Waters Protein-Pak Hi Res IEX Columns - Manual (Page 7)

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[ CARE AND USE MANUAL ]
Protein-Pak Hi Res IEX Columns and Standards
made, they likely originate from adsorption to some system
component. In that case follow the instrument manufacturer’s
recommendations. Memory effects as a source of carryover
may be reduced or eliminated in several ways. First, raising the
temperature of the separation reduces the possibility of non-
specific protein adsorption. Second, memory effects may be
more pronounced with steep gradients. Keep the gradient slope
at 10 column volumes or greater. 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. Finally, apparent memory effects may actually reflect
the solubility of the protein 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), 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. 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.
2. 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. 0.1mol/L NaOH, or
b. 20 ~ 40% acetic acid, or
c. Solution containing aqueous organic solvent such as
methanol or acetonitrile
d. Solution containing a solubilizer such as urea and
non-ionic surfactants
3. Flow reversal or back flushing 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 overnight storage, the column can remain in the IEX separation
eluent while maintaining a low flow rate (e.g., 0.1 mL/min). For
long-term storage, replace the eluent at the suggested flow rate
shown below with filtered, high purity water containing 0.05%
sodium azide that will inhibit undesired microbial growth.
<0.25 mL/min for Protein-Pak Hi Res Q, 5 µm columns
<0.5 mL/min for Protein-Pak Hi Res CM and SP, 7 µm columns
Completely seal column to avoid evaporation and drying of the bed.
VII. INT RODUCING ECORD INT ELLIGENT
CHIP T ECHNOLOGY
a. Introduction
The eCord intelligent chip is a new technology that will provide
the history of a column’s maximum backpressure, maximum
temperature and the number of injections performed throughout its
lifetime. The eCord will be permanently attached to the column to
assure that the column’s performance history is maintained in the
event that the column is moved from one instrument to another.
Figure D. Waters eCord Intelligent Chip