Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns - Manual (Page 9)

[ CARE AND USE MANUAL ]
XSelect CSH130 C
18
Columns 9
VII. ADDITIONAL INFORMATION
a. Use of Narrow-Bore Columns
This section describes how to minimize extra column effects and provides
guidelines on maximizing the performance of a narrow-bore column in an
HPLC system. A 2.1 mm i.d. column 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
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 band-
spreading.
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 columns from different suppliers).
5. Detector time constants should be shortened to less than
0.2 seconds.
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00