sample A

Rf = 5.9/7.5=0.78Rf= 0.6/7.5=0.08As the Rf values are close to that of Squalene and Cholesterol, Sample A may contain Squalene and Cholesterol

Sample B

Rf=3.4/7.5=0.45Rf=4.5/7.5=0.6Rf=6/7.5=0.8As the Rf values are close to that of Vitamin E, Squalene and Methyl stearate, Sample B may contain Vitamin E, Squalene and Methyl stearate

Sample C

Rf=0.6/7.5=0.08Rf=4.5/7.5=0.6As the Rf values are close to that of Cholesterol and Methyl stearate, Sample C may contain Cholesterol and Methyl stearate

Sample D

Rf=0.6/7.5=0.08Rf=3.4/7.5=0.45Rf=4.5/7.5=0.6As the Rf values are close to that of Cholesterol, Vitamin E and Methyl stearate, Sample D may contain Cholesterol, Vitamin E and Methyl stearate

Plate 2

(Visualizing agent used is iodine)Distance travelled by the solvent front is 7.5 cm

KNOWN SAMPLES

Sl. No.SAMPLE NAMEDistance travelled(in cm )x/7.5(in cm)RF value1Methyl oleate4.44.4/7.50.582Squalene66/7.50.83Methyl stearate4.54.5/7.50.64Cholesterol0.60.6/7.50.085Vitamin -E3.43.4/7.50.45

UNKNOWN SAMPLES

Sample ARf = 5.9/7.5=0.78Rf= 0.6/7.5=0.08As the Rf values are close to that of Squalene and Cholesterol, Sample A may contain Squalene and CholesterolSample BRf=3.4/7.5=0.45Rf=4.5/7.5=0.6Rf=6/7.5=0.8As the Rf values are close to that of Vitamin E, Squalene and Methyl stearate, Sample B may contain Vitamin E, Squalene and Methyl stearateSample CRf=0.6/7.5=0.08Rf=4.5/7.5=0.6As the Rf values are close to that of Cholesterol and Methyl stearate, Sample C may contain Cholesterol and Methyl stearateSample DRf=0.6/7.5=0.08Rf=3.4/7.5=0.45Rf=4.5/7.5=0.6As the Rf values are close to that of Cholesterol, Vitamin E and Methyl stearate, Sample D may contain Cholesterol, Vitamin E and Methyl stearate Thus, the given unklnown samples contain:

Sample - A

Squalene, Cholesterol

Sample - B

Vitamin - E, Squalene, Methylstearate

Sample - C

Cholesterol, Methylstearate

Sample - D

Cholesterol, Vitamin - E, Methylstearate

DISCUSSION:

1) COMMENT ON THE RELATIVE POLARITIES OF THE FIVE COMPONENTS

The larger Rf of a compound, the larger the distance it travels on the TLC plate. The compound with the larger Rf is less polar because it interacts less strongly with the polar adsorbent on the TLC plate.

" As polarity increases, Rf decreases "

A molecules polarity depends primarily on

1. The extent to which it can hydrogen bond

2. The number of electronegative atoms3. The polarizability of the bonds or atoms4. The net dipole moment of the molecule.

Functional Group Polarity

Alkanes least polar.

Alkenes more polar than alkenes.Conjugated Polyenes and Aromatic Compounds increasing polarity.Ethers and alkyl halides are more polar than the above

Aldehydes, Ketones, and Esters large dipole moment due to the carbonyl.

Amines and Alcohols more polar

Carboxylic Acids are the most polar functional group

Functional groups in order of increasing polarity :[ Hydrocarbons < ethers < tertiary amine < nitro < dialkyl amines < ketone < aldehyde < primary amine < alcohol < phenol < alkanoic acid < sulfonic acid ]

Relative polarity of 5 components

Methyl oleate

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