Waters Protein-Pak Hi Res IEX Columns - Manual (Page 5)

5
[ CARE AND USE MANUAL ]
Protein-Pak Hi Res IEX Columns and Standards
IV. COLUMN SPECIFICATIONS AND USE
a. Specifications
Description
Protein-Pak
Hi Res Q
Protein-Pak
Hi Res CM
Protein-Pak
Hi Res SP
Ion Exchange Strong Anion Weak Cation Strong Cation
Functional Group
Quaternary
ammonium
Carboxymethyl Sulfopropyl
Matrix
Hydrophilic
polymer
Hydrophilic
polymer
Hydrophilic
polymer
Particle Size (µm) 5 7 7
Pore Size:
i.d. x L (mm) Non porous
4.6 x 100
Non porous
4.6 x 100
Non porous
4.6 x 100
Counter Ion Cl- Na+ Na+
pH Range 3–10 3–10 3–10
Small Ion Capacity
(µeq/g dry gel)
270 100 23
pK
a
10.5 4.9 2.3
1. Approximate
Protein Binding
Capacity in mgs
per column (i.e.,
BSA for Hi Res Q
column; Lysozyme
for Hi Res CM and
Hi Res SP columns)
58 33 25
Flow Rates
0.3–0.6
mL/min
0.5–1.4
mL/min
0.5–1.4
mL/min
2. Max Pressure
across column 2175 psi
(15MPa)
1450 psi
(10Mpa)
1450 psi
(10Mpa)
Salt Concentration No limit No limit No limit
Organic
Concentration
<50%. When
switching
from aqueous
buffers to
organic, lower
flow rates to
<0.25 mL/
min.
<50%. When
switching
from aqueous
buffers to
organic, lower
flow rates to
<0.5 mL/min.
<50%. When
switching
from aqueous
buffers to
organic, lower
flow rates to
<0.5 mL/min.
Temperature (°C) 10–60 10–60 10–60
1. For optimal resolution of complex samples, do not exceed 20% of the
column’s protein binding capacity.
2. See section e. pressure for details.
To ensure the continued high performance of Protein-Pak Hi Res
IEX columns, follow these guidelines:
b. Sample Preparation
1. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that has a higher pH (anion exchange)
or lower pH (cation exchange) than the mobile phase to ensure
complete loading of the sample onto the column. The ionic
strength of the sample should also be lower or equivalent to
that of the starting buffer.
2. If the sample is not dissolved in the mobile phase, ensure that
the sample, solvent and mobile phases are miscible in order to
avoid sample and/or buffer precipitation.
c. Operating pH
The recommended operating pH range for Protein-Pak Hi Res
IEX columns is 3–10. A listing of commonly used buffers and
additives is given in Table 3. The column lifetime will vary
depending upon the operating temperature as well as the type
and concentration of buffer used.
Table 3. Buffers Commonly Used for Ion Exchange
Anion-Exchange Buffers
pH Range Additive/Buffer pK
a
(25 °C) Counter-ion Conc. (mM)
4.5–5.3 N-Methylpiperazine 4.75 Cl- 20
4.8–6.0 Piperazine 5.68 Cl-/HCOO- 20
5.8–7.0 bis-Tris 6.48 Cl- 20
6.4–7.3 Bis-tris propane 6.80 Cl- 20
6.5–7.9 MOPS 7.28 Cl- 20
7.3–8.2 Triethanolamine 7.76 Cl-/HCH
3
COO 20
7.5–8.8 Tris 8.06 Cl- 20
8.4–9.4 Diethanolamine 8.88 Cl- 20
9.0–10.0 Ethanolamine 9.50 Cl- 20
9.7–10.0 CAPS 10.40 Cl- 20
Cation-Exchange Buffers
pH Range Additive/Buffer pK
a
(25 °C) Counter-ion Conc. (mM)
3.0–4.3 Lactic acid 3.81 Na+ 20
3.3–4.3 Formic acid 3.75 Na+/Li+ 20
4.0–5.7 Acetic acid 4.76 Na+/Li+ 20
4.6–6.6 Malonic acid 5.68 Na+/Li+ 20
5.1–7.1 MES 6.10 Na+/Li+ 20
5.5–7.7 Phosphate 7.20 Na+ 20
7.0–8.0 HEPES 7.55 Na+ or Li+ 20
7.8–8.8 BICINE 8.35 Na+ 20
a Adapted from:
1. P Stanton, “Ion-Exchange Chromatography,” HPLC of Proteins and Peptide:
Methods and Protocols; Aguilar, M.-I. Ed; Method in Molecular Biology, Humana
Press, Totowa, NJ, Vol. 251, Ch 4.
2. “Buffer Reference Center,” Sigma –Aldrich, 2009. http://www.sigmaaldrich.
com/life-science/core-bior agents/biological-buffers/learning-center/buffer-
reference-center.html