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1.6 Analytical techniques used in Biology

Chromatography

1. A process used to separate out a mixture of macromolecules by using the property of their

different solubility in certain solvents.

2. A concentrated spot of the dissolved mixture of macromolecules is placed at one end of a

suitable stationary phase, such as a strip of Whatman’s chromatography paper.

3. The paper is then suspended in a jar, with one end (the ‘spotted’ end) dipped in a small amount

of solvent at the bottom of jar.

4. The solvent will be slowly absorbed by the chromatography paper and is carried up the paper,

together with the dissolved mixture of macromolecules.

Fig. 1: Asimple chromatography apparatus and chromatograms

5. Macromolecules in the mixture are separated, appearing as spots,due to

(i) their different solubility in the solvent used – more soluble

molecules are carried further up.

(ii) their size – smaller molecules are deposited further up.

(iii) their adsorption (surface attraction) to the stationary phase –

less adsorbed molecules move further up.

6. The process takes place in a closed jar/chamber so that the air

around the chromatography strip is saturated with the solvent’s

vapour.

7. The chromatography is stopped just before the solvent reaches

the top end of the paper. The distance covered by the solvent is

marked & measured, to be used in the calculation of the R f  values

of each of the separated components.

8. The R f  value, or retention factor, is the ratio of the distancemoved by a particular spot to the distance moved by the solvent.

R f  =

solvent bymoveddistance

ulemacromolec bymoveddistance 

9. R f  values obtained can be used to identify the separated

components by referring to a table of known R f  values.

Whatman Chromatography paper

Solvent

A concentrated spot of mixture to be separated.

Closed

chromatography

chamber filled

with solvent

vapour.

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Examples:

A. Protein analysis

1. The amino acids that make up a protein (e.g. albumin) can be determined by chromatography.

2. The albumin is first broken down to its amino acids by the enzyme trypsin.

3. Paper chromatography is then carried out with a suitable solvent (usually a mixture of butanol,glacial ethanoic acid and distilled water.)

4. The process is allowed to proceed undisturbed for 8 – 16 hours.

5. The separated amino acids, being uncoloured, are invisible.

6. The chromatogram is then soaked in / sprayed with ninhydrin (a poisonous locating agent) in a

fume cupboard.

7. Amino acids are coloured purple by ninhydrin and are identified by comparing their R f  valueswith a table of known R f  values.

(B) Analysis of plant pigments.

1. The pigments that make up chlorophyll in a leaf can also be identified by chromatography.

2. Several leaves are ground with a little acetone in a pestle and mortar.

3. The mixture is strained through a muslin cloth, and spotted on to a strip of chromatography

 paper.

4. Chromatography is carried out using a mixture of acetone and petroleum ether, the process

requiring several minutes.

5. Chlorophyll pigments, which separate, include chlorophyll a and b, xanthophylls, carotene and

 phaeophytin. They may be identified by their colour or R f  values.

Note:

a. Certain uncolored macromolecules may fluoresce (glow) when viewed under ultraviolet light.

 b. Spots which are not well separated may be further isolated by performing a 2 dimensional

chromatography. This is done by repeating the process with the chromatogram turned 90o aroundand with a different solvent.

c. R f  values can only be compared from tabulated values which have used a similar solvent /

solvent mixture. Different solvent mixtures will cause molecules to travel different distances.

(Why?)

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Electrophoresis

1. This is a modified form of chromatography used to separate out a mixture of charged

macromolecules by placing it in a suitable electrolyte (usually a buffer solution) and applying a

 potential difference across it.

2. Macromolecules in the mixture will movetowards the anode or cathode, depending on

their charges.

3. The rate of movement towards the

electrodes is affected by the relative sizes of

the macromolecules.

4. For biomolecules like amino acids and

 proteins, the overall charge on the molecule

depends on the pH of the electrolyte. When placed in an electrolyte with a pH equivalent to

their isoelectric points, they becomeelectrically neutral and will not migrate

towards either electrode. This property can be

used to identify specific proteins / amino acids

in a mixture.

Electrophoresis apparatus Electrophoresis strip

Example:a. Analysis of Proteins

1.  Proteins are broken down into amino acids by enzymes.

2. 

The amino acids are placed on a special support medium (e.g. chromatography paper) in a buffer solution (constant pH).

3.  An electric current is passed through the amino acids.

4.  The amino acids move at different rates and directions, according to their molecular sizes and

the charge on their R (alkyl) groups.

5.  The paper is dried after the process, and ninhydrin is sprayed on to it.

6.  Amino acids are coloured by ninhydrin.7.

 

The amino acids are identified by comparing the distances they have traveled to those traveled

 by known marker amino acids under the same conditions.

 b. Gel electrophoresis of DNA and nucleic acids

Refer to power point.