Manual

Table 2. Buffers for Anion-Exchange Chromatography
pH range Buffer Mwt pK
a
@25 °C Counter-ion ∆ pK
a
/°C
5.0 - 6.0 Piperazine 86.1 5.7 Cl
-
/HCOO
-
-0.015
5.5 - 6.0 L-Histidine 155.2 6.15 Cl
-
5.8 - 7.2 Bis-Tris 209.2 6.5 Cl
-
-0.017
6.4 - 7.3 Bis-Tris Propane 282.3 6.8, 9.0 Cl
-
7.3 - 8.3 Triethanolamine 149.2 7.8 Cl
-
/CH
3
COO
-
-0.020
7.6 - 8.6 Tris 121.1 8.1 Cl
-
-0.031
8.4 - 8.8 Diethanolamine 105.1 8.9 Cl
-
-0.025
9.0 - 9.9 Ethanolamine 61.1 9.5 Cl
-
-0.029
9.8 - 10.3 1,3-diamino-propane 74.1 10.5 Cl
-
-0.026
Table 3. Buffers for Cation-Exchange Chromatography
pH range Buffer Mwt pK
a
@25 °C Counter-ion ∆ pK
a
/°C
3.6 - 4.3 Lactic acid 90.1 3.8 Na
+
4.2 - 5.2 Citric acid 192.1 3.1 Na
+
5.5 - 6.7 MES 195.2 6.1 Na
+
-0.011
6.1 - 7.5 PIPES 302.4 6.8 Na
+
-0.009
6.5 - 7.9 MOPS 209.3 7.2 Na
+
-0.006
6.7 - 7.6 Phosphate 120 (Monobasic) 7.2 Na
+
-0.003
142 (Dibasic)
6.8 - 8.2 TES 229.2 7.4 Na
+
-0.020
6.8 - 8.2 HEPES 238.3 7.5 Na
+
-0.014
7.4 - 8.8 Tricine 179.2 8.1 Na
+
-0.021
Always use buffer components of the highest purity available as UV-absorbing impuri-
ties may cause baseline disturbances and interfere with the detection of protein peaks.
Sample Preparation
Proper adjustment of the sample pH and ionic strength is critical for consistent and repro-
ducible chromatography. For best results, the sample should be exchanged into the start buffer
or diluted to the start buffer’s concentration. Buffer exchange can be accomplished using
Bio-Spin
®
6 or Bio-Spin 30 columns, Econo-Pac
®
10DG desalting columns, Bio-Gel
®
P-6DG size exclusion gel or the Econo-Pac P6 cartridge. The choice of product depends on
sample volume. Always centrifuge or filter the sample (0.2–0.45 mm filter) to remove par-
ticulates. Application of turbid or lipid-containing samples may reduce the column lifetime.
Sample Load
The recommended sample load for each column is shown in Table 1. This amount may
vary somewhat depending on the actual sample composition. We do not recommend over-
loading the column as both resolution and column lifetime will decrease. For larger loads,
either switch to a larger column or perform several chromatographic runs with a reduced
loading. Due to the fast flow properties of the UNO gel, multiple runs may be performed in
a very short period of time. Ideally, samples should be bound in a concentrated zone at the top
of the column. Higher sample loads produce a broad application zone in which components
with less charge are displaced by more highly charged components. This may result in a shift
of certain peaks to an earlier elution position in the gradient.
3