User guide

[ Care and Use ManUal ]
XBridge
™
Columns 4
Table 2: Recommended pH and temperature Limits for XBridge
™
Columns at Ambient Temperatures
Name of Column Particle Size Pore Diameter Surface Area pH Limits
Temperature Limits
Surface Carbon Load
Low pH High pH
XBridge C
18
2.5, 3.5, 5 µm 130Å 185 m
2
/g 1-12 80 °C 60 °C 3.1 µmol/m
2
18%
XBridge C
8
2.5, 3.5, 5 µm 130Å 185 m
2
/g 1-12 60 °C 60 °C 3.1 µmol/m
2
13%
XBridge Phenyl 2.5, 3.5, 5 µm 130Å 185 m
2
/g 1-12 80 °C 60 °C 3.0 µmol/m
2
15%
XBridge Shield RP18 2.5, 3.5, 5 µm 130Å 185 m
2
/g 2-11 50 °C 45 °C 3.2 µmol/m
2
17%
XBridge HILIC 2.5, 3.5, 5 µm 130Å 185 m
2
/g 1-9 45 °C 45 °C - -
XBridge Amide 3.5 µm 130Å 185 m
2
/g 2-11 90 °C 90 °C 7.5 µmol/m
2
12%
d. Solvents
To maintain maximum column performance, use high quality chromatog-
raphy 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 poor performance.
Degas all solvents thoroughly before use to prevent bubble formation in
the pump and detector. The use of an on-line degassing unit is also recom-
mended. This is especially important when running low pressure gradients
since bubble formation can occur as a result of aqueous and organic
solvent mixing during the gradient.
e. Pressure
XBridge
™
columns can tolerate pressures of up to 6,000 psi (400 bar or 40
Mpa) although pressures greater than 4,000 – 5,000 psi should be avoided in
order to maximize column and system lifetimes.
f. Temperature
Temperatures between 20 ˚C – 80 ˚C (up to 90 ˚C for XBridge Amide columns)
are recommended for operating XBridge columns in order to enhance selectiv-
ity, lower solvent viscosity and increase mass transfer rates. However, any
temperature above ambient will have a negative effect on lifetime which will
vary depending on the pH and buffer conditions used. Under HILIC conditions
XBridge Amide columns can be used at high pH and at high temperatures with-
out issues (see recommended conditions in Getting Started with XBridge Amide
section). See Table 2 for recommended pH and temperature limits.
Additive/Buffer pKa
Buffer Range
(±1 pH unit)
Volatility 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.
Phosphate 3 12.3 11.3 - 13.3 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)
Ammonium Bicarbonate
9.2
10.3 (HCO
3
-
)
9.2 (NH
4
+
)
8.2 – 10.2
8.2 – 11.3
Volatile
Volatile
Yes
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.
Borate 9.2 8.2 – 10.2 Non-volatile No Reduce temperature/concentration and use a guard column to maximize lifetime.
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.
Triethylamine
(as acetate salt)
10.7 9.7 – 11.7 Volatile Yes Used in the 0.1-1.0% range. Volatile only when titrated with acetic acid (not hydrochloric or phosphoric).
Used as ion-pair for DNA analysis at pH 7-9.
Pyrrolidine 11.3 10.3 – 12.3 Volatile Yes Mild buffer, gives long lifetime.
Table 3: Buffer Recommendations for Using XBridge
™
Columns from pH 1 to 12
Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.