Waters ACQUITY UPLC HSS Columns - Manual (Page 4)

[ Care and Use ManUal ]
XSelect HSS HPlc columnS 4
and 1 to 8 for the HSS c
18
chemistry. A listing of commonly used
buffers and additives is given in table 2. Additionally, the column
lifetime will vary depending upon the operating temperature, the
type and concentration of buffer used.
Note: Working at the extremes of pH, temperature and/or pressure
will result in shorter column lifetimes.
c. Solvents
to maintain maximum column performance, use high quality chro-
matography grade solvents. Filter all aqueous buffers prior to use
through a 0.2 µm filter. Solvents containing suspended particulate
materials will generally clog the outside surface of the inlet distri-
bution frit of the column. this will result in higher operating pres-
sure and poorer performance. See Section V for more information.
d. Pressure
AcQuItY uPlc HSS columns can tolerate operating pressures up
to 18000 psi (1241 bar or 124 mPa).
Note: Working at the extremes of pressure, pH and/or temperature
will result in shorter column lifetimes.
e. Temperature
temperatures between 20–45 °c are recommended for operating
AcQuItY uPlc HSS columns in order to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. When operating
at high pH, lower operating temperatures are recommended for
longer column lifetime. Working at high temperatures (e.g. > 40 ˚c)
may also result in shorter column lifetimes.
Table 2: Buffer Recommendations for Using ACQUITY UPLC HSS Columns
Additive/Buffer pKa
Buffer
Range
Volatility
(±1 pH unit)
Used for
Mass Spec
Comments
TFA 0.3 Volatile Yes
Ion pair additive, can suppress mS signal, used in the
0.02-0.1% range.
Acetic Acid 4.76 Volatile Yes
maximum buffering obtained when used with ammonium acetate
salt. used in 0.1-1.0% range.
Formic Acid 3.75 Volatile Yes
maximum buffering obtained when used with ammonium formate
salt. used in 0.1-1.0% range.
Acetate
(NH
4
CH
2
COOH)
4.76 3.76 – 5.76 Volatile Yes
used in the 1-10 mm range. note that sodium or potassium
salts are not volatile.
Formate (NH
4
COOH) 3.75 2.75 – 4.75 Volatile Yes
used in the 1-10 mm range. note that sodium or potassium
salts are not volatile.
Phosphate 1 2.15 1.15 – 3.15 non-volatile no traditional low pH buffer, good uV transparency.
Phosphate 2 7.2 6.20 – 8.20 non-volatile no
Above pH 7, reduce temperature/concentration and use a guard
column to maximize lifetime.
II. Column use
to ensure the continued high performance of AcQuItY uPlc HSS
columns, follow these guidelines:
a. Sample Preparation
4. Sample impurities often contribute to column contamination.
one option to avoid this is to use Waters oasis ® solid-phase
extraction cartridges/columns or Sep-Pak ® cartridges of the
appropriate chemistry to cleanup the sample before analysis.
For more information, visit www.waters.com/sampleprep
5. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker than the mobile phase
for the best peak shape and sensitivity.
6. If the sample is not dissolved in the mobile phase, ensure that
the sample, solvent and mobile phases are miscible in order
to avoid sample and/or buffer precipitation.
Filter sample with 0.2 µm membranes to remove particulates. If
the sample is dissolved in a solvent that contains an organic modi-
fier (e.g., acetonitrile, methanol, etc.) ensure that the membrane
material does not dissolve in the solvent. contact the membrane
manufacturer with solvent compatibility questions. Alternatively,
centrifugation for 20 minutes at 8000 rpm, followed by the
transfer of the supernatant liquid to an appropriate vial, could be
considered.
b. pH Range
the recommended operating pH range for AcQuItY uPlc HSS
columns is 2 to 8 for the t3, c
18
SB, cyano and PFP chemistries,