Instruction Manual

Table 2. Buffer Recommendations for Using ACQUITY UPLC BEH Columns from pH 2 to 12
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.
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.
Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.
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