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PCX 5100 User’s Manual
Post-Column
Band-Spreading Test
Chapter 5
This test measures the amount of increased band-spreading due to the post-column
system. It exploits the fact that 1-naphthol is fluorescent without derivatization. The
general scheme is to analyze the test mixture with the post-column reagents turned off.
First, test with the column connected directly to the detector (bypassing the PCX5100);
second, test with the column effluent flowing through the post-column reactors.
This test is best performed on a system that has been shut down and at least partially
cooled down. A normal carbamate analyzer works for this test. If you have a glyphosate
analyzer, you will need to change the eluants and column.
Step 1. Conditions
Isocratic program, 40% water, 60% methanol
Flow rate: 1.0 mL/min
Sample: 10µL of Carbamate Test Mix, 1700-0063
Detector: excitation 330nm, emission 465nm
The integrator or data system should be set up similarly to normal conditions, but the
fluorescence of the 1-naphthol will only be 20% as much as the same amount under postcolumn conditions; the alkaline PC conditions enhance the native fluorescence.
Step 2. You need to decide on a criterion of peak width. Generally the width at halfmaximum is the most consistent measure. There are several ways to measure
it. If your computer data station calculates peak width, use that. The ratio of
area over height is a good approximation, and some data stations report this
number. If you are using an integrator that only reports areas, you can
measure the height in millimeters, and calculate a ratio; this number has
arbitrary units, but it is more precise than a direct measurement of the width.
However you decide, record the method and all relevant parameters (e.g.
detector sensitivity, attenuation, chart speed, chart scale).
Step 3. Install the column in the PCX5100. This can be your normal carbamate column
or any C18 column in good condition.You may have a column reserved
especially for quality control or diagnostic purposes and use it here.
Step 4. Disconnect the detector inlet from the PCX5100. Disconnect the column outlet
from the PCX 5100. Connect the detector to the outlet of the column.
Step 5. Turn ON the HPLC system. Set the “Pump” switch OFF on the PCX5100. Turn
ON the main power on the PCX5100 and press the “Reset” button.
Immediately, lower the temperature setting on the reactor to below 55˚C before
the reactor can warm up. Wait for the column to come to equilibrium.
Step 6. Collect three chromatograms of the test mixture. You should see a single peak
around 4–6 minutes. The retention times and width parameters should agree
within 2%. If not, keep trying until you have three consistent runs in sequence.
The precision of the height and area is not important for this test. Calculate
the average width (Wd).
Step 7. Turn OFF the LC pump. Restore the normal post-column connections. Turn the
LC pump back ON and press the “Reset” button. Set the reactor to the normal
operating temperature—100˚C. Wait for the entire system to equilibrate, about
15 min.
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