Subscriber access provided by - Access paid by the | UC Irvine LibrariesAnalytical Chemistry is published by the American Chemical Society. 1155 SixteenthStreet N.W., Washington, DC 20036On-the-fly fluorescence lifetime detection in HPLC using amultiharmonic Fourier transform phase-modulation spectrofluorometerMaria Brak. Smalley, Jeremy M. Shaver, and Linda B. McGownAnal. Chem., 1993, 65 (23), 3466-3472 • DOI: 10.1021/ac00071a022Downloaded from http://pubs.acs.org on January 26, 2009More About This ArticleThe permalink http://dx.doi.org/10.1021/ac00071a022 provides access to:• Links to articles and content related to this article• Copyright permission to reproduce figures and/or text from this article5400 Anal. Chem. 1999, 65, 3466-3472 This Research Contribution is in Commemoration of the Life and Science of I. M. Kolthoff (1894-1993). On-the-Fly Fluorescence Lifetime Detection in HPLC Using a Multiharmonic Fourier Transform Phase-Modulation Spectrofluorometer Maria Brak Smalley, Jeremy M. Shaver, and Linda B. McGown' Department of Chemistry, P. M. Gross Chemical Laboratory, Duke University, Box 90346, Durham, North Carolina 27708-0346 Frequency-domain fluorescence lifetime detection has been demonstrated recently as a technique for detecting and resolving overlapping peaks on-t he- fly in reversed-phase high-performance liquid chromatography (RP-HPLC). However, instru- ment limitations necessitate multiple injections of the same sample for peak resolution. The introduction of a commercially available multi- harmonic Fourier transform spectrofluorometer (MHF) eliminates this problem and offers other advantages as well. The MHF data acquired on- the-fly during a single chromatographic run con- tain all of the multifrequency information needed to determine lifetimes and to indicate and resolve overlapping peaks at intervals as short as several milliseconds. This allows essentially continuous monitoring of both lifetime and intensity during chromatographic elution. This approach to the addition of lifetime in fluorescence detection increases the information content of chromato- graphic-based analysis without increasing the time per analysis. This paper presents the first results obtained using MHF detection, including measurements of single components and simple mixtures. Optimization of instrumental and ex- perimental conditions is discussed, including the nature of the raw data and the procedures that were developed to extract and process the neces- sary lifetime and intensity information. INTRODUCTION Sensitive and selective detection in HPLC can be achieved using fluorescence techniques, which are applicable to both intrinsically fluorescent and fluorescent-derivatized com- pounds. However, traditional steady-state approaches based on measurements of intensity at one or more wavelengths have several limitations. Detection at only one or a few wavelength may be inadequate to resolve overlapping peaks, while the use of array detection to collect an entire spectrum can decrease sensitivity as aresult of dispersion. Furthermore, the techniques may not be readily able to detect the presence of minor components or matrix effects. Therefore, it is desirable to explore new approaches to fluorescence detection that are capable of identifying and resolving overlapping chromatographic peaks as well as indicating the presence of * Corresponding author. 0003-2700/93/0365-3466$04.00/0 impurities and matrix effecta. Moreover, the techniques should minimize assumptions regarding chromatographic peak shape and the identity, properties, or spectral charac- teristics of the analytes. This paper describes an approach that moves toward these goals through the addition of fluorescence lifetime to spectral information to increase the dimensionality and information content of the Chromatographic data. Fluorescence lifetime detection haa been explored previously using both time- domain and frequency-domain techniques for analysis of polycyclic aromatic hydrocarbon (PAH) compounds. In an early time-domain study, fluorescence intensity chromato- grams were measured using various delay times (0-40 ns) following the excitation pulse from an N2 laser-pumped dye laser.' In such a system, a compromise exists in choice of the optimal delay time, which should be long enough for adequate reduction of prompt background signals but short enough to obtain sufficient analyte signal, which itself decays according to the fluorescence lifetime of the analyte. Collection of several chromatograms, each at a different delay time, waa used in order to discriminate among different analytes on the basis of differences among their fluorescence lifetimes. However, the lifetime resolution in the study wae limited by the pulse width of 10 ns, which was sufficient only to distinguish between groups of shorter lived and longer lived signals but could not accomplish resolution of individual components within the groups, nor were actual lifetimes of individual components determined. A later study wed the same approach but with a subnanosecond dye laser system which improved the time resolution to the nanosecond range but still required one chromatogram per delay time.2 Mea- surements at multiple delay times in a single chromatogram were achieved in a later study by continuous monitoring of the signal resulting from pulsed excitation at a repetition rate of 20-30 Hz as the compounds were eluted. The chromatogram for a given delay time could then be con- structed from the appropriate point following each pulse in the original chromatogram in order to obtain decay curves for the various chromatographic peaks. However, the signal- to-noise ratio was not adequate for resolution of coeluting peake or recovery of the individual lifetimes of coeluting analytes from multiexponential curves. Another approach was taken to time-domain lifetime detection with pulsed laser excitation, in which the PMT anode current was split in half and one of the halves was (1) FUN@ N.; Oteuki, A. Anal. Chm. 1988,66,2407-2413. (2) Imaeaka, T.; Ishibashi, K.; Ishibashi, N. AM^. Chim. Acta 1982, (3) Ishibashi, K.; Imaeaka, T.; Ishibashi, N. Anal. Chim. Acta 1986, (4) Desilets, D. J.; Kissinger, P. T.; Lytle, F. E. Anal. Chem. 1987,69, 142,l-12. 173,166-176. 1830-1834. 0 1893 American chemical SodetyANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1983 8467