Owner's manual

[ Care and Use ManUal ]
4
III. column care & use consIderatIons
a. Column Care Considerations
• ALWAYSfiltersamplestopreventexcessivepressurebuildupdueto
particulate matter. Use a Sample Clarification Kit (Part No. 26865)
and, if necessary, centrifugation. For more details on the Waters
Sample Clarification Kit call or write for Bulletin D97.
• ALWAYS filter buffers to remove microparticulate matter. This
eliminates high pressures from plugged frits and also helps ensure
long column life. Use a Solvent Clarification Kit (Part No. 85113)
to filter solvents. Vacuum filtration or sonification may be used to
remove dissolved gasses which could affect your solvent delivery
system. For more details on the Waters Solvent Clarification Kit call
or write for Bulletin D97.
• ALWAYSusehighpuritysolventssuchasWaterssolvents.Forana-
lytical work choose high quality Waters solvents. For more details
on Waters solvents call or write for Bulletin M10.
• ALWAYS replace plugs in column fittings to prevent the column
from drying out when not in use.
b. Column Storage
• Thecolumnmaybestored1-2daysinBufferAwithoutflow.The
presence of phenol in the buffer will prevent bacterial growth.
• Forlongerstorageperiods,0.2NNaOHisstronglyrecommended.
c. Column Regeneration
For protein hydrolyzate samples, regenerate the column at least
once a week as follows:
Eluent: 0.2 N NaOH
Flow: 0.5ml/min
Temperature: 62°C
Flush Time: 30 min
Crude samples may require more frequent regeneration.
d. Troubleshooting
This section discusses some possible problems and their solutions
(refer to Table 1.)
e. Regeneration of Protein and Lipid Contaminated Column
• Largeproteinsandsomelipidscanberemovedfollowingalkaline
hydrolysis.
• Reverse direction ofbuffer flow through column. Elevate column
heatertemperature to 90°-95°Cand pump0.2NNaOHsolution
through column for at least 2 hours. Particularly difficult contamination
may require overnight treatment.
• Restore normal flow direction and temperature; reequilibrate
column with Buffer A and test column with amino acid standard
solution. If problem still exists, repeat the above treatment.
Table 1: Typical Column Problems and Solutions
Problem Cause Solution
Excess pressure buildup Column frits plugged with
particulates or mold is
growing in column or in
buffers
Reverse flow on column.
If this does not work,
follow procedure for protein
removal. If pressure is still
high, carefully remove
column inlet fitting being
sure not to disturb resin.
Clean in ultrasonic bath.
Always filter solvents and
samples.
Sampleand/orbuffer
precipitates on column
Follow procedure above.
Loss of resolution, broad
peaks, low plate counts
Contaminated column Follow procedures
for metal and protein
removal.