Excitation and emission

An instrument for measuring fluorescence that uses filters to select the excitation and emission wavelengths. 

Block diagram for molecular phosphorescence spectrometer with inset showing how choppers are used to isolate excitation and emission. 

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

Excitation and emission wavelengths are for the unconjugated fluorophore. Wavelengths for conjugates may vary according to the composition of the conjugate. 

Fluorescent and phosphorescent substances are excited into an unstable energy state by UV light. When they return to the ground state they release a part of the energy taken up in the form of radiation. The emitted radiation is less energetic than the light absorbed and usually lies in the visible part of the spectrum. Since absorption (excitation) and emission obey a linear relationship over a certain range a reduction in absorption leads to a reduction in the luminescence, too. 

Fig. 10.1.3 Fluorescence excitation and emission spectra (solid lines) and H2O2-triggered luminescence spectrum (dashed line) of Ophiopsila photoprotein (Shimomura, 1986b, revised). The dotted line indicates the in vivo bioluminescence spectrum of Ophiopsila californica plotted from the data reported by Brehm and Morin (1977).

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Requirements of Standards. The general requirements for luminescence standards have been discussed extensively (3,7-9) and include stability, purity, no overlap between excitation and emission spectra, no oxygen quenching, and a high, constant qtiantum yield independent of excitation wavelength. Specific system parameters--such as the broad or narrow excitation and emission spectra, isotropic or anisotropic emission, solubility in a specific solvent, stability (standard relative to sample), and concentration--almost require the standard to be in the same chemical and physical environment as the sample. 

Calibration. In general, standards used for instrument calibration are physical devices (standard lamps, flow meters, etc.) or pure chemical compounds in solution (solid or liquid), although some combined forms could be used (e.g., Tb + Eu in glass for wavelength calibration). Calibrated lnstr iment parameters include wavelength accuracy, detection-system spectral responsivity (to determine corrected excitation and emission spectra), and stability, among others. Fluorescence data such as corrected excitation and emission spectra, quantum yields, decay times, and polarization that are to be compared among laboratories are dependent on these calibrations. The Instrument and fluorescence parameters and various standards, reviewed recently (1,2,11), are discussed briefly below. 

Table III. Standards for Corrected Excitation and Emission Spectra...

The corrected excitation and emission spectra for quinine sulfate from previous work (17) and from a round-robin exercise (11) are given In Figure 1. The coefficients of variation are also given at each point to Indicate the amount of agreement In measured values from the ten laboratories participating In the round-robin exercise. 

All samples were monitored using a Perkin-Elmer 650-10S Fluorescence Spectrophotometer. Fluorescence excitation and emission wavelengths for the PAHs in this study were obtained from Berlman (24). The resulting spectra were analyzed using an Apple 11+ computer by integrating peak areas to determine total changes in... 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

Figures 3a-f show the emission and excitation spectra for all six humic fractions. The excitation and emission maxima are listed in Table III along with the maxima of the phase-resolved emission spectra. In each case the emission spectrum was scanned with the excitation maximum wavelength held constant, and the excitation spectrum was scanned with the emission maximum wavelength held constant. Several interesting features are noted. The two humic samples ( Figures 3a,b) each have two excitation maxima and it appears that a double peak has been merged into the emission scan as evidenced by the shoulder on the high energy side of the emission peak. Similarly it seems evident that the exaggerated shoulders in the emission spectra of all the fractions point to the inclusion of two emission peaks in each spectrum. This evidence suggests the presence of two chromophores in each humic fraction.

Table III. Humic Fractions Fluorescence Excitation and Emission Maxima and...

Figure 1. Catalog page of meta-autunite, including 3-D perspective excitation-emission plot, maximum excitation and emission spectra, and rhodamine dye equivalencies.

Amplification of the natural fluorescence of some pesticides and bathochromic shift of the excitation and emission maxima detection limits 5-100 ng. 

Also bound to the UV-Vis spectral area is fluorescence spectrometry. It is most important with respect to those fluorescent food colorants that have been incorporated into food. In detail it helps to (1) identify a colorant by the spectral pattern of fluorescence excitation and emission spectra, (2) quantify its concentration by the fluorescence emission intensity, (3) qualify the enviromnent into which the colorant molecule is embedded, and (4) perform structural research on the food matter into which the colorant is incorporated. 

To identify a colorant, its excitation and emission spectra must be measured. This can be done under standard conditions if the colorant has been extracted from a foodstuff. Usually the spectral patterns taken from real conditions will not deviate too much from standard conditions. One must be aware that the main spectral patterns are determined by the chromophore of the colorant and that further molecular identification needs to recognize special fine structures of the spectra or employ additional analytical tools. 

The excitation and emission wavelengths used for fluorescence detection in HPLC analysis are 350 and 450 nm for isoxanthopterin and 340 and 450 nm for 2,4,7-trioxopteridine, respectively." The absorption spectra of riboflavin present... 

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