Waters XBridge Columns - Manual (Page 11)

[ Care and Use ManUal ]
XBridge ™ Columns 11
Mobile Phase Considerations
1. Always maintain at least 5% polar solvent in the mobile phase or
gradient (e.g., 5% aqueous, 5% methanol or 2% aqueous/3% metha-
nol, etc.).
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in your
mobile phase or gradient.
3. At aqueous concentrations greater than 60%, lower flow rates
should be used due to high backpressure. This includes all aqueous
wash procedures.
4. Avoid phosphate salt buffers to avoid precipitation in HILIC mobile
phases. Phosphoric acid is OK.
Injection Solvents
1. If possible, injection solvents should be as close to the mobile
phase composition as possible (if isocratic) or the starting gradient
conditions.
2. A generic injection solvent is 75:25 acetonitrile:methanol. This is a
good compromise between analyte solubility and peak shape. When
separating saccharides with limited solubility in in organic solvents,
higher concentrations of aqueous solvent in the sample are accept-
able. 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.
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 XBridge Amide Columns’ recommendations described above.
2. If separating reducing sugars, please review the following information.
3. Reducing sugars can undergo mutarotation which produces the unde-
sired 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 to facilitate anomer collapse.
b. With longer columns, increased flow rates can be used. As with
all LC separations, optimal flow rates should be determined
experimentally.
c. Add triethylamine (TEA) or ammonium hydroxide (NH
4
OH)
modifiers to aqueous and organic mobile phase reservoirs at
equal concentrations.
d. For HPLC separations of mono- and/or disaccharides using
XBridge Amide columns typical isocratic conditions include:
i. 75% acetonitrile (ACN) with 0.2% TEA, 35 °C
ii. 77% acetone with 0.05% TEA, 85 °C
e. For HPLC separations of more complex sugar mixtures (e.g.,
polysaccharides) using XBridge Amide columns typical gradient
conditions include (add TEA modifier to both mobile phases A
and B):
i. Gradient going from 80% to 50% ACN with 0.2% TEA,
35 °C,
ii. 80%-55% acetone with 0.05% TEA, 85 °C
f. For HPLC/MS separations of mono- and disaccharides using
XBridge Amide columns typical isocratic conditions include:
i. 75% ACN with 0.1% NH
4
OH, 35 °C