Waters XBridge Peptide BEH C18 130A and 300A Columns - Manual (Page 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
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February 2014 Rev C 715001443EN KP-PDF