9780521516358book.pdf

Viruses, enzymes, hormones, antibodies, nucleic acids and polysaccharides have all

been separated and purified by use of appropriate gels or glass granules.

Relative molecular mass determination

The elution volumes of globular proteins are determined largely by their relative

molecular mass (Mr). It has been shown that, over a considerable range of relative

molecular masses, the elution volume orKdis an approximately linear function of the

logarithm ofMr. Hence the construction of a calibration curve, with proteins of a

similar shape and knownMr, enables theMrvalues of other proteins, even in crude

preparations, to be estimated (See Example 2, p. 463).

Solution concentration

Solutions of highMrsubstances can be concentrated by the addition of dry Sephadex

G-25 (coarse). The swelling gel absorbs water and lowMrsubstances, whereas the high

Mrsubstances remain in solution. After 10 min the gel is removed by centrifugation,

leaving the highMrmaterial in a solution whose concentration has increased but

whose pH and ionic strength are unaltered.

Desalting

By use of a column of, for example, Sephadex G-25, solutions of highMrcompounds

may be desalted, i.e. removed from contaminants such as salts, detergents, lipids and

chaotropic agents. The highMrcompounds move with the void volume, whereas the

lowMrcompounds are distributed between the mobile and stationary phases and

hence move slowly. This method of desalting is faster and more efficient than dialysis.

Applications include removal of phenol from nucleic acid preparations, ammonium

sulphate from protein preparations and salt from samples eluted from ion-exchange

chromatography columns.

11.8 Affinity chromatography

11.8.1 Principle

Separation and purification of analytes by affinity chromatography is unlike most

other forms of chromatography and such techniques as electrophoresis and

Table 11.4 (cont.)

Polymer Trade name

Fractionation rangea

(Mr 10 –3)

Dextran linked to cross-linked agarose Superdex

75 3–70

200 10–600

Note:aDetermined for globular proteins. The range is approximately the same for single-stranded

nucleic acids and smaller for fibrous proteins and double-stranded DNA.

465 11.8 Affinity chromatography