Bio-Rad Bio-Scale™ CHT™ Type I Columns - Manual (Page 6)

Fig. 1. Schematic gradient for separation on Bio-Scale CHT2-I column.
2.4 Use of Detergents
Most of the common detergents used for protein isolation may be used with the Bio-Scale
CHT-I support. We recommend the use of the reduced form of Triton ® X-100 to minimize UV
absorption artifacts. It is essential to thoroughly equilibrate the column with the detergent-
containing buffer prior to sample application. Pay particular attention to pH, which influences
the solubility of the various classes of detergents. Problems may arise during gradient elu-
tion due to the formation of micelles. This may cause a sudden increase in the UV baseline
as the micelles themselves scatter light. We recommend using a concentration of detergent
above the critical micelle concentration (CMC) during gradient elution. If subsequent chro-
matographic steps (e.g. hydrophobic interaction) or other biochemical manipulations will be
affected by the presence of the detergent, then the initial choice of detergent should be influ-
enced by the availability of a protocol for its removal. See Refs. 1, 4, and 5 for a more detailed
explanation of the characteristics and use of detergents in biology and biochemistry and their
removal from biological samples.
2.5 Effect of Calcium Chloride
CaCl
2
may be added to the buffers to increase the binding of acidic proteins. The buffers
should contain calcium and phosphate at the level of the solubility product for calcium phos-
phate. For example, at pH 7.0, the solubility product is 3x10
-6 M. Hence, if a 10 mM to 500
mM phosphate gradient is used, the 10 mM phosphate buffer should be 0.3 mM in calcium
and the 500 mM phosphate buffer should be 0.006 mM in calcium.
Note: At pH 6.0, the solubility product is for calcium phosphate is 3 x 10 -5 M and at pH 8.0
the solubility product is 3 x 10 -7 M.
2.6 Chromatography Pre-Runs
Prior to loading the sample, we recommend running a blank gradient to check that the
column is clean. In addition, detergents and other buffer components may themselves elute as
0
0.5
1
[Buffer B] M
0 6 12 18 24 30 36 42 48
Volume (ml)
4
Scale-up
Recommended gradient volumes:
CHT2-I 20 to 40 ml
CHT5-I 50 to 100 ml
CHT10-I 100 to 200 ml
CHT20-I 200 to 400 ml
load
sample