Fluorescence excitation spectrum concentration

In pure crystals, singlet excitons can be created by mutual annihilation of triplet excitons. The intensity of the singlet exciton fluorescence depends quadratically on the triplet exciton concentration and is therefore proportional to the square of the singlet-triplet extinction coefficient. It is interesting to compare such a delayed fluorescence excitation spectrum, observed by Avakian et cd. 52) on naphthalene, with a corresponding phosphorescence excitation spectrum (Fig. 22). 

Measurements of the intensity of fluorescence at any wavelength vs the wavelength of monochromatic light used to excite the fluorescence give a fluorescence excitation spectrum. The excitation spectrum is an example of an action spectrum, which is a measure of any response to absorbed light. At very low concentrations of pure substances, action spectra tend to be identical to absorption spectra. However, since the observed response (fluorescence in this case) is proportional to light absorbed, action spectra should be compared to plots of 1-T (where T = transmittance, Section B,l) vs wavelength rather than to plots of e vs X. The two plots are proportional at low concentrations. For a discussion of action spectra see Clayton.123... 

Figure 23-13 (A) Corrected emission and excitation spectra of riboflavin tetrabutyrate in w-heptane. Concentration, about 0.4 mg I-1. Curve 1 excitation spectrum emission at 525 nm. Curve 2 emission spectrum excitation at 345 nm. FromKotaki and Yagi.128 (B) Indole in cyclohexane, T = 196 K. 1, Fluorescence excitation spectrum 2, fluorescence spectrum and 3, phosphorescence spectrum. From Konev.125...

Since the sensitivity of fluorescence is higher than absorption, the fluorescence excitation spectrum of a pure molecule can be obtained at low concentrations compared to the absorption spectrum. 

For example, we studied the interaction between the fluorescent probe calcofluor and a 1-acid glycoprotein at two concentrations of calcofluor. Also, we recorded the fluorescence excitation spectrum of the protein so that to find out whether binding of calcofluor to the protein modifies its structure or not. 

Fig. 3.11 Influence of molecule addition on the fluorescence excitation spectrum (recorded at a fixed emission wavelength of 580nm)ofdi-8-ANEPPS difference spectra. Molecule addition at 288.75 pM in all cases except 43 with PC7o%Chol3o% when the concentration was 247.50 pM. a Difference spectra in presence of 400 p.M PCioo% PLVs. b Difference spectra in presence of 400 p.M PC7o%Chol3o% PLVs...

While a laser beam can be used for traditional absorption spectroscopy by measuring / and 7q, the strength of laser spectroscopy lies in more specialized experiments which often do not lend themselves to such measurements. Other techniques are connnonly used to detect the absorption of light from the laser beam. A coimnon one is to observe fluorescence excited by the laser. The total fluorescence produced is nonnally proportional to the amount of light absorbed. It can be used as a measurement of concentration to detect species present in extremely small amounts. Or a measurement of the fluorescence intensity as the laser frequency is scaimed can give an absorption spectrum. This may allow much higher resolution than is easily obtained with a traditional absorption spectrometer. In other experiments the fluorescence may be dispersed and its spectrum detennined with a traditional spectrometer. In suitable cases this could be the emission from a single electronic-vibrational-rotational level of a molecule and the experimenter can study how the spectrum varies with level. 

Fig. 4.1.3 Absorption spectra of aequorin (A), spent solution of aequorin after Ca2+-triggered luminescence (B), and the chromophore of aequorin (C). Fluorescence emission spectrum of the spent solution of aequorin after Ca2+-triggered bioluminescence, excited at 340 nm (D). Luminescence spectrum of aequorin triggered with Ca2+ (E). Curve C is a differential spectrum between aequorin and the protein residue (Shimomura et al., 1974b) protein concentration 0.5 mg/ml for A and B, 1.0 mg/ml for C. From Shimomura and Johnson, 1976.

The excitation spectrum of a molecule is similar to its absorption spectrum, while the fluorescence and phosphorescence emission occur at longer wavelengths than the absorbed light. The intensity of the emitted light allows quantitative measurement since, for dilute solutions, the emitted intensity is proportional to concentration. The excitation and emission spectra are characteristic of the molecule and allow qualitative measurements to be made. The inherent advantages of the techniques, particularly fluorescence, are ... 

Inner-filter effects. The absorption of the fluorescence excitation and emission by the specimen is referred to as the "inner-filter" effect this effect has been treated in the literature (24-27). The inner-filter effect reduces the signal levels and distorts the emission spectrum and the intensity-concentration relationship. The effect is more pronounced in right-angle fluorescence measurements (27) than in the "front face" configuration in which the fluorescence is viewed from the same side as the excitation beam. 

