Wavelength-resolved fluorescence detection

In wavelength-resolved fluorescence detection, a complete emission spectrum is generated for each time point. The resulting data are plotted as a three-dimensional electropherogram, with axes... 

Timperman, A. T., Khatib, K., and Sweedler, J. V., Wavelength-resolved fluorescence detection in capillary electrophoresis, Anal. Chem., 67,139,1995. 

Timperman, A.T. and Sweedler, J.V., Capillary electrophoresis with wavelength-resolved fluorescence detection, Ana/yif, 121, 45R, 1996. 

Beekman, M.C., Lingeman, H., Brinkman, U.A.Th., and Gooijer, C., Determination of the isoflavone formononetin in red clover (Trifolium pratense L.) by micellar electrokinetic chromatography combined with deep-UV laser-induced wavelength-resolved fluorescence detection, J. Microcolumn Sep., 11, 347-350, 1999. 

Fig. 4.4 Schematic diagram of a typical arrangment for laser induced fluorescence measurements of lifetimes. A pulsed laser beam (or beams) passes through a heated glass cell containing alkali vapor and the time and wavelength resolved fluorescence is detected...

In the present paper we describe an apparatus for recording transient emission spectra that yields data which approach the ideal multidimensional case. We emphasize in the discussion the advantages of multichannel detection for transient emission data. We also briefly compare our approach to alternative methods for recording time and wavelength resolved fluorescence data on the picosecond time scale. 

The possibilities of molecular beam spectroscopy can be enhanced by allowing for spectrally resolved fluorescence detection or for resonant two-photon ionization in combination with a mass spectrometer. Such a molecular beam apparatus is shown in Fig. 4.5. The photomultiplier PMl monitors the total fluorescence /r(A.l) as a function of the laser wavelength Xl (excitation spectrum. Sect. 1.3). Photomultiplier PM2 records the dispersed fluorescence spectrum excited at a fixed laser... 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Fig. 2 (a) Fluorescence up-conversion transients of TAB dissolved in n-heptane. Detection wavelengths in nm are indicated in circles (b) reconstructed time-resolved fluorescence spectra of TAB in n-heptane... 

Time-resolved fluorescence was used to detect rhodamine 6G (R6G), sulfor-hodamine 101 (SR101), and rhodamine B (RB). A Ti-sapphire laser (800 nm, 50 fs) was used, but the excitation wavelength has been converted to 400 nm (for R6G) or 532 nm (for SR 101 or RB) by an optical parametric amplifier or by second harmonic generation [676], In another report, fluorescence burst detection was used for detection of single chromophore molecules [677]. 

Figure 1.13. Time-resolved fluorescence decays of P-CIP4 with fits at 600 nm and 700 nm detection wavelengths. The upper panel shows the weighted distribution of residuals (Rt) and the lower panel represents the autocorrelation (ac) function for the decays. Inset reports on a shorter time scale.

We developed two kinds of multidimensional fluorescence spectroscopic systems the time-gated excitation-emission matrix spectroscopic system and the time- and spectrally resolved fluorescence microscopic system. The former acquires the fluorescence intensities as a function of excitation wavelength (Ex), emission wavelength (Em), and delay time (x) after impulsive photoexcitation, while the latter acquires the fluorescence intensities as a function of Em, x, and spatial localization (%-, y-positions). In both methods, efficient acquisition of a whole data set is achieved based on line illumination by the laser beam and detection of the fluorescence image by a 2D image sensor, that is, a charge-coupled device (CCD) camera. 

A few reports have appeared on combining the streak camera temporal dispersion with polychromators and three dimensional optical multichannel detection. This approach yields three dimensional fluorescence data for each laser pulse. With the present technological limitations of three dimensional detectors and streak cameras, however, data of this type suffer from low wavelength resolution. As detector and streak camera technology improve, this technique may become the method of choice for time and wavelength resolved emission spectra. 

Figure 33. Time-resolved fluorescence and dispersed fluorescence spectra for v, excitation of the alkylanilines. The excitation frequencies of the spectra have been marked by an asterisk and aligned for comparison. The detection wavelengths were different from the excitation wavelengths for all decays. R for the decays were 2.4 A for aniline, 3.2 A for butylaniline, and 1.6 A for all others.

Figure 34. Time-resolved fluorescence and dispersed fluorescence spectra for v, excitation of the gauche and trans conformers of propylaniline. An asterisk marks the excitation frequency in each spectrum, while an arrow indicates the detection frequency associated with the decay at left. For the trans species, complementary build-ups at different detection wavelengths were observed. For the gauche species, the Fourier transform of the decay is very rich.

Figure 4.6 Principle of phase-resolved fluorescence. If the excitation source is modulated, the time delay in fluorescence causes a phase lag in the detected light signal, depending on the wavelength of the emitted light. With appropriate signal processing, it is possible to detect phase lags for multiple fluorescent indicators.

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