Care and use manual – Waters XBridge BEH Glycan, 2.5 μm XP and 3.5 μm Columns and Standards User Manual

Page 6

exacerbated by high flow rates. Reduce the flow rate by one
half while doubling the gradient time to maintain a constant
slope. Finally, apparent memory effects may actually reflect
the solubility of the sample in the mobile phase. Reducing the
amount injected may eliminate the effect.

Note: Useful general information on column troubleshooting
problems may be found in HPLC Columns Theory, Technology
and Practice, U.D. Neue, Wiley-VCH, 1997 (P/N WAT038216),
the Waters HPLC Troubleshooting Guide (Literature code #
720000181EN), or visit www.waters.com.

V. 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. It is reasonable to conduct cleaning at 70 ˚C.

2. It may be useful to conduct cleaning procedures at one-half

the flow rate typically 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

gradients from 0–100% water. Be sure to reduce the flow rate
for gradients with higher than 75% aqueous content. Columns
of 150 mm length should be operated at 250 µL per minute or
less during washes. The gradients can be as short as 5 column
volumes and 3–5 repetitions may be effective.

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.

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 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 (<50% 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
the column to avoid evaporation and drying out 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.

Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
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F: 1 508 872 1990
www.waters.com

[ CARE AND USE MANUAL ]

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