Waters XSelect CSH HPLC Columns - Manual (Page 8)

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8 XSelect CSH HPLC Columns
5. Measure the peak width at 4.4% of peak height (5-sigma method):
5-sigma Band-spreading (µL) = Peak Width (min) x Flow Rate (mL/min) x
(1000 µL/1 mL)
System Variance (µL 2 ) = (5-sigma band-spreading) 2 / 25
Figure 7: Determination of System Band-spreading Volume Using 5-Sigma Method
In a typical HPLC system, the Band-spreading Volume should be
no greater than 100 µL ± 30 µL (or Variance of 400 µL
2 ± 36 µL 2 ).
In a microbore (2.1 mm i.d.) system, the Band-spreading Volume
should be no greater than 20 to 40 µL (or Variance no greater
than 16 µL
2 to 64 µL 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.
Figure 8: Determination of Gradient Delay Volume
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
tg
) (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
tg
) 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 system modifications. A 2.1 mm
i.d. column, however, requires modifications to the HPLC system
in order to eliminate excessive system band-spreading 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