Care and use manual, B. bandspreading minimization, D. measuring system volume – Waters SunFire Columns User Manual
Page 8: Figure 6: effect of connecting tubing on system, 36 μl, Figure 9: determination of gradient delay volume
[ Care and Use ManUal ]
8
b. Bandspreading minimization
Figure 6 shows the influence of tubing internal diameter on system
bandspreading and peak shape. As can be seen, the larger tubing
diameter causes excessive peak broadening and lower sensitivity.
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
Figure 6: effect of Connecting tubing on System
c. 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 Part Number
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) x Flow Rate (mL/min) x (1000 μL/1 mL)
System Variance (μL
2
) = (5-sigma bandspreading)
h
/ 25
System Volume
4.4 %h
5
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
).
d. 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).
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
Figure 9: determination of Gradient delay Volume