Waters CORTECS 1.6 um Columns - Manual (Page 6)

6
CORTECS Columns
III. CoLUMn CLeAnInG, ReGeneRAtInG
AnD stoRAGe
a. Cleaning and regeneration
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. Flushing
with a neat organic solvent, taking care not to precipitate buffers,
is usually sufficient to remove the 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 solvent.
Increasing column temperature increases cleaning efficiency. If
the column performance is poor after regenerating and cleaning,
call your local Waters office for additional support.
Flush CORTECS HILIC Columns with 50:50 acetonitrile/water to
remove polar contaminants. If this flushing procedure does not
solve the problem, purge the column with 5:95 acetonitrile/water.
table 4. reversed-Phase Column Cleaning Sequence
Polar Samples
non-polar
Samples**
Proteinaceous Samples
1. water
1. isoproanol (or
an appropriate
isopropanol/water
mixture*) Option 1: Inject
repeated aliquots of
dimethylsulfoxide
(DMSO)
2. methanol
2. tetrahydrofuran
(THF) Option 2: gradient of
10% to 90% B where:
A = 0.1% trifluoroacetic
acid (TFA) in water
B = 0.1% trifluoroacetic
acid (TFA) in acetonitrile
(CH
3
CN)
3. tetrahydrofuran
(THF)
3. dichloromethane
4. methanol 4. hexane
5. water
5. isopropanol (fol-
lowed by an appro-
priate 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.
** Unless a Hexane Tetrahydrofuran Compatibility Kit (P/N 205000464) has been
installed, running solvents such as THF or hexane should only be considered when
the column cannot be cleaning by running neat, reversed-phase organic solvents
such as acetonitrile. Reduce flow rate, lower operating temperatures and limit
system exposure to THF and/or hexane.
b. Storage
For periods longer than four days at room temperature,
store reversed-phase and CORTECS HILIC Columns in 100%
acetonitrile. For elevated temperature applications, 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 column
volumes of HPLC grade water (see Table 1 for common column
volumes) and replace with 100% acetonitrile for storage. Failure
to perform this intermediate step could result in precipitation
of the buffer salt in the column when 100% acetonitrile is
introduced. Completely seal column to avoid evaporation and
drying out of the bed.
For periods longer than four days, store CORTECS HILIC Columns
in 95:5 acetonitrile/water. Do not store in buffered solvent. If
the mobile phase contained a buffered salt, flush the column with
10 column volumes of 95:5 acetonitrile/water (see Table 1 for
common column volumes).
Note: If a column has been run with a mobile phase that contains
formate (e.g., ammonium formate, formic acid, etc.) and is then
flushed with 100% acetonitrile, slightly longer equilibration times
may be necessary when the column is re-installed and run again
with a formate-containing mobile phase.