Waters XBridge Columns - Manual (Page 9)

[ Care and Use ManUal ]
XBridge ™ Columns 9
b. Impact of Bandspreading Volume on 2.1 mm i.d. Column
Performance
System with 70 µL bandspreading: 10,000 plates
System with 130 µL bandspreading: 8,000 plates (same column)
Note: Flow splitters after the column will introduce additional
bandspreading.
System optimization, especially in a system that contains a flow
splitter, can have dramatic effects on sensitivity and resolution.
Optimization includes using correct ferrule depths and minimizing
tubing inner diameters and lengths. An example is given in Figure 9
where system optimization resulted in a doubling of sensitivity and
resolution of the metabolite in an LC/MS/MS system.
Figure 9: Non-Optimized vs. Optimized LC/MS/MS System
c. Non-Optimized vs. Optimized LC/MS/MS System: System
Modification Recommendations
1. Use a microbore detector flow cell with 2.1 mm i.d. columns.
Note: Detector sensitivity is reduced with the shorter flow cell path length in
order to achieve lower bandspreading volume.
2. Minimize injector sample loop volume.
3. Use 0.009 inch (0.25 mm) tubing for rest of connections in standard
systems and 0.005 inch (0.12 mm) tubing for narrowbore (2.1 mm
i.d.) systems.
4. Use perfect (pre-cut) connections (with a variable depth inlet if using col-
umns from different suppliers).
5. Detector time constants should be shortened to less than
0.2 seconds.
d. Waters Small Particle Size (2.5 µm) Columns –
Fast Chromatography
Waters columns that contain 2.5 µm particles provide faster and more effi-
cient separations without sacrificing column lifetime. This section describes
five parameters to consider when performing separations with columns
containing 2.5 µm particles.
Note: Columns that contain 2.5 µm particles have smaller outlet frits to retain
packing material. These columns should not be backflushed.
1. Flow Rate—Compared with the 5 µm columns, columns with 2.5 µm par-
ticles have higher optimum flow rates. These columns are used when high
efficiency and short analysis times are required. These higher flow rates,
however, lead to increased backpressure.
Note: Use a flow rate that is practical for your system.
2. Backpressure—Backpressures for columns with 2.5 µm particles are higher
than for 5 µm columns with the same dimensions. Waters suggests using a
shorter column to compensate for increased backpressure and to obtain a
shorter analysis time.
3. Temperature—Use a higher temperature to reduce backpressure caused by
smaller particle sizes. The recommended temperature range for XBridge ™
columns is 20 °C to 60 °C. See Column Use section for a discussion of
elevated temperature use with XBridge
™ columns.
4. Sampling Rate—Use a sampling rate of about 10 points per second
or higher. A minimum of 20 points across the earliest eluting peak of
interest is needed for optimum reproducibility.
5. Detector Time Constant—Use a time constant of 0.1 seconds or lower
for fast analyses.
e. Getting Started With XBridge HILIC Columns
1. Because XBridge HILIC columns do not posses a bonded phase,
the pH operating range is 1 to 9, and they can be operated at
temperatures up to 45 °C.
2. As with any LC column, operating at the extremes of pH, pressures
and temperatures will result in decreased column lifetime.
Column Equilibration
1. When column is first received, flush in 50% acetonitrile: 50% water
with 10 mM final buffer concentration for 50 column volumes.
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00