6
CORTECS and CORTECS Premier 2.7
µ
m Columns
[ CARE AND USE MANUAL ]
IV. 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.
Flush 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 3. Recommended pH and Temperature Limits for CORTECS Columns
Name of
column
Ligand type
Surface
charge
modification
End cap
style
Carbon
load
Ligand
density
pH
limits
Temp.
limits
C
18
Trifunctional C
18
None
Proprietary
6.6%
2.7 µmol/m2
2–8
45 °C
C
18
+
Trifunctional C
18
+
Proprietary
5.7%
2.4 µmol/m2
2–8
45 °C
C
8
Trifunctional C
8
None
Proprietary
4.5%
3.4 µmol/m2
2–8
45 °C
Phenyl
Trifunctional phenyl
None
Proprietary
5.9%
3.2 µmol/m2
2–8
45 °C
HILIC
None
None
None
Unbonded
n/a
1–5
45 °C
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 (followed by an appropriate
isopropanol/water mixture**)
Option 3: Flush column with 7 M guanidine
hydrochloride or 7 M urea
6. Mobile phase
6. Mobile phase
* Prior to using THF or hexane, ensure your system is compatible with these solvents. 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.
** Use low organic solvent content to avoid precipitating buffers.