Waters Spherisorb Columns - Manual (Page 10)

[ Care and Use ManUal ]
Waters Spherisorb Columns 10
1. Remove column.
2. Use acetonitrile as mobile phase A, and acetonitrile with 0.05 mg/mL
uracil as mobile phase B (eliminates non-additive mixing and viscosity
problems).
3. Set UV detector at 254 nm.
4. Use the flow rate in the original method and the intended flow rate on
the target instrument.
5. Collect 100% A baseline for 5 minutes.
6. Program a step change at 5 minutes to 100% B, and collect data for an
additional 5 minutes.
7. Measure absorbance difference between 100% A and 100% B.
8. Measure time at 50% of that absorbance difference.
9. Calculate time difference between start of step and 50% point.
10. Multiply time difference by flow rate.
vii. 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.
7 8 min 7.5 7 8 min 7.5
System with 130 ìL
bandspreading: 8,000 plates System with 70 ìL bandspreading:
10,000 plates (same column)
Non-optimized LC/MS/MS System Optimized 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.