Waters Symmetry Columns - Manual (Page 8)

[ Care and Use ManUal ]
Symmetry Columns 8
d. Measuring System Bandspreading Volume and System
Variance
This test should be performed on an HPLC system with a single
wavelength 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
sensitivity of 0.5 - 1.0 AUFS (system start up test mix can be
used which contains uracil, ethyl and propyl parabens; Waters
P/N WAT034544).
4. Inject 2 to 5 μL of this solution.
5. Measure the peak width at 4.4% of peak height
(5-sigma method):
5-sigma Bandspreading (μL) = Peak Width (min) Flow Rate (mL/min x (1000 μL/1 mL)
System Variance (μL 2 ) = (5-sigma bandspreading) 2 / 25
Figure 8: Determination of System Bandspreading Volume Using
5-Sigma Method
In a typical HPLC system, the Bandspreading Volume should be
100 μL ± 30 μL (or Variance of 400 μL
2 +/- 36 μL 2 ).
In a microbore (2.1 mm i.d.) system, the Bandspreading Volume
should be no greater than 20 to 40 μL (or Variance no greater than
16 μL
2 to 64 μL 2 ).
e. Measuring System Volume
System volume is important in scaling separations because it
creates an isocratic hold at the start of every run. This hold is often
several column volumes on a small scale, but a fraction of the
volume of a prep column. Compensation for this volume must be
included in planning a scaling experiment to avoid distorting the
chromatography (Figure 9).
Figure 9: Determination of Gradient Delay Volume
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.
5.69 minutes
- 5.00 minutes
0.69 minutes
50% Time = 5.69 minutes
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0 2 4 6 8 10 12 14 16 18 20 min
Programmed time = 5.00 minutes
System Volume:
0.69 min x 1.5 mL/min = 1.04 mL
Flow Rate = 1.5 mL/min
AU
System Volume
4.4% h