Ul

Effluent volume Ve

Sample Excluded injection molecules

Test molecules

Figure 3.36 Gel permeation chromatogram. All molecules larger than the exclusion limit of the gel appear at Vu (the void volume). Molecules which can gain access to the gel structure to varying degrees are eluted in order of decreasing size.

> The void volume is the amount of liquid that is outside the gel struc-

similarly those molecules that can gain access to all parts of the gel matrix will have an effluent volume equal to the total bed volume (Vt). Molecules of an intermediate molecular size will be eluted in volumes between Vt and V0 (Figure 3.36).

The measurement of effluent volume is not very reliable because of the effect of the geometry and packing characteristics of any column. It is often more useful to use a reduced parameter, such as VJVQ, which is not so dependent upon column characteristics and is comparable with the calculation for RF values in thin-layer chromatography.

It is possible to calculate a partition constant (Kd) for a solute in terms of the access gained to the gel matrix:

Ve = V0 + KáX Vj where V¡ is the gel inner volume. Hence V - V

However, because it is difficult to measure Vi precisely, the equation may be modified to determine the 'available' part of the resin, K.dv:

av vt-v0

In order to separate two solutes satisfactorily, their values for K&y must be significantly different from each other.

> Desalting is the name given to the technique ot removing unwanted small molecules from preparations of macro molecules.

Applications of gel permeation chromatography

Gel permeation chromatography is commonly used in the fractionation of mixtures of substances that vary in their relative molecular mass and it is extremely useful for labile molecules, such as enzymes.

The elution volume of a solute is determined mainly by its relative molecular mass and it has been shown that the elution volume is approximately a linear function of the logarithm of the relative molecular mass. It is possible to determine the relative molecular mass of a test molecule using a calibration curve prepared from the elution volumes of several reference substances of known relative molecular mass. This should be done using the same column and conditions (Figure 3.37) and in practice it may be possible to calibrate the column and separate the test substance at the same time by incorporating the reference compounds in the sample. Such a method is rapid and inexpensive and does not demand a highly purified sample, provided that there is a specific method for detecting the molecule in the eluate.

Solutes of low relative molecular mass may be removed from preparations of macromolecules, often called desalting, using a small column of a gel which excludes the macromolecules but retains the small solutes. The macro-molecules will be eluted in the void volume, which for a small column will be only 2-3 ml.

A dry gel may also be used to concentrate a dilute aqueous solution of a macromolecule. When it is added to the solution, it will swell as it takes up water but will exclude the larger molecules. Hence, after sedimentation the supernatant fluid will contain the macromolecule, leaving the majority of the water in the pores of the gel. Such a technique is quicker and more effective than dialysis.

Elution time (hours)

Figure 3.37 Determination of relative molecular mass by gel permeation chromatography. A mixture of proteins (approximately 10 ¿ig of each) was separated on an UltroPak TSK SW column and the elution volume for each protein plotted against the logarithm of its relative molecular mass (RMM). (Reproduced by permission of LKB, Stockholm, Sweden.)

Figure 3.37 Determination of relative molecular mass by gel permeation chromatography. A mixture of proteins (approximately 10 ¿ig of each) was separated on an UltroPak TSK SW column and the elution volume for each protein plotted against the logarithm of its relative molecular mass (RMM). (Reproduced by permission of LKB, Stockholm, Sweden.)

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