Care and use manual – Waters XBridge Protein BEH SEC Columns and Standards User Manual

Page 6

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[ CARE AND USE MANUAL ]

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V. COLUMN CLEANING, REGENERATION
AND STORAGE

a. Cleaning and Regeneration

Changes in peak shape, such as increased tailing, shoulders on
the peak, shifts in retention, change in resolution, ghost peaks,
or increased backpressure may indicate contamination of the
column. Choose a cleaning option that may be expected to
dissolve the suspected contaminant.

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.

Recommended cleaning solvents:

a. A concentrated salt solution at low pH (e.g. 0.5 M Na SO, pH 2.7).

b. A low concentration of methanol (e.g., 20%) in HPLC-grade water.

c. Use of ionic detergents and other surfactants should be

avoided if the SEC column is to be subsequently used to
analyze native proteins.

Note: Choose a cleaning solvent based on sample properties, e.g.
use (a) to remove basic protein and (b) to remove hydrophobic
proteins. Chaotrophic agents can solvate strongly adsorbed
proteins via hydrogen bond disruption.

As a last resort, flow reversal or back flushing can be tried
at a low flow rate (e.g., 0.1 mL/min). However, this approach
may further damage the column or only provide short-lived
improvement in performance.

b. Storage

For overnight storage, continuously flush the column with the
mobile phase at 20% of the maximum recommended flow rate.
Store the column in the HPLC-grade water when it will be used
within 24 hours or in 20% methanol for long term storage.

Note: Working at extremes of pressure, pH and/or temperature may
result in shorter column lifetime.