Waters CORTECS 2.7 um Columns - Manual (Page 3)

3 CORTECS 2.7 µm Columns
Table 1. Expected System Band Spread Volumes for Waters
LC Systems
Recommended
CORTECS Column i.d.
System
Band
Spread
1st
Choice
2nd
Choice
Alliance ® 2695 HPLC 29 µL 4.6 mm 3.0 mm
ACQUITY UPLC 12 µL 2.1 mm 3.0 mm
ACQUITY UPLC H-Class 9 µL 2.1 mm 3.0 mm
ACQUITY UPLC I-Class (FTN) 7.5 µL 2.1 mm 3.0 mm
ACQUITY UPLC I-Class (FL) 5.5 µL 2.1 mm 1.0 mm
Non-Waters UHPLC System 15–25 µL 3.0 mm 2.1 mm
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
value is necessary when transferring a method between different
chromatographic 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 containing
0.05 mg/mL uracil as mobile phase B.
3. Use the flow rate in the original method and the intended flow
rate on the target instrument.
4. Collect detector baseline using 100% mobile phase A for
5 minutes.
5. At 5.00 minutes, program a step to 100% mobile phase B,
and collect data for an additional 5 minutes.
6. Measure absorbance difference between 100% A and 100% B.
7. Measure time at 50% of that absorbance difference.
8. Calculate time difference between start of step and 50% point.
9. Multiply time difference by the flow rate to calculate
system volume.
5.69 minutes
- 5.00 minutes
0.69 minutes
50% Time = 5.69 minutes
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0 2 4 6 8 10 12 14 16 18 20 min
Programmed time = 5.00 minutes
System Volume:
0.69 min x 1.5 mL/min = 1.04 mL
Flow Rate = 1.5 mL/min
AU
Figure 3. Measuring System Dwell Volume.
Table 2. Expected System Dwell Volumes for Waters Systems
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
CORTECS 2.7 µm Columns are shipped in 100% acetonitrile. It is
important to ensure mobile phase compatibility before changing
to a different mobile phase system. To avoid precipitating mobile
phase buffers within the column or system, flush the column with
five column volumes of a waters/organic solvent mixture, using
the same, or lower, solvent content as in the desired buffered
mobile phase (for example, flush the column and system with 60%
methanol in water prior to introducing 60% methanol/40% buffer
mobile phase).
Note. If mobile phase additives (i.e., ion-pairing reagents) are
present in low concentrations (<0.2% v/v), 100 to 200 column
volumes may be required for complete equilibration. In addition,
mobile phases that contain formate (i.e., ammonium formate,
formic acid) may require extended equilibration times.
For reversed-phase separations, 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 and a
stable detector baseline is achieved.