User guide

3
[ CARE AND USE MANUAL ]
ACQUITY UPLC Peptide BEH C
18
, 130Å and 300Å Columns
III. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC Peptide
BEH C
18
, 130Å and 300Å Columns follow these guidelines:
a. Sample Preparation
1. Sample must be dissolved in a diluent compatiable with initial
strength of mobile phase.
2. Sample must be completely in solution and free of particulates.
3. To remove particulates the sample may be filted with a 0.2 µm
membrane. If the sample is dissolved in a solvent that contained
an organic modifier (e.g., acetronitrile, 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
8,000 rpm, followed by the transfer of the supernatant liquid to
an appropriate vial, could be considere
d.
b. pH Range
The recommended operating pH range for ACQUITY UPLC BEH 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, the type and concentration 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.
Table 2. Buffer Recommendations for Using ACQUITY UPLC Peptide BEH C
18
, 130Å and 300Å Columns from pH 1 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.
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.
Triethylamine 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).
(as acetate salt) Pyrrolidine 11.3 10.3 – 12.3 Volatile Yes
Used as ion-pair for DNA analysis at pH 7-9 Mild buffer, gives
long lifetime.