Biomedical Engineering Reference
In-Depth Information
Table 3.1.
Constants of proportionality for Sephadex GPC media
µ
Gel type
C
Particle diameter (
m)
G-10
19
55 - 165
G-25 superfine
9
17 - 70
G-25 fine
30
35 - 140
G-25 medium
80
85 - 260
G-25 coarse
290
170 - 520
G-50 fine
36
40 - 160
Data from Amersham Biosciences Handbook, Gel
filtration: Principles and methods 18-1022-18.
Edition AI
The best column design for GPC as well as for affinity chro-
matography is a column with adaptors at both ends (Fig. 3.3C).
This column type guarantees even surfaces of the gel bed, symmet-
rical application of sample, and regular flow of the eluent.
The quality of separation depends on several factors. One point
is the size and size distribution of the matrix beads. Small beads of
same size increase fractionation efficacy, but also increase the flow
resistance; therefore, fractionations should be made with beads
characterized as “medium,” “fine,” or “superfine,” whereas group
separations are mostly successful with “coarse” media.
A second point of view is the geometry of the column. For
fractionations the ratio of bed height to bed diameter should be
at least 50:1, but this value is a rule of thumb and may be varied
depending on the geometry of adaptors and size distribution of
matrix beads.
The third point concerns the size of the sample. For group
separations the sample volume may be up to 1/3 of the bed volume,
whereas for fractionations the sample volume should be on the
orderof1/50ofthebedvolume.
A fourth factor is the flow rate of the eluent (mobile phase).
A too high rate decreases resolution because there is no time for
molecules to diffuse into the pores of the matrix. In contrast, a very
slow migration of solvent decreases the resolution by remixing the
components by diffusion. The effect of diffusion is minimized if
the chromatography is done at low temperature. If short separa-
tion times are necessary, pre-packed columns, elevated pressure,
or HPLC columns are indispensable.
3.3.1 Selection of Supports
The main criterion for selection of a GPC matrix is the fraction-
ation range. Manufacturers of GPC media declare a fractionation
range and avoid molar weight. These values are mostly obtained
