1.6 analytical techniques
TRANSCRIPT
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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.