Waters XSelect HSS XP 2.5 µm Columns - Manual (Page 3)

XSelect HSS 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 gradient 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 50% 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
XSelect HSS 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.