Waters XTerra and XTerra Prep Columns - Manual (Page 4)

[ Care and Use ManUal ]
XTerra Columns 4
Figure 7: Determination of Dwell Volume
h. Use of Narrow-Bore Columns – ( ≤3.0 mm i.d.)
This section describes how to minimize extra column effects and
gives some guidelines on how to maximize the advantages of your
narrow-bore column. The 3.0 mm i.d. narrow-bore column usually
requires no system modifications. With the 2.1 mm i.d. column,
however, modifications to your HPLC system may be required in order
to eliminate excessive system bandspread volume. Without proper
system modifications, excessive system bandspread volume causes
peak broadening and has a large impact on peak width as peak
volume decreases.
i. I
mpact of bandspreading on column performance
(2.1 mm i.d. column)
System with 70 µL bandspread >> 10,000 plates
System with 130 µL bandspread >> ~8,000 plates (same column)
Note flow splitters after the column will introduce additional
bandspreading. Optimizing a system, especially one using flow split-
ters can have a dramatic effect on sensitivity and resolution. Use of
correct ferrule depth connectors and minimizing tubing diameter and
lengths showed a doubling of sensitivity and enabled resolution of the
metabolite on this LC/MS/MS system.
Time
1/2 Vertical
Distance
1.0
0.8
0.6
0.4
Au
0.2
0.0
t
1/2
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00
j. Modification Guidelines
1. Use a microbore detector flow cell with the 2.1 mm columns.
Recall that due to the shorter pathlength, detector sensitivity is
reduced to achieve lower band spread volume.
2. Injector sample loop should be reduced to minimum.
3. Use 0.009” (0.25 mm) tubing between pump and injector.
4. Use 0.009” (0.25 mm) tubing for rest of connections in
standard systems and 0.005” (0.12 mm) tubing for narrowbore
(≤2.1 mm i.d.) systems.
5. Use perfect (pre-cut) connections (with a variable depth inlet if
using columns from different suppliers).
6. Time constants should be shortened <0.2.
k. Waters Small Particle Size (2.5 µm and 3.5 µm) Columns
– Fast Chromatography
The Waters columns with 2.5 µm and 3.5 µm packings provide faster
and more efficient separations without sacrificing column lifetime.
This section describes five parameters to consider when performing
separations on the 2.5 µm and 3.5 µm columns.
Note: All 3.5 µm and 2.5 mm materials have smaller outlet frits to
retain packing material. These columns should not be backflushed.
1. Flow Rate — Compared with the 5 µm columns, the 2.5 µm and
3.5 µm columns have a higher optimum flow rate. These columns
are used for high efficiency and short analysis times. The higher
flow rates, however, lead to increased backpressure. Use a flow
rate that is practical for your system.
2. Backpressure — The backpressures on the 2.5 µm and 3.5
µm columns are higher than for the 5 µm columns of the same
dimension. Use a shorter column to compensate for increased
backpressure and obtain a shorter analysis time.
3. Temperature — Use a higher temperature to reduce backpres-
sure caused by smaller particle sizes (see Column Care and Use
temperatures recommended in Section III. e.).
4. Sampling Rate — Use a sampling rate of about 10 points per
second.
5. Detector Time Constant – Use a time constant of 0.1 seconds for
fast analysis.
Figure 8: Determination of Gradient Delay Volume