Waters XBridge Peptide BEH C18 130A and 300A Columns - Manual (Page 8)

b. 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)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
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 ).
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).
Figure 8. Determination of Gradient Delay Volume.
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.
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
8
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XBridge Peptide BEH C
18
, 130Å and 300Å Columns