THE EVALUATION OF PHASE FLUORIMETRY AS AN INSTRUMENTAL TECHNIQUE FOR QUANTITATIVE ANALYSIS
Abstract
A 0.1-50 MHz laser-based phase fluorimeter, using lock-in amplifier detection, has been built for the ultimate purpose of ascertaining if the phase experiment can be confidently applied to the determination of low levels of emitters in the presence of strong luminescence backgrounds. The performance of the instrument was initially evaluated with respect to the measurement of small rf delays and fluorescence lifetimes. It was found that the instrument, particularly the phase sensitive detector, is subject to appreciable amplitude- and frequency dependencies which have a profound effect on the accuracy and precision of measurements. Lifetimes over the range of (TURNEQ)1.5 to 25 nsec could be determined with a relative accuracy and precision of only 5-20%, in sharp contrast to the degree of confidence commonly associated with phase measurements but in agreement with theory. Binary mixtures were analyzed by both phase-resolved fluorescence spectroscopy (PRFS) and multi-frequency phase analysis. The former method was found to be extremely sensitive to the instrumental errors observed for the lifetime measurements. Systematic errors in the phase null adjustment resulted in signal errors of >20% below and the 20% relative concentration level in a best-case situation of well-separated lifetimes, suggesting that background suppression by phase resolution is not an acceptable approach for the determination of low levels of emitters. Multi-frequency phase analysis improved the accuracy in the 5-20% relative concentration range by a factor of 3-10. Instrumental uncertainties in the phase measurements were found to preclude making a reasonable fit to a two-component model below this range.
Degree
Ph.D.
Subject Area
Analytical chemistry
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