Waters XSelect HSS XP 2.5 µm Columns - Manual (Page 6)

XSelect HSS XP 2.5 µm Columns 6
[ CARE AND USE MANUAL ]
Table 5: Buffer Recommendations for
XSelect HSS XP 2.5 µm Columns
Additive or
Buffer
pKa
Buffer
Range
(±1 pH
unit)
Volatility
Used for
Mass
Spec?
Comments
TFA 0.3 – Volatile Yes
Ion pair additive, can suppress
MS signal. Used in the 0.01-
0.1% range.
Formic
Acid
3.75 – Volatile Yes
Maximum buffering obtained
when used with ammonium
formate salt. Used in 0.1-1.0%
range.
Acetic Acid 4.76 – Volatile Yes
Maximum buffering obtained
when used with ammonium
acetate salt. Used in 0.1-1.0%
range.
Formate
(HCOO-)
3.75
2.75 –
4.75
Volatile Yes
Used in the 1-10 mM range.
Note: sodium or potassium salts
are not volatile.
Acetate
(CH
3
COO-)
4.76
3.76 –
5.76
Volatile Yes
Used in the 1-10 mM range.
Note: sodium or potassium salts
are not volatile.
Phosphate 1 2.15
1.15 –
3.15
Non-
volatile
No
Traditional low-pH buffer, good
UV transparency.
Phosphate 2 7.2
6.20 –
8.20
Non-
volatile
No
Much shorter colum lifetimes
will be realized using phosphate
at pH 7.
c. Solvents
To maintain maximum column performance, use high quality HPLC
or MS grade solvents. Filter all aqueous buffers prior to use through
a 0.2 µm filter. Solvents containing suspended particulate materials
will generally clog the outside surface of the inlet of the column. This
may result in higher backpressure or distorted peak shape.
d. Pressure
XSelect HSS XP 2.5 µm Columns are compatible with HPLC, UHPLC
and UPLC pressures. Table 6 depicts the maximum operation pressure
Table 6: Maximum Operation Pressure
Column ID Pressure Range
2.1 mm 18,000 psi [1034 bar]
3.0 mm 18,000 psi [1034 bar]
4.6 mm 9000 psi [620 bar]
e. Temperature
XSelect HSS XP 2.5 µm Columns can be used up at intermediate
temperatures to enhance selectivity, reduce solvent viscosity and
increase mass transfer rates.
Chemistry Temperature Limit
XSelect HSS Cyano 45 °C
XSelect HSS PFP 45 °C
XSelect HSS T3 45 °C
XSelect HSS C
18
SB 45 °C
XSelect HSS C
18
45 °C
Note: Working in combinations of extreme pH, temperature and pressure may
result in reduced column lifetime.
IV. COLUMN CLEANING, REGENERATION AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shouldering peaks, shifts
in retention, change in resolution or increasing backpressure may
indicate contamination of the column. Flush with a neat organic
solvent to remove the non-polar contaminant(s), taking care not to
precipitate any buffered mobile-phase components. If this flushing
procedure does not solve the problem, purge the column with the
following cleaning and regeneration procedures.
Use a cleaning routine that matches the properties of the samples,
stationary-phase type (reversed-phase, normal-phase or HILIC) and
will solubilize the suspected contaminate. Flush with 20 column
volumes of solvent at an intermediate temperature of 45 °C. Return
to the initial mobile-phase conditions by reversing the sequence.
If using a reversed-phase column, purge the column with a sequence
of progressively more non-polar solvents (i.e., water-to-methanol-to-
tetrahydrofuran-to-methylene chloride).
If using a HILIC column, purge the column with a
sequence of progressively more polar-organic solvents
(i.e., acetonitrile-to-acetonitrile/methanol-to-acetonitrile/
water-to-water).
If column performance has not improved after regeneration/
cleaning procedures, contact your local Waters representative
for additional support.