Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 7)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 7
e. Column Efficiency Test Condition
Perform the following test sample run for your new column and
record the results (retention time and the settings used) before
attempting the first analysis. Use these results for comparison
throughout the life of your column.
The detector should monitor 254 nm or 280 nm (UV). Use
instrument settings that produce an acceptable test peak (this may
mean using a lower sensitivity on the UV detector than normally
practiced for a specific analysis).
Note: Be sure to record results and instrument settings (and
configurations) to allow exact future reproduction and comparison.
Table 3: Column Test Conditions
Refer to Table 6, Typical Column Problems and Solutions, if a change
is observed in any of the following:
• Retention of a particular compound
• Resolution between two compounds
• Peak shape
• Backpressure
When the problem is solved, use the test in Sections IV, d, Column
Efficiency and V, a, Troubleshooting, to monitor the continued high
performance of your column.
V. Care and MaIntenanCe
a. Troubleshooting
To monitor the continued high performance of your column, perform
a functional test for each type of column using the conditions
outlined in Tables 4 and 5, and Figure 6. Refer to Table 6 for
troubleshooting column and packing material performance.
Table 4: Functional Test Conditions
*AP Mini column - Injection volume: 13 µL; Flow rate: 0.2 mL/min
Create the sample protein mix using the concentrations in Table 5.
The concentration of the adenosine solution is 0.5 mg/mL. All pro-
tein solutions are made at a concentration of 5 mg/mL. The protein
mixtures are made by combining the aliquots of each protein solution
given in Table 5.
Column Name Mobile Phase Flow Rate
(mL/min)
Test Sample
Mini (5 mm x 50 mm) Milli-Q Water 0.2 1 µL Acetone
AP-1 (10 mm x 100 mm) Milli-Q Water 1 2 µL Acetone
AP-2 (20 mm x 100 mm) Milli-Q Water 4 8 µL Acetone
AP-5 (50 mm x 100 mm) Milli-Q Water 25 50 µL. Acetone
Conditions DEAE 8HR/Q 8HR SP BHR/CM 8HR
Sample proteins
(Table 5) Protein Mix Dissolved in
Buffer A Protein Mix Dissolved in
Buffer A
Injection volume,
mL and total mg
applied 100 µL 500 µg protein* 100 µL 375 µg protein
Buffers Buffer A: 20 mM Tris HCI
pH 8.2
Buffer B: Buffer A + 1 M
NaCl Buffer A: 20 mM Sodium
Phosphate pH 7.0
Buffer B: Buffer A + 1 M
NaCl
Flow Rate 1.56 mL/min or 2.4 cm/
min* 1.56 mL/min or 2.4 cm/
min
Linear gradient
duration 38 min to 25% Buffer A 78 min to 50% Buffer B
Detector 280 nm at 0.16 AUFS 280 nm at 0.16 AUFS
Column AP-1 (10 mm x 100 mm) AP-1 (10 mm x 100 mm)