Waters ACQUITY UPC2 BEH, HSS, and CSH Columns - Manual (Page 6)

6
[ CARE AND USE MANUAL ]
ACQUITY UPC 2 Torus Columns Care & Use
volumes of solvent. Increasing column temperature increases
cleaning efficiency. If the column performance is poor after
cleaning and regenerating, call your local Waters office for
additional support.
Note: Using an ACQUITY UPC 2 System is not recommended for
the cleaning and regeneration procedures because recommended
solvents and high concentrations of water are immiscible with
supercritical CO
2
. Rather, the use of an ACQUITY UPLC System is
recommended for cleaning and regeneration.
Polar Samples Non-Polar Samples
1. Water (could also be
a mixture of acetonitrile
and water)
1. Isopropanol (or an
appropriate water/
Isopropanol mixture)
2. Methanol 2. Tetrahydrofuran
3. Tetrahydrofuran 3. Dichloromethane
4. Methanol 4. Hexane
5. Water
5. Isopropanol (followed
by an appropriate water/
isopropanol mixture)
6. Mobile Phase 6. Mobile Phase
Table 4: Column Cleaning Sequence.
Retest the column after using the cleaning procedure to
determine if the specific problem has been fixed. If so, continue
using the column, avoiding samples and solvents that may clog
the column inlet.
b. Aging
Research at the Waters Corporation confirms that there may be
changes in retention with SFC columns due to packing material
surface changes. This is commonly referred to by SFC industry
experts as “Aging.” Mobile-phase additives and methanol can
cause changes to the silica or hybrid surface resulting in retention
shifts. When working with a new column, the column can be
flushed with CO
2
/methanol to eliminate or reduce further shifts
in retention. Storage in 100% CO
2
is recommended to ensure the
column retention and selectivity will be the same from the end of
one test to the beginning of another. The 100% CO
2
will halt the
aging process and results in no additional retention shifts.
c. Regeneration
If you have noticed a significant change in retention or selectivity,
an additional regeneration process may restore your column’s
performance. There are two main sources of retention or
selectivity change for ACQUITY UPC
2 Columns, assuming no
damage has been done to the column bed. Mobile-phase additives
and methanol can cause changes to the silica or hybrid surface.
The effects can be gradual or expeditious and may be permanent.
Columns showing peak shape, retention or selectivity changes
are best cleaned and regenerated on an ACQUITY UPLC System.
Before placing the column to be regenerated on a LC system,
it should be free of mobile-phase co-solvents and additives.
To do this, flow 100% CO
2
through the column (3.0 x 100 mm) at
1 mL/min for five minutes (40 °C and 2000 psi at the ABPR).
For other column dimensions, the flow rate should be recalculated
appropriately to avoid overpressuring the system.
Based on the stationary phase to be regenerated, different rinsing
agents should be used as to not damage the packing material.
High-pH rinsing agents (pH > 8) should be avoided for cleaning
silica-based materials. Low-pH rinsing agents (pH < 1) should be
avoided for cleaning silica-based materials with ligand bondings.
Step 1. Unbonded and bonded silica-based materials
should be rinsed with 200+ column volumes of pure water.
All hybrid-based materials (e.g., BEH and CSH) can be
rinsed with either 200+ column volumes of pure water or
30+ column volumes of 0.1% N,N-Dimethylbutylamine
(or similar tertiary amine) in water.
Step 2. All columns should be rinsed with an additional
10-column volumes of 90/10 IPA/water to remove the mobile-
phase additives.
Step 3. All columns should be rinsed with 10-column
volumes of isopropanol to remove any residual water before
returning the column to an ACQUITY UPC
2 System.
After regeneration, please reinstall and equilibrate the ACQUITY
UPC
2 Column on the ACQUITY UPC 2 System. Retest the column
after using the cleaning procedure to determine if the specific
problem has been fixed.