Waters XBridge XP 2.5 µm Columns - Manual (Page 4)

XBridge 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 water/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.
For XBridge HILIC XP 2.5 µm Columns, flush with 50 column volumes
of 50:50 acetonitrile:water with 10 mM final buffer concentration.
For XBridge Amide XP 2.5 µm Columns, flush with 50 column
volumes of 60:40 acetonitrile:water with 10 mM final buffer
concentration. Prior to the first injection, equilibrate with 20 column
volumes of initial mobile-phase conditions (refer to Table 3 for a
list of column volumes). See “Getting Started with XBridge HILIC
Columns” or “Getting Started with XBridge Amide Columns” sections
for additional information.
g. 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 device 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.
h. 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.
i. 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 2.5 µm Column.
Note: VanGuard Pre-Columns are shipped with a collet and ferrule that are NOT
pre-swaged. This enables the end user to mate the VanGuard Pre-Column to a
specific XP 2.5 µm Column and ensures void-free and leak-free connections.
Care must be taken when removing the O-ring that holds these two pieces on the
pre-column tubing.
XP 2.5 µm Column VanGuard Pre-Column
Place wrench here
Ferrule
Collet
Place wrench here
Flow
Figure 4: Installing a VanGuard Pre-Column.