Waters ACQUITY UPLC BEH Glycan, 1.7 µm Columns, Glycan Performance Test Standard - Manual (Page 5)

[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Glycan, 1.7 �m Columns 5
III. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC 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 AQUITY UPLC 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.
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. If filtering, Acrodisc
® filters are
recommended. Solvents containing suspended particulate materials
can damage the fluidic components of the UPLC system and will
generally clog the inlet distribution frit of the column. This will result
in higher operating pressure and poor performance.
d. Pressure
The ACQUITY UPLC BEH Glycan columns will have greatly increased
backpressure when operated in 90-100% aqueous mobile phases.
As shown in the gradient table for Figure 1, the flow rate needs
to be lowered to 0.25 mL/min when washing a 2.1 x 150 mm
Glycan column in 100% A. ACQUITY UPLC BEH Glycan columns
can tolerate pressures of up to 15,000 psi (1034 bar or 103 Mpa),
although pressures greater than 13,000 psi should be avoided in
order to maximize column and system lifetimes.
Note: Working at the extremes of pressure, pH and/or temperature
will result in shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C – 90 ˚C are recommended for operating
ACQUITY UPLC BEH Glycan columns in order to enhance selectivity,
lower solvent viscosity, and increase mass transfer rates. However,
higher temperature will have a negative effect on lifetime that will
vary depending on the pH and buffer conditions used.
Additive/Buffer pKa
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.
Table 2: Buffer Recommendations for Using ACQUITY UPLC BEH Glycan columns from pH 3 to 8