Waters Amino Acid Analysis Liquid Chromatography Column - Manual (Page 7)

[ Care and Use ManUal ]
7
All peaks should be sharp and symmetrical. Backpressure should be
less than 2,000 psi. If you suspect that the quality of your column
has decreased, the above test is a rapid method of determining the
efficiency of your separation. Peaks which are not symmetrical could
in¬dicate partially plugged frits are at fault or foreign material has
accumulated on the resin packing. Generally this phenomenon is also
ac¬companied by increased backpressure (refer to Table 2 for correc-
tive action). Alternatively, resin packing material may have shifted,
voided or become uneven. Careful removal of top column packing nut
and visual inspection of the resin bed is the best diagnostic procedure
to determine whether this is the cause. Since removal of the nut could,
itself, disturb the packing, this should be a last resort measure. If resin
bed is not smooth and completely full, please contact your nearest
Waters representative for instructions.
If backpressure is normal but resolution is poor or peaks are very broad
to nonexistent, the problem could be heavy metal contamination of the
resin (refer to Table 1 for corrective action).
It should always be kept in mind that column efficiency is affected by
the liquid chromatography (LC) system as well as buffers and reagents.
Care should be taken to make sure the LC system is working normally,
that tubing and post-column reaction coil(s) are properly connected
and leak¬free, and that buffers and reagents are fresh, filtered and
tree from contamination.
Whenever problems persist after making the above tests and checks
on your system, con¬tact your local Waters representative for further
assistance.
V. column end fIt tIngs
The column is symmetrical, having the same kind of fittings and
frits at both ends. Flow of buffer can occur in either direction
without harmful effects. However, the end bearing and column
label should be considered the inlet since this was the direction of
flow during packing and testing procedures. Use of the same flow
direction for analysis is encouraged, and it will prove helpful during
any necessary cleaning procedures.
Both end fittings are made up of a smaller and a larger hex nut. The
larger hex nuts contain a non-removable stainless steel frit to both
retain the resin packing and to “catch” particulates before they reach
the resin. In normal operation particulates from the sample or buffer
should be filtered out using a 0.45 µm membrane filter (refer to
Section III d.). If the backpressure of the column should rise above 2500
psi, reversing the direction of flow on the column for a few minutes will
usually flush particulates away. If flushing and cleaning procedures
(see Troubleshooting, Section III d.) do not work, the frits may be
cleaned ultrasonically following their removal from the column.
a. end fitting removal Procedure
CAUTION: Removal of end fittings may disturb the resin packing
material and cause skewed or broad peaks. Therefore, this procedure
should be used only as a last resort measure.
1. Work only in a clean area where dust, dirt and debris will not
contaminate the exposed packing material.
2. Secure column in a vice. Hold in an upright position by clamping
lightly (do not over¬tighten) onto the smaller hex fitting.
3. Remove only one fitting at a time to avoid loss of packing material.
Always begin by removing column inlet (end with column label) first.
4. Use 5/8 inch (16 mm) wrench, turning counterclockwise slowly
until fitting is removed.
5. With a clean spatula or similar tool, remove any resin packing which
has adhered to the frit and place smoothly and carefully back on¬to
resin bed at end of column tube.
6. Fritted fitting may now be cleaned in ultrasonic cleaner using
appropriate solvent. 6 N nitric acid (HNO,) often works well but be
very cautious to avoid contact with skin.
7. Column fittings should be reassembled as soon as possible after
inspection and cleaning to avoid drying and contamination of resin.