Waters 2475, Operator'S Manual, Page: 20 / 228

1-6 Theory of Operation
Gain
Raman
and Counts
Raman
= values from the most recent execution of
the normalize units function
PMTCounts
t
and Gain
t
= values at the time of data collection
Normalizing the emission units results in a water/Raman signal strength, at
E
x
350 nm/E
m
397 nm, of 100 emission units. The xenon spectrum output is
not uniform over the detector’s operating range, and low-UV wavelengths can
degrade faster than normalization wavelengths.
Energy units
The alternative to emission units is energy units, which are similar to those
used by traditional HPLC fluorescence detectors. They directly correlate to
the anode current of the PMT, so they are directly influenced by the gain
setting. All instrumental variables, such as lamp intensity, optics efficiency,
and gain, directly influence the fluorescence emission signal strength. As a
result, energy units are less reliable. Nonetheless, when you must calculate
energy units to conform to established protocols, use the following equation:
EU = PMTCounts × K × (ReferenceCounts
0
/ ReferenceCounts
t
)
where K scales the maximum detectable fluorescence signal to 10,000 units.
References
Consult the following texts for additional information on fluorescence
detection:
N. Ichinose, G. Schwedt, F. M. Schnepel, and K. Adachi,
Fluorometric
Analysis in Biomedical Chemistry , Chapter 5, Wiley-Interscience: New York,
1991.
E. S. Yeung, ed.,
Detectors for Liquid Chromatography , Chapter 5, Wiley: New
York,1986.
W. R. Seitz, in
Treatise on Analytical Chemistry , 2nd ed., P. J. Elving, E. J.
Meehan, I. M. Kolthoff, eds., Part I, Vol. 7, Chapter 4, Wiley: New York, 1981.
J. R. Lakowicz,
Principles of Fluorescence Spectroscopy , Plenum: New York,
1983.