Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 4)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 4
Adjusting gradient delay volume
The gradient may need additional adjustment due to a delay volume
between the mixing point of the gradient and the top of the column.
This delay volume results in an isocratic separation until the delay
volume is purged.
The effect is more pronounced on a small-volume column than on a
large-volume column. You may need to simulate the short isocratic
section on the large column. The additional time delay (td) can be
calculated from the following formula. Incorporate td into the gradi-
ent on the preparative column.
td = additional isocratic delay time for the preparative gradient
Vd = system delay volume of the instrument
Vp = volume of the preparative column
Va = volume of the analytical column
Fp = preparative flow rate
Note: Omit correction for the gradient time delay if a consistent
offset in retention times from analytical to preparative separations is
acceptable.
Determine the system delay volume as follows:
1. Install a connector union in place of the column.
2. Fill two reservoirs labeled A and B with 100 mL each of your standard
buffers.
3. Add 2 µL/mL acetone to reservoir B.
4. Equilibrate the system with reservoir B buffer for 5 minutes.
5. Equilibrate the system with reservoir A buffer for 5 minutes.
6. Set the system flow rate to 1 mL/min. Set the detector to 254 nm.
7. Start a timer and immediately switch to reservoir B. Stop the timer
when the acetone is detected, seen as a sharp rise in the baseline.
8. Enter the results in the system delay volume equation as follows:
System Delay Volume = (Acetone Detection Time - Buffer B Initiation Time)
x Flow Rate
Finally, use the time delay to make the preparative peak retention
time correspond to the analytical retention time by either:
• Delaying the initiation of the preparative gradient after making the
preparative injection
• Delaying the preparative injection after initiating the preparative
gradient
II. InstallatIon
Install the pre-packed Protein-Pak HR series of ion exchange glass columns
on any HPLC or medium-pressure system. Refer to the appropriate
operator’s manual for installation information. Refer to Table 6 for part
numbers and specific adapter information. Install the columns in a vertical
position for optimum performance.
a. Cautions
• When using preparative columns (AP-2 and AP-5) at elevated flow
rates, do not exceed the pressure limits of the column. Use a detector
flow cell configuration which will accommodate higher flow rates
without generating excessive backpressure.
• Non-metallic fittings should be hand-tightened only. Overtightening
does not improve performance and may damage the threaded parts. If
leakage occurs, check for damage to the threaded parts, ferrules, and
0-rings, or for foreign matter on the sealing surfaces.
• Do not adjust the piston on the column while there is flow through the
column. This could result in damage to the glass column and 0-rings.
See the Waters Advanced Purification Glass Column Care & Use
Manual for details.
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