Waters Analytical Columns and Cartridges - Manual (Page 2)

[ Care and Use ManUal ]
Analytical Columns and Cartridges 2
II. start-up test proCedure
Follow the four step start-up procedure for successful operation of
your Waters column or cartridge: A. The pH range for the mobile
phase is pH 2 to 8. However, phosphate buffer at pH 8 in combination
with high temperatures can lead to shorter column life. For high
temperature work, this buffer at neutral pH is preferred.
a. Measuring Instrument Band Spreading
Poor column performance can sometimes be attributed to excessive
instrument band spreading. Test the band spreading of your HPLC
system before you install your column or cartridge. Band spreading
of a properly operating analytical system should be less than
100 μL and <40 μL for 2.1 mm i.d. microbore columns.
Test the instrument band spreading using the procedure below.
1. Remove the column or cartridge from the system. Connect the
inlet and outlet tubing with a low-dead-volume union.
2. Inject the test sample used for a plate count determination (see
Section II. d.) using a fast chart speed or sampling rate. You may
need to adjust the detector sensitivity or dilute the sample to
keep the peak on scale.
3. Use the 5-Sigma method shown in Figure 2 to obtain the band
spreading value in μL.
Figure 1: Installing a Cartridge Column with a Sentry TM Guard Column
Determine the width of the peak at 4.4% of the peak maximum.
Determine how many microliters per minute your eluent is flowing.
Multiply the two terms to yield the band spread of your instrument.
If the band spreading is excessive, check all tubing and fittings. If it
is necessary to repair fittings, refer to the procedure below. If you
have a 2.1 mm i.d. microbore column, you may need to modify your
HPLC system. Refer to the System Modifications Guidelines outlined
in Section III.
Repair a damaged compression screw or a worn ferrule assembly
using the procedure below.
1. Scribe the circumference of the tubing at the desired break point
using a tube cutter or a file with a cutting edge.
2. Grasp the tubing on both sides of the scribe mark with cloth
covered pliers. Gently work the tube back and forth until it sepa-
rates at the scribe mark. Ensure that the tubing end is straight,
open and free of burrs.
3. Slide the compression screw, followed by the ferrule (large end
of the taper first), over the tube. Seat the tubing in the fitting. If
the tubing is not completely seated, the resulting dead volume
can lead to poor chromatographic results.
Spacer 0.25 mm (0.009 inch) Tubing
Sentry Guard
Column
C-Clip
Cartridge Column
End Connector