Waters ACQUITY UPLC BEH Columns - Manual (Page 12)

in the sample are acceptable. 50:50 acetonitrile:water can
provide satisfactory results.
3. The injection solvent’s influence on peak shape should be
determined experimentally. In some cases, injections of water
(or highly aqueous solutions) may not adversely affect peak
shape.
Miscellaneous Tips
1. For initial scouting conditions, run a gradient from 95%
acetonitrile to 50% acetonitrile. If no retention occurs, run
isocratically with 95:3:2 acetonitrile:methanol:aqueous
buffer.
2. Alternate polar solvents such as methanol, acetone or
isopropanol can also be used in place of water to increase
retention.
3. Ensure that your weak needle wash solvent/purge solvent is
your starting mobile phase (i.e., high organic), or your peak
shapes will suffer. Typical needle wash conditions: 800 µL
Strong wash in 20:80 ACN/H
2
O, 500 µL Weak wash in 75:25
ACN/H
2
O.
4. Acetone should not be used as a sample solvent/diluent
unless a Hexane Tetrahydrofuran Compatibility Kit (P/N:
205000464) has been installed.
Tips for Separating Sugars/Saccharides/Carbohydrates
1. If separating sugars or sugar-containing compounds that
do not include reducing sugars (see below) follow generic
‘Getting Started with ACQUITY UPLC BEH Amide Columns’
recommendations described above.
2. If separating reducing sugars, please review the following
information.
3. Reducing sugars can undergo mutarotation which produces the
undesired separation of the α and β ring forms (anomers).
4. Collapsing anomers into one peak is accomplished through the
use of a combination of elevated temperature and high pH:
a. Use of 35 °C with high pH (0.2% triethylamine (TEA) or
0.1% ammonium hydroxide (NH
4
OH)) and/or
b. Use of >80 °C with 0.05% TEA high temperature (>80 °C)
5. When separating reducing sugars (e.g., fructose, glucose,
maltose, lactose, arabinose, glyceraldehyde, etc.) please pay
attention to the following suggestions. Failure to do so will
result in the appearance of split peaks (anomer separation) for
these analytes:
a. Operate at a slow flow rate (e.g., 0.10 - 0.13 mL/min on
2.1 x 50 mm column) to facilitate anomer collapse.
b. With longer columns, increased flow rates (e.g., up to
0.3 mL/min) can be used. As with all LC separations,
optimal flow rates should be determined experimentally.
c. Add triethylamine (TEA) or ammonium hydroxide (NH4OH)
modifiers to both mobile phase (e.g., A2, B2, etc) reservoirs.
d. For UPLC/ELSD separations of mono- and/or disaccharides,
typical isocratic UPLC conditions include:
i. 75% acetonitrile (ACN) 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% ACN with 0.2% TEA, 35 °C, 0.2mL/min,
2.1 x 100 mm BEH Amide column.
e. For UPLC/ELSD separations of more complex sugar
mixtures (e.g., polysaccharides), typical gradient UPLC
conditions include (add TEA modifier to both mobile
phases A and B):
i. Gradient going from 80% to 50% ACN with 0.2% TEA in
10 min, 35 °C, 0.13 mL/min, 2.1 x 100 mm BEH Amide
column or up to 0.3 mL/min flow rate with 2.1 x 150 mm
BEH Amide column;
ii. 80%-55% Acetone with 0.05% TEA in 10 min,
85 °C, 0.15 mL/min, 2.1 x 100 mm BEH Amide column.
f. For UPLC/MS separations of mono- and disaccharides,
typical isocratic UPLC conditions include:
i. 75% ACN with 0.1% NH
4
OH, 35 °C, 0.13 mL/min,
2.1 x 50 mm BEH Amide column.
g. For UPLC/MS separations of more complex sugar mixtures
(e.g., polysaccharides), typical gradient UPLC conditions
include (add NH
4
OH modifier to both mobile phases A
and B):
i. Gradient going from 75% to 45% ACN with 0.1% NH
4
OH
in 10 min, 35 °C, 0.2 mL/min, 2.1 x 100 mm BEH Amide
column.
6. More complex sample mixtures may require the use of gradient
conditions and/or longer UPLC column lengths.
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