Waters XBridge XP 2.5 µm Columns - Manual (Page 13)

XBridge XP 2.5 µm Columns 13
[ CARE AND USE MANUAL ]
c. Add triethylamine (TEA) or ammonium hydroxide
(NH
4
OH) modifiers to both mobile-phase (e.g., A2,
B2, etc.) reservoirs.
d. For LC/ELSD separations of mono- and/or disaccharides, typical
isocratic conditions include:
i. 75% (acetonitrile) with 0.2% TEA, 35 °C,
0.13 mL/min, 2.1 x 50 mm BEH Amide column;
ii. 77% acetone with 0.05% TEA, 85 °C,
0.15 mL/min, 2.1 x 50 mm BEH Amide column;
iii. 75% acetonitrile with 0.2% TEA, 35 °C,
0.2mL/min, 2.1 x 100 mm BEH Amide column.
e. For LC/ELSD separations of more complex sugar mixtures (e.g.,
polysaccharides), typical gradient conditions include (add TEA
modifier to both mobile-phases A and B):
i. Gradient going from 80% - 50% acetonitrile
with 0.2% TEA in 10 minutes, 35 °C, 0.13 mL/min,
2.1 x 100 mm BEH Amide column;
ii. 80% - 55% acetone with 0.05% TEA in
10 minutes, 85 °C, 0.15 mL/min, 2.1 x 100 mm BEH
Amide column.
f. For LC/MS separations of mono- and disaccharides, typical
isocratic conditions include:
i. 75% acetonitrile with 0.1% ammonium
hydroxide, 35 °C, 0.13 mL/min, 2.1 x 50 mm
BEH Amide column.
g. For LC/MS separations of more complex sugar mixtures (e.g.,
polysaccharides), typical gradient conditions include (add NH
4
OH
modifier to both mobile-phases A and B):
i. Gradient going from 75% - 45% acetonitrile
with 0.1% ammonium hydroxide in 10 minutes,
35 °C, 0.2 mL/min, 2.1 x 100 mm BEH Amide
column.
5. More complex sample mixtures may require the use of gradient
conditions and/or longer column lengths.
6. If acetone is used in one or more mobile-phases, do not use acetone
as a sample diluent or needle wash solvent. Refer to injection solvents
section for sample diluent recommendations and miscellaneous tip
(#3) for needle wash solvent/purge solvent recommendations.
7. Typical sample preparation suggestions for samples that contain
sugars /saccharides /carbohydrates:
a. Liquid Samples
i. Dilute with 50/50 acetonitrile/water
ii. Filter using 0.45 μm or 0.22 μm syringe filter
(if necessary).
b. Solid Samples
i. Weigh out sample (~3 g) into 50 mL centrifuge tube
ii. Add 25 mL of 50/50 acetonitrile/water and
homogenize (mechanically)
iii. Centrifuge at 3200 rpm for 30 minutes
iv. Collect supernatant and filter using 0.45 μm or
0.22 μm syringe filter (if necessary).
c. Depending on sample and/or analyte concentrations, additional
sample dilutions may be necessary.
d. More complex samples and/or lower analyte concentrations may
require additional sample preparation steps and/or procedures
such as solid-phase extraction (SPE).
e. Consider VanGuard BEH Amide Pre-Columns for UPLC Column
protection.