Waters ACQUITY UPLC HSS Columns - Manual (Page 5)

[ Care and Use ManUal ]
XSelect HSS HPlc columnS 5
Note: Working at the extremes of temperature, pressure and/or pH
will result in shorter column lifetimes.
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 3). 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.
Table 3. Reversed-Phase Column Cleaning Sequence
* use low organic solvent content to avoid precipitating buffers.
** unless a Hexane tetrahydrofuran compatibility Kit (Pn 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.
Polar Samples
Non-polar
Samples**
Proteinaceous Samples
1. water
1. isoproanol (or
an appropriate
isopropanol/ water
mixture*)
option 1: Inject repeated
aliquots of dimethylsulf-
oxide (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
(cH3cn)
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 hydro-
chloride, or 7m urea
6. mobile phase 6. mobile phase
b. For Normal-Phase Conditions
the HSS cyano column can be used for both reversed-phase sepa-
rations as well as normal-phase separations. the column is origi-
nally shipped in acetonitrile and is ready to use for reversed-phase
conditions.
If you intend to use the column for normal-phase applications, you
will need to condition the column with the following procedure:
1. Flush the column with a minimum of 20 column volumes of
100% methanol using a low flow rate to avoid overpressuring
the lc system. Refer to table 1 for minimum solvent volume.
2. Flush the column with a minimum of 20 column volumes of
100% isopropanol using a low flow rate to avoid overpressuring
the lc system. Refer to table 1 for the minimum solvent volume.
3. Flush the column with a minimum of 20 column volumes of
100% dichloromethane using a low flow rate to avoid over-
pressuring the lc system. Refer to table 1 for the minimum
solvent volume.
4. Flush the column with the intended mobile-phase conditions
until a stable baseline is achieved.
c. Storage
For periods longer than four days at room temperature, store
reversed-phase AcQuItY uPlc HSS 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 buf-
fer 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.
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.