Waters Peptide Separation Technology ACQUITY UPLC BEH130 and BEH300 - Manual (Page 4)

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[ CARE AND USE MANUAL ]
ACQUITY UPLC Peptide BEH C
18
, 130Å and 300Å Columns
Table 3. Column Cleaning Sequence
Polar
Samples Proteinaceous Samples
1. Water Option 1: Inject repeated 100 µL aliquots of
dimethylsulfoxide (DMSO) using a reduced flow
rate delivering 50% Eluent A and 50% Eluent B
2. Methanol Option 2: gradient of 10% to 90% B where:
A = 0.1% trifluoroacetic acid (TFA) in water
B = 0.1% trifluoroacetic acid (TFA) in
acetonitrile (CH
3
CN)
3. Isopropanol
Option 3: Flush column with 7M guanidine
hydrochloride, or 7M urea
Note: To avoid potentially damaging precipitation within your column (e.g., if
your separation eluent contains phosphate buffer), be certain to flush column
with 5–10 column volumes of water BEFORE using suggested organic eluent
column wash procedures.
b. Storage
For periods longer than four days at room temperature, store
the column in 100% acetonitrile. For elevated temperature
applications, store immediately after use in 100% acetonitrile
for the best column lifetime. Do not store columns in buffered
eluents. 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 when
100% acetonitrile is introduced. Completely seal column to avoid
evaporation and drying out of 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.
V. eCORD
a. Introduction
The eCord intelligent chip is a new technology that will provide
the history of a column’s performance throughout its lifetime. The
eCord is 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.
Waters eCord intelligent chip
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior to
use. Pall Gelman Laboratory Acrodisc
® filters are recommended.
Solvents containing suspended particulate materials will
generally clog the outside surface of the inlet distribution frit
of the column. This will result in higher operating pressure and
poorer performance.
d. Pressure
ACQUITY UPLC BEH130 and BEH300 Columns can tolerate pressures of
up to 15,000 psi (1034 bar or 103 Mpa).
e. Temperature
Temperatures between 20 ˚C – 55 ˚C are recommended for operating
ACQUITY UPLC Peptide BEH C
18
, 130Å and 300Å Columns in order
to enhance selectivity, lower solvent viscosity and increase mass
transfer rates. Operating at the extremes of pH, temperature, and/or
pressure will result in a shortened column lifetime.
IV. COLUMN CLEANING, REGENERATING
AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the peak, shifts
in retention, change in resolution or increasing backpressure may
indicate contamination of the column. Flushing with a neat organic
solvent, taking care not to precipitate buffers, is usually sufficient
to remove the contaminant. If the flushing procedure does not solve
the problem, purge the column using the following cleaning and
regeneration procedures.
Use the cleaning routine that matches the properties of the
samples and/or what you believe is contaminating the column (see
Table 3 below). Flush columns with 20 column volumes of HPLC-
grade solvents. Increasing mobile phase temperature to 35–55 ˚C
increases cleaning efficiency. If the column performance is poor
after regenerating and cleaning, call your local Waters office for
additional support.