Waters PAH Columns - Manual (Page 4)

[ Care and Use ManUal ]
Name of Document 4
b. SLIPFREE Connectors
Guarantees a void-free connection because it pushes the tubing into the
endfitting; This design comes installed on the tubing. Fingertight to
10,000 psi – never needs wrenches. Readjusts to all column endfit-
tings. Compatible with all commercially available endfittings. Unique
design separates tube-holding function from sealing function.
c. Minimization of Bandspreading
The following figure shows the influence of tubing internal diameter on
system band spreading and peak shape. As can be seen, the larger tubing
diameter causes excessive peak broadening and lower sensitivity.
Figure 3: Effect of Connecting Tubing on System
d. Measuring System Bandspread Volume
1. Disconnect column from system and replace with a zero dead volume
union.
2. Flow rate 1 mL/min. This should be performed on a single wavelength
detector (not a PDA/DAD).
3. Dilute a test mix in mobile phase to give a detector sensitivity 0.5-1.0
AUFS (can use the system start up test mix which contains uracil, ethyl
and propyl parabens; Waters part number WAT034544).
4. Inject 2 to 5 mL of this solution.
In a typical LC bandspreading volume system should be 100 mL ±
30 mL (or variance of 400 μL2 +/- 36 μL2)
Microbore (2.1 mm i.d. and smaller) system should be no greater than
20-40 mL
Figure 4: Determination of System Bandspread Volume using the
5-Sigma Method
e. Measuring Gradient Delay Volume
1. Replace the column with a zero dead volume union.
2. Determine the gradient-delay or dwell volume for your system by
performing the following test. Prepare eluent A (pure solvent, such
as methanol) and eluent B (solvent plus sample, such as 5.6 mg/mL
propylparaben in methanol).
3. Equilibrate the system with eluent A until a stable baseline is achieved.
Switch to 100% eluent B and record the half height of the step. Refer to
Figure 5 for an illustration.
The dwell volume should be less than 1 mL. If this is not the case, see section
on System Modifications (below) to reduce your system volume.
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches