Bio-Rad Media Sampler Pack User Manual
Page 9
7
Peaks should be symmetrical. The best achievable asymmetry factor is 1. Values
of 0.8 to 1.8 are excellent for large columns >30 cm in diameter. If functional
performance is acceptable, A
s
factors more distant from 1 put less demand on
the column packing procedure in production. A change in the shape of the peak is
usually the first indication of deteriorating performance. Peak asymmetry factor
calculation:
A
s
= b/a
a = Front section of peak width at 10% of peak height bisected by line denoting V
e
.
b = Latter section of peak width at 10% of peak height bisected by line denoting V
e
.
Section 8
Operation and Maintenance
Macro-Prep media were designed to remove trace contaminants while operating
at high throughput. Macro-Prep media should be run at the highest linear
velocities and loading capacities while staying within the pressure limits of the
column and chromatography system. For flow-through mode, a linear flow rate of
300 cm/hr in a 20 cm bed is a reasonable starting point. If the product is being
retained on the Macro-Prep, flow rates of 100–200 cm/hr are recommended.
Optimization can be achieved by changing the pH, the ionic strength, or additives
in the elution buffer.
All buffers commonly used for cation exchange chromatography can be used with
Macro-Prep HIC media (see Table 2).
Table 2. Common Buffers for Macro-Prep HIC.
Buffer
Useful pH Range
Citric acid
2.6–3.6
Lactic acid
3.6–4.3
Formic acid
3.8–4.3
Acetic acid
4.8–5.2
MES 5.5–6.7
PIPES
6.1–7.5
MOPS 6.5–7.9
Phosphate 6.7–7.6
Bicine
8.2–8.7
HEPES 6.8–8.2
TES 6.8–8.2
Tricine 7.4–8.8
Column Equilibration
Higher salt concentrations are used to enhance hydrophobic interactions.
Normally, a column is equilibrated in a high-salt buffer, and the sample is loaded
onto the column at the same pH and salt concentration. At small scale, the
following salts are commonly used: ammonium sulfate (
scale, sodium citrate is a very practical salt. This salt can function as both a buffer
and a salt during HIC. Lower molar quantities are required to reach specific ionic
concentrations and the somewhat hydrophobic nature of the citrate ion often
LIT486C.qxd 11/2/2004 3:12 PM Page 7