Waters ACQUITY UPLC CSH Columns - Manual (Page 5)

[ Care and Use ManUal ]
ACQUITY UPLC CSH Columns 5
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
to use through a 0.2 µm filter. Solvents containing suspended
particulate materials will generally clog the outside surface of
the inlet distribution frit of the column. This will result in higher
operating pressure and poorer performance. See Section V for more
information.
d. Pressure
ACQUITY UPLC CSH columns can tolerate operating pressures up to
18000 psi (1241 bar or 124 MPa).
Note: Working at the extremes of pressure, pH and/or temperature will result in shorter
column lifetimes.
e. Temperature
Temperatures up to 80 ˚C are recommended for operating ACQUITY
UPLC CSH columns in order to enhance selectivity, lower solvent
viscosity and increase mass transfer rates. When operating at high
pH, lower operating temperatures are recommended for longer
column lifetime. Working at high temperatures (e.g. > 70 °C) may
also result in shorter column lifetimes. See Table 2 above for more
information on suggested operating temperatures and pH ranges.
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 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.
Table 4. Reversed-Phase Column Cleaning Sequence
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. tetrahydrofuran
(THF)
2. methanol 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
(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.
** Unless a Hexane Tetrahydrofuran Compatibility Kit (Part Number 205000464) has
been installed, running solvents such as THF or hexane should only be considered when
the column cannot be cleaned 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
ACQUITY UPLC CSH 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 ACQUITY UPLC CSH columns 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.