1.

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 Whatmans 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.

Whatman Chromatography paper

Closed
chromatography
chamber filled
with solvent
vapour.

A concentrated spot of mixture to

be separated.

Solvent

Fig. 1: A
simple 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 solvents
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 Rf values
of each of the separated components.
8. The Rf value, or retention factor, is the ratio of the distance
moved by a particular spot to the distance moved by the solvent.
Rf =

distance moved by macromolecule

distance moved by solvent

9. Rf values obtained can be used to identify the separated

components by referring to a table of known Rf values.

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 Rf values
with a table of known Rf 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 Rf 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 around
and with a different solvent.
c. Rf 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?)

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 move
towards 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 become
electrically 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.