Waters Protein Separation Technology ACQUITY UPLC BEH300, C4, 1.7 µm Columns - Manual (Page 6)

[ Care and Use ManUal ]
Protein Separation Technology ACQUITY UPLC BEH300, C
4
, 1.7 �m
6
b. Operating pH Limits
The recommended operating pH range for ACQUITY UPLC BEH300, C
4
, 1.7 µm columns is 1 to 12. A listing of commonly used buffers and
additives is given in Table 2. Additionally, the column lifetime will vary depending upon the operating temperature as well as the type and
concentration of buffer used.
Table 2: Buffer Recommendations for Using ACQUITY UPLC BEH300, C
4
columns from pH 1 to 12
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-Methylm-
orpholine
~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
(HCO3 - )
9.2
(NH4
+
)
8.2 – 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 CO3)
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-Methyl-
piperidine 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 phos-
phoric).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.