Waters XSelect HSS XP 2.5 µm Columns - Manual (Page 4)

XSelect HSS XP 2.5 µm Columns 4
[ CARE AND USE MANUAL ]
Table 3: Column Volumes (mL)
Column Length
(mm)
Internal Diameter
2.1 mm 3.0 mm 4.6 mm
30 0.10 0.21 0.50
50 0.17 0.35 0.83
75 0.26 0.53 1.25
100 0.35 0.71 1.66
To avoid precipitating mobile-phase buffers within the column or
system, flush the column with five column volumes of a waters/
organic solvent mixture using the same, or lower, solvent content
as in the desired buffered mobile phase (i.e., flush the column and
system with 60% methanol in water prior to introducing 60%
methanol/40% buffer mobile phase).
Note: If mobile-phase additives (i.e., ion-pairing reagents) are present in low
concentrations (<0.2% v/v), 100 to 200 column volumes may be required for
complete equilibration. In addition, mobile phases that contain formate (i.e.,
ammonium formate, formic acid) may require extended equilibration times.
g. Solvent Conversion for Normal-Phase Use
XSelect HSS Cyano XP 2.5 µm Columns can be used for both reversed-
phase as well as normal-phase separations. The column is shipped in
100% acetonitrile and is ready for use in reversed-phase conditions.
If you intend to use the XSelect HSS Cyano Column for normal-
phase applications, the following solvent conversion procedure
must be followed:
1. Flush the column with a minimum of 20 column volumes of 100%
methanol using a low flow rate to avoid overpressuring the LC system.
Refer to Table 3 for column volume information.
2. Flush the column with a minimum of 20 column volumes of
100% isopropanol using a low flow rate to avoid overpressuring
the LC system. Refer to Table 3 for column volume information.
3. Flush the column with a minimum of 20 column volumes of 100%
dichloromethane using a low flow rate to avoid overpressuring the
LC system. Refer to Table 3 for column volume information.
4. Flush the column with the intended mobile-phase conditions
until a stable baseline is achieved.
h. eCord Installation
eCord Technology represents a significant advancement in column
usage tracking management which can be realized if the column is
installed on an ACQUITY UPLC System. The eCord can be read
by connecting the yellow fob to the reader/writer located on the
right-hand side of the ACQUITY UPLC Column heater module.
Embedded information such as the column manufacturing QC data
and Certificates of Analysis may then be accessed via the ACQUITY
UPLC console.
i. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it to track
column performance over time. This test may consist of:
a. An analyte test mixture that is commonly used in your laboratory
b. An analyte mixture as found on the ‘Performance Test
Chromatogram’ which can be accessed via the eCord.
Note: If (b) is performed, the isocratic efficiencies measured in your laboratory may
be less than those given on the Waters Performance Test Chromatogram.
This is normal and expected. The Waters isocratic column testing systems
have been modified in order to achieve extremely low system dispersion.
This presents a more challenging test of how well the column was packed. This also
guarantees the highest quality packed column. These special testing systems have
been modified to such an extent that they are not commercially viable and have
limited method flexibility other than isocratic column testing.
2. Determine the number of theoretical plates (N) and use this value for
periodic comparisons.
3. Repeat the test periodically to track column performance over time.
Slight variations may be obtained on different LC systems due to the
quality of the connections, operating environment, system electronics,
reagent quality, column condition and operator technique.
j. VanGuard Pre-Columns
VanGuard ™ Pre-Columns are 2.1 mm ID x 5 mm length guard column
devices designed specifically to protect an analytical column
while minimizing the negative dispersion impact of utilizing such a
device. VanGuard Pre-Columns are packed with the same stationary
phases as the XP 2.5 µm Column offering. VanGuard Pre-Columns
are designed to be directly attached to the inlet of a eXtended
Performance [XP] 2.5 µm Column.