Cleaning And Regeneration - Waters ACQUITY UPLC Care And Use Manual

[ CARE AND USE MANUAL ]
c. Recommended Injector Wash Solvents
Between analyses, the ACQUITY UPLC System injector and
seals can and should be washed with two separate solvents.
A 90% water/10% acetonitrile mixture is the recommended
strong solvent injector wash solution for the TEAA ion-pairing
based method.
A 90% water/10% methanol mixture is the recommended strong
solvent injector wash solution for the TEA-HFIP based method.
0.20 µm membrane filtered, LC grade water is the recommended
weak wash solvent solution for all ACQUITY oligonucleotide
separation methods.
Note: Do not use oligonucleotide separation mobile phases
A and B for the respective weak and strong injector wash
solvents especially with TEA-HFIP ion pairing systems due
to seal incompatibility issues with HFIP.
d. pH Range
The recommended operating pH range for ACQUITY UPLC and
ACQUITY Premier Oligonucleotide BEH C
e. Pressure
ACQUITY UPLC and ACQUITY Premier Oligonucleotide
BEH C Columns can tolerate pressures of up to 15,000 psi
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(1034 bar or 103 Mpa).
f. Temperature
Temperatures between 20 °C–90 °C are recommended
for operating ACQUITY UPLC and ACQUITY Premier
Oligonucleotide BEH C Columns in order to enhance selectivity,
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lower solvent viscosity, and increase mass transfer rates.
Note: Operating at elevated pH, temperature, and/or
pressure may potentially result in shortened column life.
g. ACQUITY UPLC Mixer Options
The standard Waters ACQUITY UPLC System is equipped
with 50 µL in-line mobile phase mixer. For demanding
biopolymer separations (e.g., peptide mapping), use of a
shallow gradients (e.g., 0.15% mobile phase B change per
minute) is required. In these situations, it is recommended
that the organic solvent concentration in mobile phase B be
reduced by "premixing" with a measured amount of mobile
phase A (e.g., mobile phase A= 0.1 M TEAA and mobile phase
B= acetonitrile/0.1 M TEAA, 20/80, v/v).
Use of a 425 µL mixer (p/n: 205000403) specifically designed
for shallow UPLC gradient separations is recommended when
the solvent premixing technique (detailed above) is not used
and when mobile phase B contains either 100% acetonitrile
(for TEAA ion-pairing method) or 100% methanol (for TEA-
HFIP ion-pairing method).
Columns is 1 to 12.
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ACQUITY UPLC and ACQUITY Premier Oligonucleotide BEH C
In addition, the Solvent Deliver System Outlet Tube Assembly
(p/n: 430001486) is required for 425 µL mixer installation onto
a standard ACQUITY UPLC System.
Note: The 425 µL mixer introduces an additional delay volume
to gradient separations. For ultra-fast oligonucleotide analyses,
the smaller 50 µL mixer should be used with the described
premixed mobile-phase technique.
h. Flow Rate
The recommended flow rate for oligonucleotide separations
performed on a 2.1 x 50 mm ACQUITY UPLC and ACQUITY
Premier Oligonucleotide BEH C
faster flow rates are desired for separations, use of the 425 µL
mixer with installed Outlet Tubing Assembly is recommended.
IV. 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 or increasing
backpressure may indicate contamination of the column.
Flushing with approximately 20-column volumes of 0.2 µm
membrane filtered, neat organic solvent (e.g., acetonitrile
with the TEAA method of methanol with the TEA/HFIP
protocol) is usually sufficient to remove the contaminant. If
the neat organic solvent flushing procedure does not solve
the problem, purge the column with 20-column volumes
of oligonucleotide mobile phase A followed by 20-column
volumes of either 7 M guanidine hydrochloride or 7 M urea.
Be sure to flush column with an additional 20-column volumes
of 0.2 µm membrane filtered, LC-grade water prior to reuse of
oligonucleotide mobile phases. If the column performance is
poor after regenerating and cleaning, call your local Waters
office for additional support.
b. Storage
For periods longer than four days at room temperature,
store the column in 100% acetonitrile. Immediately after use
at elevated temperature and/or pH, store column in 100%
acetonitrile for the best column lifetime. Do not store column
in highly aqueous (<20% organic) mobile phase, as this may
promote bacterial growth. Completely seal column to avoid
evaporation and drying out of the packed bed.
Column is 0.2 mL/min. When
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Columns
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