Waters XBridge BEH Glycan, 2.5 μm XP and 3.5 μm Columns and Standards - Manual (Page 4)

LC Systems with different system volumes would affect the final
recorded results.
Figure 2. Comparative scaled separations (based on column particle size
differences) using Waters Glycan Performance Test Standard (P/N 186006349)
on ACQUITY H-Class UPLC System with ACQUITY BEH Glycan, 1.7 μm, XBridge
Glycan 2.5 μm XP, and XBridge Glycan 3.5 μm XP columns.
III. COLUMN USE
To ensure the continued high performance of XBridge BEH Glycan
Columns, observe the following guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
Samples should be free of particles before injection into
the system.
2. In most separations it is preferable to prepare the sample
in the gradient initial composition. However, the 2-AB
labeled glycans are often insoluble in the high acetonitrile
concentrations which typify HILIC initial conditions. Since
small volume injections are being made, the sample diluents
may contain higher aqueous content (e.g. 50%) than the initial
composition.
3. If the sample is not dissolved in the mobile-phase or solvent
combinations specified in this manual, ensure that the sample,
solvent, and mobile phases are miscible in order to avoid
sample and/or buffer precipitation. Preparation of 2-AB
labeled glycans involves one or two steps of solid-phase
extraction. As a result, protein precipitate has typically been
removed. If not, remove protein particles by centrifugation at
>10,000 rpm for more than 2 minutes.
b. Operating pH Limits
The recommended operating pH range for the XBridge BEH
Glycan Column is 3 to 8. A listing of commonly used buffers and
additives is given in Table 2. Additionally, the column lifetime
will vary depending on the operating temperature as well as the
type and concentration of buffer used.
Table 2: Buffer Recommendations for Using XBridge BEH Glycan Columns from pH 3 to 8
Additive/Buffer pK
a
Buffer Range
(±1 pH unit)
Volatility
Used for
Mass Spec Comments
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.
Ammonium (Acetate) 9.20 8.2–10.2 Volatile Yes Up to 100 mM.
Ammonium
(Formate)
9.20 8.2–10.2 Volatile Yes Up to 250 mM.
Triethylamine
(as acetate salt)
10.70 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.
15 16
ACQUITY BEH Glycan 1.7 µm
2.1 x 150 mm
0.50 mL/min
XBRIDGE BEH Glycan 2.5 µm XP
2.1 x 150 mm
0.34 mL/min
7.4
5.0
XBRIDGE BEH Glycan 3.5 µm
2.1 x 150 mm
0.24 mL/min
10.3
1 2
3
4
5
6
7
89
10
11
12 13
14
8700 psi (Column, Max)
3300 psi (Column, Max)
900 psi (Column, Max)
30.0 min
44.3 min
62.1 min
N-Acetylglucosamine
Manose Galactose
Fucose
Sialic Acid
1
2
3
4
5
6,7
8
9
11
12
13
14
10
4
[ CARE AND USE MANUAL ]
XBridge BEH Glycan, 2.5 µm XP and 3.5 µm Columns and Standards