1.6 analytical techniques

7/29/2019 1.6 Analytical Techniques

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Outline :

1) Basic principle of paperchromatography in pigmentseparation

2) Basic principle of electrophoresis for

protein and nucleic acid separation


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A technique for separating mixtures in very

small samples into its components. These separated components can then be

isolated, identified and used for furtherinvestigation.

Ideal for separating a mixture ofmacromolecules such as proteins &photosynthetic pigments.


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The mixture is dissolved in a suitable solvent ormobile phase & allowed to pass over a stationaryphase : a porous chromatography paper which

restricts the movement of macromolecules. Basic principle is the differences in - molecular size - solubility

- adhesion of the macromolecules to the chromatography paper Cause the component molecules to move

through the pores of the paper at different

speeds. Components with a higher affinity towards the

solvent will move further up the paper.


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1) Chlorophyll is first extracted from leaves using

acetone.

2) A strip of absorptive paper is used as the

stationary phase.

3) The chlorophyll extract is then applied repeatedly to

form a fine concentrated spot on the base line drawn

earlier on one edge of the paper.4) The spot is hen air-dried.

5) The edge of the paper is immersed in a solvent

(petroleum ether) in a boiling tube & then closed

with a cork stopper.6) As the solvent moves up, the chlorophyll

components dissolve in the solvent and are carried

along at different rates.


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7) Before the solvent front reaches the top

end of the chromatography paper, theprocess is stopped and the

chromatogram is air-dried.

8) The location of the colour pigments are

then determined and marked.

9) Molecules in the mixture can be identified

by their Rf (retardation factor) values,wherebyRf= distance travelled by compound

distance travelled by solvent front


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The Rfvalue for each compound in amixture is constant for the same solventused. This value can be used as acomparison to identify unknowncompounds in a mixture.

Pigment Colour Rf valueCarotene Yellow-orange 0.95

Phaeophytin Grey 0.83

Xanthophyll Yellow 0.71

Chlorophyll a Blue-green 0.65Chlorophyll b Green 0.45


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Is used for a more complex mixture.

A complete separation of its compounds issometimes not obtained by one-directionchromatography.

A square sheet of filter paper is used for the firstseparation.

Then, the chromatogram is taken out driedrotated at 900 and a second separation is carriedout with the same or a different solvent.

This separates out the various components withtheir different properties.

The chromatogram is removed and the solutesare identified by treating with a suitable reagent.Example : The separated amino acids are sprayedwith ninhydrin to produce purple spots on thechromatogram.


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Is used to separate a mixture of charged

molecules such as amino acids, proteins &fragments of DNA using an electric field.

The overall charge for any type of aminoaid depends on the pH of the mobilephase. At a certain pH, the overall chargeof the amino acid is zero and the moleculeconcerned neither move towards the

cathode nor the anode which is called theisoelectric point.


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When the pH of a solution is above its

isoelectric point, the amino acid or proteinmolecule will be ve charged & will movetowards the anode.

When the pH of a solution is below itsisoelectric point, the molecule will be +vecharged & will move towards the cathode.

2 factors which affect the speed of

charged molecules are the amount ofcharge & the size of the molecule.

1.6 analytical techniques

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