Waters Symmetry Columns - Manual (Page 9)

[ Care and Use ManUal ]
Symmetry Columns 9
Viii. additional information
a. Use of Narrow-bore (<3.0 mm i.d.) Columns
This section describes how to minimize extra column effects and
provides guidelines on maximizing the performance of a narrowbore
column in an HPLC system. A 3.0 mm i.d. narrow-bore column
usually requires no system modifications. A 2.1 mm i.d. column,
however, requires modifications to the HPLC system in order to
eliminate excessive system bandspreading volume. Without proper
system modifications, excessive system bandspreading volume
causes peak broadening and has a large impact on peak width as
peak volume decreases.
b. Impact of Bandspreading Volume on 2.1 mm i.d.
Column Performance
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-depth ferrules and minimizing
tubing diameter and lengths. An example is given in Figure 10
where system optimization resulted in a doubling of sensitivity and
resolution of the metabolite in an LC/MS/MS system.
Figure 10: 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 between pump and injector.
4. 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.
5. Use perfect (pre-cut) connections (with a variable depth inlet
if using columns from different suppliers).
6. Detector time constants should be shortened to less than
0.2 seconds.
d. Waters Small Particle Size (3.5 μm) Columns –
Fast Chromatography
Waters columns that contain 3.5 μm particles provide faster and
more efficient separations without sacrificing column lifetime. This
section describes five parameters to consider when performing
separations with columns containing 3.5 μm particles.
Note: Columns that contain 3.5 μm particles have smaller outlet frits
to retain packing material. These columns should not be backflushed.
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00
System with 130 µL
bandspreading: 8,000 plates System with 70 µL bandspreading:
10,000 plates (same column)
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00