Vanguard Pre-Columns; Installation Instructions; Column Use; Sample Preparation - Waters ACQUITY UPLC Peptide HSS T3 100A Care And Use Manual

[ CARE AND USE MANUAL ]
f. VanGuard Pre-Columns
VanGuard™ Pre-columns are 2.1 mm I.D. x 5 mm length guard
column devices designed specifically for use in the ACQUITY
UPLC® System. VanGuard Pre-columns are packed with the
same UPLC Chemistries and frits as our 2.1 mm I.D. UPLC
Columns. VanGuard Pre-columns are designed to be attached
directly to the inlet side of an ACQUITY UPLC Column.
Note: In order to ensure void-free and leak-free connections, the VanGuard
Pre-column is shipped with the collet and ferrule NOT permanently attached.
Care must be taken when removing the O-ring that holds these two pieces
on the pre-column tubing.
ACQUITY UPLC Column
Place wrench here
Ferrule
g. Installation Instructions
1. Remove the VanGuard Pre-column from its box and
shipping tube and remove plastic plug.
2. Orient the pre-column so that male end is facing up and
carefully remove rubber O-ring that holds collet and ferrule
in place during shipping (collet and ferrule are not yet
permanently attached).
3. Orient the ACQUITY UPLC Peptide HSS T3 Column
perpendicular to the work surface so that column inlet is
on the bottom (column outlet on top).
4. From below, insert the VanGuard Pre-column into the
ACQUITY UPLC Peptide HSS T3 Column inlet and hand-
tighten (collet and ferrule are not yet permanently attached).
Table 2. Buffer Recommendations for Using ACQUITY UPLC Peptide HSS T3, 100Å Columns from pH 2 to 8
Additive/Buffer pKa
TFA 0.30
Acetic acid 4.76
Formic acid 3.75
Acetate (NH CH COOH) 4.76
4 2
Formate (NH COOH) 3.75
4
Phosphate 1 2.15
VanGuard Pre-Column
Collet
Place wrench here
Flow
Volatility
Buffer range
(±1 pH unit)
— Volatile
— Volatile
— Volatile
3.76–5.76 Volatile
2.75–4.75 Volatile
1.15–3.15 Non-volatile
5. While pushing the VanGuard Pre-column into the column
inlet, turn assembled column and pre-column 180˚ so that
the pre-column is now on top.
6. Tighten with two 5/16" wrenches placed onto the
ACQUITY UPLC Column flats and the VanGuard
Pre-column hex nut (male end) as shown.
7. Tighten 1/4 turn to set collet and ferrule.
8. Check that the ferrule is set by loosening the connection
and inspecting the ferrule depth. A properly set ferrule
depth will resemble other connections in the ACQUITY
UPLC System.
9. Reattach pre-column, apply mobile-phase flow, and
inspect for leaks.
II. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC
Peptide HSS T3 Columns, follow these guidelines:
a. Sample Preparation
1. Sample must be dissolved in a diluent compatible
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 filtered
with a 0.2 µm membrane.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, 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
15,000 rpm, followed by the transfer of the supernatant
liquid to an appropriate vial, could be considered.
Used for
Comments
mass spec
Yes Ion pair additive, can suppress MS signal, used in the 0.02–0.1% range.
Maximum buffering obtained when used with ammonium
Yes
acetate salt. Used in 0.1–1.0% range.
Maximum buffering obtained when used with ammonium
Yes
formate salt. Used in 0.1–1.0% range.
Yes Used in the 1–10 mM range. Note that sodium or potassium salts are not volatile.
Yes Used in the 1–10 mM range. Note that sodium or potassium salts are not volatile.
No Traditional low pH buffer, good UV transparency.
ACQUITY UPLC Peptide HSS T3 Columns 3