Waters Atlantis T3, DC18 and HILIC Silica Columns - Manual (Page 4)

[ Care and Use ManUal ]
Atlantis Columns 4
e. Measuring System Bandspread Volume
This test should be performed on an HPLC system with a single wave-
length UV detector (not a Photodiode Array (PDA)).
1. Disconnect column from system and replace with a zero dead
volume union.
2. Set flow rate to 1 mL/min.
3. Dilute a test mix in mobile phase to give a detector sensitiv-
ity 0.5-1.0 AUFS (system start up test mix can be used which
contains uracil, ethyl and propyl parabens; Waters Part Number
WAT034544).
4. Inject 2 to 5 µL of this solution.
5. Using 5 sigma method measure the peak width at 4.4% of peak
height:
Band Spreading (µL) = Peak Width (min) x Flow Rate (µL/min)
= 0.1 min x 1000 µL/min
= 100 µL
Figure 8: Determination of System Bandspread
Volume Using 5-Sigma Method
In a typical HPLC system, the Bandspread Volume should be 100 µL ±
30 µL.
In a microbore (for 2.1 mm i.d. columns) system, the Bandspread
Volume should be no greater than 20 to 40 µL.
f. Measuring Gradient Delay Volume
1. Replace the column with a zero dead volume union.
2. Prepare eluent A (pure solvent, such as methanol) and eluent
B (solvent plus sample, such as 5.6 mg/mL propylparaben in
methanol).
3. Equilibrate the system with eluent A until a stable baseline is
achieved.
4. Switch to 100% eluent B.
5. Record the half height of the step and determine dwell volume
(Figure 9).
Figure 9: Determination of Dwell Volume
Time
Inflection
point time
1.0
0.8
0.6
0.4
Au
0.2
0.0
The dwell volume should be less than 1 mL. If the dwell volume is
greater than 1 mL, see
“
System Modification Recommendations
“
for
how to reduce system volume.
g. Use of Smaller i.d. Columns
A 3.0 mm i.d. narrow-bore column usually requires no system modifi-
cations. A 2.1 mm i.d. microbore column, however, requires modifica-
tions to the HPLC system 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.
h. Impact of Bandspread Volume on 2.1 mm i.d. Column
Performance
System with 70 µL bandspread: 10,000 plates
System with 130 µL bandspread: 8,000 plates (same column)
Attention: Flow splitters after the column will introduce additional band
spreading which will reduce sensitivity and resolution. Loss of sensitiv-
ity or resolution may affect the accuracy and/or precision of results.
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. System optimization results in a doubling of sensitivity and
resolution of the metabolite in an LC/MS/MS system (Figure 10).