Waters ACQUITY UPLC CSH130 C18 1.7 μm and XP 2.5 μm Columns - Manual (Page 4)

[ CARE AND USE MANUAL ]
ACQUITY UPLC CSH130 C
18
Columns 4
b. pH Range
The recommended operating pH range for ACQUITY UPLC CSH130
C
18
columns is 1 to 11. 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 con-
centration of buffer used. For example, the use of phosphate buffer at
pH 8 or above in combination with elevated temperatures will lead to
shorter column lifetimes.
c. Solvents
To maintain maximum column performance, use high quality chromat-
ography 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 CSH130 C
18
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 up to 80 ˚C are recommended for operating ACQUITY UPLC
CSH130 C
18
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. > 70 °C) may also result in shorter column lifetimes.
Note: Working at the extremes of temperature, pressure and/or pH will result
in shorter column lifetimes.
III. 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.
Table 2. Buffer Recommendations for Using ACQUITY UPLC CSH130 C
18
Columns from pH 1 to 11
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.
4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generally used at 10 mM or less.
Ammonia (NH
4
OH) 9.2 8.2 – 10.2 Volatile Yes Keep concentration below 10 mM and temperatures below 30 ˚C.
Ammonium Bicarbonate
10.3 (HCO
3 -
)
9.2 (NH
4 +
)
6.3 (H
2
CO
3
)
6.8 – 11.3 Volatile Yes
Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with ammonium hydroxide or acetic acid.
Good buffering capacity at pH 10.
Note: use ammonium bicarbonate (NH
4
HCO
3
), not ammonium carbonate ((NH
4
)
2
CO
3
).
Ammonium (Acetate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Ammonium (Formate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
CAPSO 9.7 8.7 – 10.7 Non-Volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.
Glycine 2.4, 9.8 8.8 – 10.8 Non-Volatile No Zwitterionic buffer, can give longer lifetimes than borate buffer.
1-Methylpiperidine 10.2 9.3 – 11.3 Volatile Yes Used in the 1-10 mM range.
CAPS 10.4 9.5 – 11.5 Non-Volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.