Waters XBridge Columns - Manual (Page 8)

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XBridge ™ 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 400µL
2 ± 36µL 2 ).
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 16µL
2 to 64µL 2 ).
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 (t
1/2
) (half the vertical distance between the initial and final
isocratic segments as shown in Figure 8).
7. Subtract half the gradient time ( 1 /
2
t
g
) (10 min/2 = 5 min in this
example) from the gradient midpoint (t
1/2
) to obtain the dwell time (t
D
).
8. Convert the dwell time (t
D
) to the dwell volume (V
D
) 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.
VII. addItIonal InformatIon
a. Use of Narrow-Bore (3.0 mm i.d.) Columns
This section describes how to minimize extra column effects and provides
guidelines on maximizing the performance of a narrow-bore column in an
HPLC system. A 3.0 mm i.d. narrow-bore column usually requires no sys-
tem modifications. A 2.1 mm i.d. column, however, requires modifications
to the HPLC system in order to eliminate excessive system bandspreading
volume. Without proper system modifications, excessive system band-
spreading volume causes peak broadening and has a large impact on peak
width as peak volume decreases.
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