Waters PAH Columns - Manual (Page 5)

[ Care and Use ManUal ]
Name of Document 5
Figure 5: Determination of Dwell Volume
f. 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 modifica-
tions. 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.
g. Impact of Bandspreading on Column Performance
(2.1 mm i.d. column)
System with 70 mL bandspread >> 10,000 plates
System with 130 mL bandspread >> ~8,000 plates (same column)
Note: Flow splitters after the column will introduce additional bandspreading.
Figure 6: Impact of Bandspreading
Optimizing a system, especially one using flow splitters can have a dramatic
effect on sensitivity andresolution. 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.
h. System 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 inch (0.25 mm) tubing between pump and injector.
4. Use 0.009 inch (0.25 mm) tubing for rest of connections in
standardsystems 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. Time constants should be shortened <0.2 Column Equilibration.
III. COLUMN EqUILIBRATION
Waters delivers the column in 100% acetonitrile. It is important to ensure
solvent compatibility before changing to a new solvent. Equilibrate your
column with a minimum of 10 times its internal volume with the mobile
phase to be used (refer to Table 1 for some standard column volumes).
1. Purge your pumping system and then connect the inlet end of the
column to the injector outlet. Turn on the pump flow at 0.1 mL/min.
and increase to 1 mL/min over 5 minutes.
2. When the solvent is flowing freely from the column outlet, attach the
column to the detector. This procedure prevents entry of air into the
detection system and gives more rapid equilibration.
3. When the mobile phase is changed, gradually increase the flow rate of
the new mobile phase from zero mL/min to 1.0 mL/min in 0.1 mL/min
increments.
4. Once a steady backpressure and baseline have been achieved, the
column is ready to be used.
Volum e (µL)
Dw ell
Volum e
1.0
0.8
0.6
0.4
Height
0.2
0.0
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00 7.00 7.50 8.00