Waters Spherisorb Columns - Manual (Page 6)

[ Care and Use ManUal ]
Waters Spherisorb Columns 6
iv. t roubleshootinG
Changes in retention time, resolution, or backpressure are often due
to column contamination. See “Column Cleaning, Regenerating and
Storage”. Information on column troubleshooting problems may be
found in the current Waters Chromatography Columns and Supplies
Catalog. You can also download a copy of the HPLC Troubleshooting
Guide at www.waters.com, and in the Search Box, enter WA20769.
v. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
Changes in peak shape, peak splitting, shoulders on the peak, shifts in
retention, change in resolution or increasing backpressure may indicate
contamination of the column. Changing the guard column being used
will often restore column performance. If not (or if no guard column
is being used), follow the procedures detailed below. To prevent
potential contamination from affecting detector performance, it is
recommended that any detector(s) be disconnected from the effluent
flow of the column during cleaning. Reversing the direction of the
flow through the column (backflushing) may sometimes improve the
effectiveness of any cleaning procedure.
1. Reversed-Phase Columns
Flushing with a neat organic solvent, taking care not to precipitate
buffers, is usually sufficient to remove most contaminant. If the
flushing procedure does not solve the problem, purge the column
using the following cleaning and regeneration procedures. Use the
cleaning routine that matches the properties of the samples and/or
what you believe is contaminating the column (see Table 4). Flush
columns with 20-column volumes of HPLC-grade solvents (e.g.,
80 mL total for 4.6 x 250 mm column). Increasing mobile-phase
temperature to 35-55 ˚C increases cleaning efficiency. If the column
performance is poor after regenerating and cleaning, call your local
Waters office for additional support.
Table 4: Column Sequence or Options
Polar Samples Non-polar Samples Proteinaceous Samples
1. Water
1. Isopropanol (or an
appropriate isopropanol/
water mixture*) Option 1: Inject repeated
aliquots of dimethyl
sulfoxide (DMSO)
2. Methanol 2. Tetrahydrofuran (THF)
Option 2: gradient of
10-90% B where:
A= 0.1% trifluoroacetic
acid (TFA) in water
B= 0.1% trifluoroacetic acid
(TFA) in acetonitrile (CH
3
CN)
3. Tetrahydrofuran 3. Dichloromethane
4. Methanol 4. Hexane
5. Water
5. Isopropanol (followed by
an appropriate isopropanol/
water mixture*) Option 3: Flush column with
7M guanidine hydrochloride
or 7M urea
6. Mobile Phase 6. Mobile Phase
* Use low organic solvent content to avoid precipitating buffers.
2. Normal-Phase Columns
To regenerate, pump 20-30 column volumes each of
dichloromethane and isopropanol through the column. Other wash
solvents such as tetrahydrofurane (THF) may also be selected based
on the suspected contamination.
Guard columns need to be replaced at regular intervals, as
determined by sample contamination. When system backpressure
steadily increases above a set pressure limit, it is usually an
indication that the guard column should be replaced. A sudden
appearance of split peaks is also indicative of a need to replace the
guard column.
b. Storage
Completely seal the column to avoid evaporation and drying out of
the bed.
1. Reversed-Phase Columns
For periods longer than four days at room temperature, store the
column (with the exception of cyano chemistry columns) in 100%
acetonitrile at room temperature. For elevated temperature applica-
tions, store immediately after use in 100% acetonitrile for the best
column lifetime. Do not store columns in buffered eluents. If the
mobile phase contained a buffer salt, flush the column with 10 col-
umn volumes of HPLC grade water (see Table 2 for common column
volumes) and replace with 100% acetonitrile for storage. Failure to
perform this intermediate step could result in precipitation of the