Equation (2) also shows how the intensity of fluorescence varies when the frequency of the exciting light varies. For a given solution the fluorescence intensity is proportional to 7oe0 and for many substances in solution the fluorescence efficiency () is approximately independent of the excitation frequency. Thus, if the intensity of exciting light is kept constant as the frequency is varied, the fluorescence intensity will be proportional to e, the molecular extinction coefficient of the solute. Hence the true excitation spectrum frequently corresponds closely to the absorption spectrum of the compound (see Fig. 2). Spectrofluorimetry can thus be used to measure the absorption spectra of fluorescent solutes, but at concentrations far lower than could be measured directly with an absorption spectrophotometer. It has the further advantage that the... 

Fig. 22. Sensitized delayed fluorescence spectra of anthracene in 10 SM phenan-threne solution.38 Intensity of exciting light was approximately 2.7 X 10-8 einstein cm. 2 sec. 1 at 3.19m-1 (313 dim)- Delayed emission spectra with anthracene concentrations of (1) 10 8Af, (2) 5 X 10 W, (3) 10-W, (4) 10- M, (5) 10 9M. Curve (6) Fluorescence emission spectrum of solution 1 at 260 times less sensitivity. (Owing to variation in the shapes of the cylindrical optical cells, the relative intensities of the delayed emission are only approximately proportional to 9A/fF.)...

Simonson et al. [148] demonstrated remote detection of explosives in soil by combining distributed sensor particles with UV/vis fluorescence LIDAR technology. The key to this approach is that the fluorescence emission spectrum of the distributed particles is strongly affected by absorption of nitroaromatic explosives from the surrounding environment. Remote sensing of the fluorescence quenching by TNT or DNT is achieved by fluorescence LIDAR - the emission spectra were excited in field LIDAR measurements by a frequency-tripled Nd YAG laser at 355 nm and the fluorescence collected with a telescope and various detector systems housed in a 10 x 50 trailer. TNT has been detected in the ppm range at a standoff distance of 0.5 km with this system (Fig. 16). An important limitation to this technique is the pre-concentration of the explosives on the sensor particles, which requires the presence of water to facilitate the transport of the explosive from the surface of the soil particles to the sensor particles. 

The same studies were performed with calf thymus DNA. In addition to the absorption and emission spectra, we have recorded the fluorescence excitation spectra of ethidium bromide at different DNA concentrations (Figure 12.12). One can see that the increase in fluorescence intensity stops when the stoichiometry of the complex is reached. Binding parameters (n = 2.6 and Krl = 3 x 105 M-1) determined from the fluorescence excitation intensities are found to be equal to those calculated with the fluorescence emission and the OD variations. Figure 12.13 shows the normalized intensity increase in both fluorescence emission and excitation intensity peaks of ethidium bromide with the function of DNA. The intensity increase in the excitation spectrum of ethidium bromide in the presence of DNA is the result of binding of the fluorophore to DNA and thus of the increase in the number of excited bound fluorophores. 

Figure 12.12 Fluorescence excitation spectra of 23 /,iM ethidium bromide (spectrum 1) with increasing concentrations of calf thymus DNA (spectra 2-12) (A.em = 600 nm).

Explain why it is important to record fluorescence spectra at low product concentrations. R3. A high concentration may increase the optical density at the excitation and/or the emission wavelengths. This will distort the fluorescence emission spectrum by decreasing the real fluorescence intensity and by shifting the emission peak. 

In the bulk, the low concentration of ground-state pairs excludes their observation by absorption. The formation of the excited-state complex, termed exciplex, is a collisional process electronic excitation of either the acceptor or the donor leads to the formation of a locally excited state (for instance, in hydrocarbon molecules, it is a nn state). During the lifetime of this state, a collision with the other partner (which is in the ground state) leads to the formation of the exciplex. This mechanism is compatible with the fact that the absorption and fluorescence excitation spectra of the system are identical with those obtained by superimposing the spectra of the individual components. At the same time, the fluorescence emission spectrum changes drastically—a broad band, red shifted with respect to the bare molecule s emission spectrum, appears. It is usually devoid of vibrational structure, and is shifted to longer wavelengths as the solvent polarity increases [1],... 

In hydrocarbon solution, the thlazole shows absorption maxima at 30,300 and 31,250 cm l and fluorescence with a maximum near 20,000 cm l (19,350 in the solid) (42). In ethanol, the intensity of these absorption bands decreases and a new, concentration-dependent absorption near 25,600 cm appears. Together with this change, an additional fluorescence band appears at 27,000 cm. The excitation spectrum of the long-wavelength fluorescence coincides with the long-wavelength absorption and that of the short-wavelength fluorescence with the absorption... 

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