Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 8)

8
[ CARE AND USE MANUAL ]
XBridge Protein BEH C
4
, 300Å, 3.5, 5, 10 �m
reduces the possibility of incomplete elution of the protein from
the column. Second, memory effects may be more pronounced with
steep gradients. Keep the gradient slope at 1% per column volume
or less. Third, memory effects may be exacerbated by high flow
rates. Reduce the flow rate by one half while doubling the gradient
time to maintain a constant slope. Fourth, memory effects may be
reduced by changing the organic solvent to incorporate propanol,
typically 70% propanol:30% acetonitrile as strong solvent.
Fifth, carryover may be reduced in routine assays with a
regeneration step including a series of fast gradients from 0–100%
acetonitrile. The gradients can be as short as 2 column volumes and
3–5 repetitions may be effective. This “sawtooth” gradient may be
appended to each injection. Finally, apparent memory effects may
actually reflect the solubility of the protein in the mobile phase.
Reducing the amount injected may eliminate the effect.
5. Recovery is often improved by elevating the column
temperature.
Note: Useful general information on column troubleshooting problems may
be found in HPLC Columns Theory, Technology and Practice, U.D. Neue,
(Wiley-VCH, 1997), the Waters HPLC Troubleshooting Guide (Literature code
# 720000181EN) or visit the Waters Corporation website for information on
seminars (www.waters.com).
VI. COLUMN CLEANING, REGENERATION,
AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the peak,
shifts in retention, change in resolution, carryover, ghost peaks,
or increasing backpressure may indicate contamination of the
column. Choose a cleaning option that may be expected to
dissolve the suspected contaminant.
1. All cleaning procedures will be more effective at higher
temperatures. The BEH300 C
4
can be routinely operated at
temperatures as high as 90 °C so it is reasonable to conduct
cleaning at 70–90 °C.
2. It may be useful to conduct cleaning procedures at one-half
the flow rate typical used with that column. In this way the
possibility of high pressure events is reduced.
3. The first and simplest cleaning procedure is to run a series of
fast gradients from 0–100% acetonitrile. The gradients can
be as short as 2 column volumes and 3–5 repetitions may be
effective. This “sawtooth” gradient may be appended to each
injection to stabilize routine assays.
4. Several different cleaning solutions may be injected to
strip strongly adsorbed material or particulates from the
column. Make the largest injection possible with the system
configuration. With such strong cleaning solutions, it is best to
disconnect the detector from the column and to direct the flow
to waste.
a. An injection of 1% formic acid.
b. An injection of 10% formic acid.
c. An injection of either 4M urea or 6M guanidine-HCl.
d. If contamination with lipids is suspected, a strong
cleaning option is an injection of tetrahydrofuran.
5. Flow reversal or backflushing is often suggested as part of a
cleaning procedure. This should be reserved as a last resort.
It may further damage the column or provide a short-lived
improvement in performance.
b. Storage
For short-term storage, the column should be stored in the mobile
phase with 20–50% acetonitrile. For periods longer than four
days at room temperature, store the column in 100% acetonitrile.
Immediately after use with elevated temperatures and/or at pH
extremes, store in 100% acetonitrile for the best column lifetime.
Do not store columns in highly aqueous (<20% organic) mobile
phases, as this may promote bacterial growth. If the mobile
phase contained a buffer 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 or system when 100% acetonitrile
is introduced. Completely seal column to avoid evaporation and
drying out of the bed.
V. CONNECTING T HE COLUMN TO T HE HPLC
a. Column Connectors and System Tubing Considerations
Tools needed:
§ 3/8-inch wrench
§ 5/16-inch wrench
Handle the column with care. Do not drop or hit the column on a
hard surface as it may disturb the bed and affect its performance.