Waters Advanced Purification Glass Columns - Manual (Page 5)

[ Care and Use ManUal ]
Waters Advanced Purification Columns 5
Emptying the column
To remove packing material from the column:
1. Reverse the direction of flow through the column by connecting the
column outlet to the pump.
2. Remove the inlet connector by unscrewing the fine adjustment knob
from the inlet cap.
3. Screw the packing funnel into the inlet cap. Position a beaker under the
mouth of the funnel.
4. Start flow. Gradually increase the flow until the packed bed begins
to move. Continue flow until all packing material washes out of the
column.
5. Remove the funnel and clean the column. Make sure the threads are
clean.
6. Repack the column (see “Slurry-packing the column”, or “Dry-packing
the column”, for repacking instructions).
d. Installing the Column
The AP Column can be installed in:
•
Waters 650 Advanced Protein Purification System
• Waters 625 system
• Pharmacia ® FPLC ® systems
• Other high performance liquid chromatography (HPLC) systems (refer
to Table 4 for adapters and unions used to connect the AP column to
selected systems)
Refer to the appropriate system user’s guide for specific column instal-
lation information.
Tightening fittings
Finger-tighten plastic fittings. Do not overtighten. Overtightening
may damage the threaded parts of the column. Overtightening does
not improve performance.
End plugs
Save the column end plugs for future column storage.
e. Equilibrating the Column
Before performing a test run, equilibrate the column:
1. Gradually increase the flow in 0.1 mL/min increments until you attain
the desired flow rate.
2. While equilibrating the column, check for voids; if present, gradually
reduce flow to 0 mL/min, Adjust the inlet connector until the void is
eliminated. (If this is unsuccessful, see “Eliminating Voids”).
3. Equilibrate the column with 10 to 20 column volumes of mobile phase,
or as recommended by the manufacturer of the packing material.
f. Eliminating Voids
Voids may develop in the column during normal use. To eliminate
voids:
1. Stop flow.
2. Disconnect the inlet column tubing from the system to allow displace-
ment of fluid while adjusting the plunger.
3. Deactivate the O-ring seal by turning the seal adjustment nut
counterclockwise.
4. Move the slide to the fine (down) position and turn it slowly to eliminate
the void.
If the fine adjustment knob is all the way down, back if off a few turns.
Move the slide to the coarse adjustment position and move the plunger
down a short distance. To eliminate the void, return the slide to the fine
adjustment position and turn it slowly.