Step 1: Check The Lc System For Impurities - Agilent Technologies 1200 Series User Manual

4 First Steps with the Detector

Step 1: Check the LC system for impurities

A critical issue in trace level fluorescence detection is to have an LC system
free of fluorescent contamination. Most contaminants derive from impure
solvents. Taking a fluorescence scan is a convenient way to check the quality
of the solvent in a few minutes. This can be done, for example, by filling the
FLD cuvette directly with the solvent for an offline measurement even before
the start of a chromatographic run. The result can be displayed as an
isofluorescence plot or a three-dimensional plot. Different colors reflect
different intensities.
Figure 28
as mobile phase. The area where fluorescence of the contaminated water
sample can be seen is between the stray light areas: the first- and second-order
Raleigh stray light and Raman stray light.
A pure water
sample was put
into the flow cell.
Spectra were
recorded at 5 nm
step sizes.
Figure 28
Since "excitation" and "emission" wavelength are the same for Raleigh stray
light, the area of first-order Raleigh stray light is visible in the left upper area
of the diagram. The Raman bands of water are seen below the first-order
Raleigh stray light. Since the cut-off filter cuts off light below 280 nm, the
second-order Raleigh stray light starts above 560 nm.
64
shows a sample of slightly impure water which was planned for use
Impurity 1. order
Isofluorescence plot of a mobile phase
Raman 2. order
1200 Series FLD User Manual