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For two analytes of different relative molecular mass andKdvalues,K^0 dandK^00 d,the

difference in their elution volumes,VS, can be shown to be:

VS¼ðKd^0 K^00 dÞVi ð 11 : 14 Þ

whereViis the inner volume within the particle available to a compound whoseKd¼1.

In practice, deviations from ideal behaviour, for example owing to poor packing of

the column, make it advisable to reduce the sample volume below the value ofVS

because the ratio between sample volume and inside particle volume affects both the

sharpness of the separation and the degree of dilution of the sample.

The stationary phases for exclusion separations are generally based on silica,

polymethacrylate or polyvinyl acetate or chloride or on cross-linked dextran or

agarose (Table 11.4). All are available in a range of pore sizes. They are generally

used where the eluent is an organic system. The supports for affinity separations are

similar to those for exclusion separations.

11.7.2 Applications

Purification

The main application of exclusion chromatography is in the purification of biological

macromolecules by facilitating their separation from larger and smaller molecules.

Example 2ESTIMATION OF RELATIVE MOLECULAR MASS

Question The relative molecular mass (Mr) of a protein was investigated by exclusion
chromatography using a Sephacryl S300 column and using aldolase, catalase,
ferritin, thyroglobulin and Blue Dextran as standard. The following elution data
were obtained.
Retention volume
Mr Vr(cm^3 )

Aldolase 158 000 22.5

Catalase 210 000 21.4

Ferritin 444 000 18.2

Thyroglobulin 669 000 16.4

Blue Dextran 2 000 000 13.6

Unknown 19.5

What is the approximateMrof the unknown protein?

Answer A plot of the logarithm of the relative molecular mass of individual proteins versus

their retention volume has a linear section from which it can be deduced that the

unknown protein with a retention volume of 19.5 cm^3 must have a relative

molecular mass of 330 000.

463 11.7 Molecular (size) exclusion chromatography