CHROMATOGRAPHY

Lab # 5

introduction• Chromatography is a laboratory technique for the

separation of mixtures.• It involves passing a mixture dissolved in a mobile phase

through a stationary phase, which separates the analyte.

• Different chemicals in a mixture have different degrees of

dissolving in a liquid (mobile phase) or sticking to a solid surface (stationary phase).

Chromatography terms • The analyte: is the substance to be separated during

chromatography.

• The eluate: is the mobile phase leaving the column. • The eluent: is the solvent that will carry the analyte.

• The mobile phase: is the phase which moves in a definite direction, It may be a liquid (LC), a gas (GC).

• The stationary phase: is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.

Principle of chromatography

• Chromatography involves a sample being dissolved in a mobile phase (which may be a gas or a liquid).The mobile phase is then forced through an immobile, immiscible stationary phase.

• The phases are chosen such that components of the sample have differing solubilities in each phase.

Principle of chromatography

• A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase.

• As a result of these differences in mobilities , sample components will become separated from each other as they travel through the stationary phase.

Steps of chromatography

1. Compound is placed on stationary phase.

2. Mobile phase passes through the stationary phase.

3. Mobile phase solubilizes the components.

4. Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases.

5. The molecules in the mixture that adsorbs the most to the stationary phase is moving slowest through the particle bed.

Types of chromatography

1. Column chromatography.

2. Planar chromatography.

a. Paper chromatography.

b. Thin layer chromatography

Column chromatography

• In column chromatography, the stationary phase (solid adsorbent) is placed in a vertical glass column and the mixture is added to the top and the mobile phase will flows down through the column so, the substance with less ability to adsorb in the stationary phase will be separated first .

Column chromatography

Column chromatography

Planar chromatography

• A separation technique in which the stationary phase is present as or on a plane.

• The plane can be a paper, or a layer of solid particles spread on a support e.g. a glass plate (TLC).

Thin Layer Chromatography

• Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction.

• It is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material (0.25mm), usually silica gel, aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase.

Thin Layer Chromatography

Retardation Factor

• Rf is constant value for a substance (if it meager in the same condition) so, it can be use to identify the substance as one of its physical proprieties.

Amino acids

• Proteins consist of amino acids interconnected by peptide bonds.

• Mixtures of amino acids can be separated on chromatographic sheet.

• The separated amino acids are visualized using solution of ninhydrin.

• Purple color develops upon reaction of amino acid with ninhydrin.

Amino acids

Amino acid Rf value

alanine 0.38

arginine 0.20

asparagine 0.5

aspartic acid 0.24

Turmeric• Active constituent:

1. 1-5% orange yellow volatile oil

2. 0.3% of yellow crystalline dye (curcumin)

3. 30-50% starch

4. resin

Separation of Turmeric using TLC

• Procedure:

1. in the TLC, draw 2 horizontal lines (the first one at 1.5cm from the bottom edge and the other one at 1cm from top edge)

2. draw one spot in the bottom line with around 8mm from the edge of the plate

3. by a capillary tube add one spot of the sample in the line of bottom ,wait to dry then repeat this process three times

4. if the spots not dry use the hotplate to dry it.

Separation of Turmeric using TLC

5. fill the chamber with 10 ml of solvent ( make sure that the height of solvent lower than the lower line of the sheet)

solvent system: ethanol: chloroform : ethanol : acetic acid (94+5+1)

5. pot the sheet in a chamber, close the it and wait until the solvent reach the top line of sheet.

6. remove the sheet from the chamber AND move the plate over ammonia vapour

7. circle the spots and calculate the Rf factor for the samples

Separation of Turmeric using TLC

curcumin

desmethoxy curcumin

bisdesmethoxy curcumin

sample

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Front of solvent

Result

Thank you