Waters ACQUITY UPLC BEH Columns - Manual (Page 6)

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 below). 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 ACQUITY UPLC BEH 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.
To clean polar contaminants from ACQUITY UPLC BEH Amide
Columns, run a 10 minute gradient from 0-100% water. Please
note that as aqueous concentration increases, backpressure will
rapidly increase as well. Reduce flow rate when operating at
greater than 60% aqueous. Repeat if necessary.
Table 3: Recommended pH and Temperature Limits for ACQUITY UPLC BEH Columns
Name of Column Particle Size Pore Diameter Surface Area pH Limits
Temperature Limits
Surface Carbon %
Low pH High pH
BEH C
18
1.7 µm 130Å 185 m 2 /g 1-12 80 °C 60 °C 3.1 µmol/m 2 17.7
BEH C
8
1.7 µm 130Å 185 m 2 /g 1-12 60 °C 60 °C 3.1 µmol/m 2 12.8
BEH Phenyl 1.7 µm 130Å 185 m 2 /g 1-12 80 °C 60 °C 3.0 µmol/m 2 14.5
BEH Shield RP
18
1.7 µm 130Å 185 m 2 /g 2-11 50 °C 45 °C 3.2 µmol/m 2 16.6
BEH HILIC 1.7 µm 130Å 185 m 2 /g 1-9 60 °C 45 °C — —
BEH Amide 1.7 µm 130Å 185 m 2 /g 2-11 90 °C 90 °C 7.5 µmol/m 2 12
Table 4. Reversed-Phase Column Cleaning Sequence
* 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.
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 (followed by an appropriate
isopropanol/water mixture*) Option 3: Flush column with 7M guanidine
hydrochloride, or 7M urea
6. mobile phase 6. mobile phase
6
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