Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns - Manual (Page 8)

[ Care and Use ManUal ]
Protein Separation Technology ACQUITY UPLC BEH300, C
4
, 1.7 �m
8
3. Change in peak shape, resolution, or relative retention of peaks.
Follow the same steps as for loss of retention (Symptom 2).
4. Carryover and memory effects are defined as the appearance
of the constituents of one sample in the next gradient analysis.
First determine whether the column or the system is the
source of carryover. Define a gradient method that includes an
“internal gradient”. That is, the analytical gradient is repeated
within a single method. If the protein peaks appear in both
gradients, at the same time after start, the protein came from
the column in what is often described as a “memory effect”. If
the protein peaks only appear when an injection is made, they
likely originate from adsorption to some system component. In
that case follow the instrument manufacturer’s recommenda-
tions. 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 non-specific protein
adsorption. 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.
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 possibil-
ity 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 configura-
tion. 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.