Manual

Table 2. UNO S Column Characteristics

S-1 S-6 S-12

Column volume (ml) 1.3 6 12

Recommended max. protein loading (mg) 20 80 160

Recommended flow-rates (ml/min) 0.5 to 6.0 0.5 to 8.0 0.5 to 10.0

Column Dimensions (mm) 7 x 35 12 x 53 15 x 68

Maximum operating pressure (psi/MPa/bar) 700/4.5/48 700/4.5/48 700/4.5/48

Preparation for Initial Use

The columns are supplied in a storage buffer of 0.1 M NaCl + 20% ethanol. The counter

ion for the Q column is Cl

and Na

for the S column. Prior to initial use and after extended

storage periods, each column should be conditioned as described below (steps 1–4). Always

use HPLC grade reagent, and filter and degas the buffers. During this operation do not exceed

the following flow rates:

S-1 or Q-1 1 ml/min

S-6 or Q-6 2 ml/min

S-12 or Q-12 3 ml/min

1. Wash with 5 column volumes of water. Elevated backpressures may occur when wash-

ing with deionized water. Do not exceed 700 psi.

2. Wash with 5 column volumes of low ionic strength start buffer [e.g. 20 mM Tris-HCl (Q)

or 20 mM Sodium Phosphate (S)].

3. Wash with 5 column volumes of high ionic strength elution buffer (e.g. Starting buffer +

1.0 M NaCl).

4. Wash with 5 column volumes of low ionic strength equilibration buffer [e.g. 20 mM

Tris-HCl (Q) or 20 mM Sodium Phosphate (S)].

The column may now be further equilibrated in start buffer at the desired flow rate.

Buffer Selection

Table 2 and 3 lists commonly-used buffers for anion and cation-exchange chromatogra-

phy. The choice of whether to use an anion- or cation exchanger is determined mainly by (a)

the isoelectric point (pI) and, (b) the relationship between pH and the activity/stability of the

protein of interest. Once the type of ion-exchanger is determined, the choice of buffer and

pH is also determined by the pH-activity relationship. As a general rule, the pH used should

be within ± 0.5 pH units of the pK

of the chosen buffer. This permits use of the lowest pos-

sible buffer concentration while maintaining maximum buffering capacity. In any case, a

buffer concentration of 20 mM is recommended.

As can be seen in Table 2 and 3, the pK

and hence the pH of the buffer, changes with tem-

perature. Therefore the pH of the buffer must be adjusted at the working temperature.