Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns - Manual (Page 8)

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XSelect CSH130 C
18
Columns 8
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
Figure 7: Determination of System Bandspreading Volume Using
5-Sigma Method
In
a typical HPLC system, the Bandspreading Volume should be no greater than
100 µL ± 30 µL (or Variance of 400 µL ± 36 µL).
In a microbore (2.1mm i.d.) system, the Bandspreading Volume should be no
greater than 20 to 40 µL (or Variance no greater than 16 µL to 64 µL).
c. Measuring Gradient Delay Volume (or Dwell Volume)
For successful gradient-method transfers the gradient delay volumes should
be measured using the same method on both HPLC systems. The procedure
below describes a method for determining the gradient delay volumes.
1. Replace the column with a zero dead volume union.
2. Prepare mobile phase A (pure solvent, such as methanol) and
mobile phase B (mobile phase A with a UV absorbing sample,
such as (v/v) 0.1% acetone in methanol).
3. Equilibrate the system with mobile phase A until a stable
baseline is achieved.
4. Set the detector wavelength to the absorbance maximum of the
probe (265 nm for acetone).
5. Program a 0-100% B linear gradient in 10 min at 2 mL/min
(the exact conditions are not critical; just make sure the gradient
volume is at least 20 mL) with a hold at 100% B.
6. Determine the dwell time by first locating the time at the
midpoint of the formed gradient (t1/2) (half the vertical distance
between the initial and final isocratic segments as shown in
Figure 8).
7. Subtract half the gradient time (1/2 tg) (10 min/2 = 5 min in
this example) from the gradient midpoint (t1/2) to obtain the
dwell time (tD).
8. Convert the dwell time (tD) to the dwell volume (VD) by
multiplying by the flow rate (F).
Dwell Volume V
D
= (t
1/2
—1/2 t
g
) x F
For fast gradient methods, the gradient delay volume (or dwell volume)
should be less than 1 mL. If the gradient delay volume is greater than 1 mL,
see System Modification Recommendations section on how to reduce
system volume.
System Volume
4.4 %h
5
Time
1/2 Vertical
Distance
1.0
0.8
0.6
0.4
Au
0.2
0.0
t
1/2
Figure 8: Determination of Gradient Delay Volume