Waters XBridge XP 2.5 µm Columns - Manual (Page 3)

XBridge XP 2.5 µm Columns 3
[ CARE AND USE MANUAL ]
System Volume
4.4 %h
5
Figure 2: Determination of system band spread volume using 5-Sigma Method.
Table 1: Expected System Band Spread Volumes
System Band Spread
Alliance 2695 HPLC 29 µL
Vendor A HPLC 41 µL
Vendor B UHPLC (600 bar) 28 µL
Vendor C UHPLC 21 µL
Vendor D UHPLC 17 µL
ACQUITY UPLC 12 µL
ACQUITY UPLC H-Class 9 µL
ACQUITY UPLC I-Class (FTN) 7.5 µL
ACQUITY UPLC I-Class (FL) 5.5 µL
e. Measuring System Dwell Volume
Dwell volume is different than system band spreading. System dwell
volume is a measurement of the volume it takes for the initial gradi-
ent conditions to reach the head of the column. This calculation is
particularly useful when it is necessary to transfer a method between
different LC systems.
1. Disconnect the column from the system and replace with a zero
dead volume union.
2. Use acetonitrile as mobile-phase A, and acetonitrile with
0.05 mg/mL uracil as mobile-phase B.
3. Monitor UV at 254 nm.
4. Use the flow rate in the original method and the intended flow
rate on the target instrument.
5. Collect 100% A baseline for 5 minutes.
6. At 5 minutes, program a step to 100% B, and collect data for an
additional 5 minutes.
7. Measure absorbance difference between 100% A and 100% B.
8. Measure time at 5% of that absorbance difference.
9. Calculate time difference between start of step and 50% point.
10. Multiply time difference by flow rate to calculate system volume.
Programmed time = 5 minutes
50% Absorbance = 0.35852 AU
Time = 5.6953 minutes
100% Asymptotic
Total absorbance = 0.7164 AU
5.69
5.00
0.69 min
= Programmed Gradient
= Actual Gradient
System Volume
0.69 min x 1.5 mL/min = 1.04 mL
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
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Figure 3: Measuring system band spread volume.
Table 2: Expected System Dwell Volumes
System Dwell Volume
Alliance 2695 HPLC 900 µL
ACQUITY UPLC 120 µL
ACQUITY UPLC H-Class 350 µL
ACQUITY UPLC I-Class (FTN) 100 µL
ACQUITY UPLC I-Class (FL) 95 µL
f. Column Equilibration
XBridge XP 2.5 µm Columns are shipped in 100% acetonitrile. It is
important to ensure mobile-phase compatibility before changing to a
different mobile-phase system. Equilibrate the column with a minimum
of 10 column volumes of the mobile-phase to be used (refer to Table
3 for a list of column volumes). The column may be considered fully
equilibrated once a constant backpressure is achieved.