Waters Spherisorb Columns - Manual (Page 9)

[ Care and Use ManUal ]
Waters Spherisorb Columns 9
b. Band Spreading Minimization
Figure 6 shows the influence of tubing internal diameter on system
band spreading 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 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) x Flow Rate (mL/min) x (1000 μL/1 mL)
System Variance (μL 2 ) = (5-sigma bandspreading) 2 / 25
System Volume
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 preparative 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
Programmed time = 5 min
Flow Rate = 1.5 mL/min
5.69 min
-5.00 min
0.69 min
Time = 5.69 minutes
System Volume:
0.69 min x 1.5 mL/min = 1.04 mL
50%
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
AU
0 2 4 6 8 10 12 14 16 18 20 Min.