Relationship between fluorescence and concentration

For solutions, the quantum yield of fluorescence Of (between 0 and 1 inclusive), which is independent of the intensity emitted by the light source, is defined by the ratio of the number of photons emitted to the number of photons absorbed, this latter being equivalent to the ratio of fluorescence intensity 7f over that absorbed 7g (expression 11.2). 

Accepting that I = Iq — I I representing the transmitted light intensity), the following reasoning allows to relate 7f to the concentration C, of the compound  

Knowing that the absorbance A is equal to log Iq/I, expression 11.3 becomes  

If the solution is diluted, the term A is close to 0 and the term 10 is therefore close to 1 — 2.3A. The expression (11.4) can then be simplified, becoming  

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The relationship between fluorescence and concentration is virtually linear at very low concentrations where the absorbance is less than 0.02 and is approximated by... 

The excitation light may be absorbed so strongly by the fluorophore that fluorescence is reduced along the excitation path, one aspect of the inner filter effect (1, 2). Such an effect should be suspected if dilution of a sample leads, initially, to a fluorescence increase. This could arise, for example, if the detector observes a part of the sample reached by the excitation beam only in dilute solutions. The expected linear relationship between fluorescence and concentration occurs only with dilute (<10 m) preparations (1). Remember that it is not only the fluorophore that may absorb the excitation and that it is the overall absorbance that must be kept low to avoid any inner filter effects. 

Relationship between fluorescence and concentration. The Beer-Lambert law is valid only in the absence of fluorescence a highly fluorescent compound will give erroneous absorption data when measured in the usual type of photometer. 

It is often experimentally convenient to use an analytical method that provides an instrumental signal that is proportional to concentration, rather than providing an absolute concentration, and such methods readily yield the ratio clc°. Solution absorbance, fluorescence intensity, and conductance are examples of this type of instrument response. The requirements are that the reactants and products both give a signal that is directly proportional to their concentrations and that there be an experimentally usable change in the observed property as the reactants are transformed into the products. We take absorption spectroscopy as an example, so that Beer s law is the functional relationship between absorbance and concentration. Let A be the reactant and Z the product. We then require that Ea ez, where e signifies a molar absorptivity. As initial conditions (t = 0) we set Ca = ca and cz = 0. The mass balance relationship Eq. (2-47) relates Ca and cz, where c is the product concentration at infinity time, that is, when the reaction is essentially complete. 

Kowalczuk, R, Cooper, W.J., Durako, M J., Kahn, A.E., Gonsior, M., andYoung, H. (2010). Characterization of dissolved organic matter fluorescence in the South Atlantic Bight with use of PARAFAC model Relationships between fluorescence and its components, absorption coefficients and organic carbon concentrations. Mar. Chem., 118,... 

For analytical applications, when a linear relationship between fluorescence intensity and concentration is desirable, a correction curve must be built up under the same conditions as those that will be used for the actual experiment. 

Figure 2. The relationship between fluorescence of ANS and concentration of LiFCB in 6ED-LiFCB mixed system. The fixed concentrations are ImM, 0.9mM, O.SmM, 0.6mM and 0.4mM.

A check on the consistency of the constants Kh K2, and K3 can be obtained from the measurements of the normal fluorescence quenching constant for the monomer (Table VII). By considering the stationary concentration of P for normal fluorescence, and applying the relationship between P2 and P given by eq. (43), it can be shown that... 

The relationship between the weight concentration of an element and the intensity of one of its characteristic lines is complex. Several models have been developed to correlate fluorescence to weak, atomic concentrations. Many corrections have to be made due to inter-element interactions, preferential excitation, self-absorption, and fluorescence yield (heavy elements relax more quickly by internal conversion without emission of photons). All of these factors require the reference sample to be practically the same structure and atomic composition for all elements present as the... 

ANALYZER (Reagent-Tape). The key to chemical analysis by this method is a tape (paper or fabric) that has been impregnated with a chemical substance that reacts with the unknown to form a reaction product on the tape which lias some special characteristic, e.g., color, increased or decreased opacity, change in electrical conductance, or increased or lessened fluorescence. Small pieces of paper treated with lead acetate, for example, have, been used manually by chemists for many years to determine the presence of hydrogen sulfide in a solution or in the atmosphere. This basic concept forms the foundation for a number of sophisticated instruments that may pietreat a sample gas, pass it over a cyclically advanced tape, and, for example, photo-metrically sense the color of the exposed tape, to establish a relationship between color and gas concentration. Depending upon tile type uf reactiun involved, the tape may he wet or dry and it may be advanced continuously or periodically. Obviously, there are many possible variations within the framework of this general concept. 

The fluorescence properties of two fulvic acids, one derived from the soil and the other from river water, were studied. The maximum emission intensity occurred at 445-450 nm upon excitation at 350 nm, and the intensity varied with pH, reaching a maximum at pH 5.0 and decreasing rapidly as the pH dropped below 4. Neither oxygen nor electrolyte concentration affected the fluorescence of the fulvic acid derived from the soil. Complexes of fulvic acid with copper, lead, cobalt, nickel and manganese were examined and it was found that bound copper II ions quench fulvic acid fluorescence. Ion-selective electrode potentiometry was used to demonstrate the close relationship between fluorescence quenching and fulvic acid complexation of cupric ions. It is suggested that fluorescence and ion-selective electrode analysis may not be measuring the same complexation phenomenon in the cases of nickel and cobalt complexes with fulvic acid. 

The system is standardized in the absence of cells with known amounts of peroxide either generated as H202 or from glucose in the medium and glucose oxidase or added directly as ethyl peroxide. The relationship between fluorescence intensity of 2 pM scopoletin and peroxide concentration is shown in Fig. 3.13. 

Although a linear relationship between the analyte concentration and the measured signal will exist for most methods used in pharmaceutical analysis, there are some exceptions such as TLC and fluorescence detection. Therefore, the term analytical response would be more appropriate. 

Luminescence spectroscopy is used more often in quantitative analysis than in any other application. The quantitative relationship between fluorescence intensity F and analyte concentration C is derived from the Beer-Lambert law ... 

The linear model predicts the relationship between fluorescence intensity, /, and analyte concentration, x, to be of the form. 

The quantitative relationship between fluorescence power and analyte concentration can be derived from the number of molecules in the excited state and the radiant power absorbed by the processed sample ... 

Fig. 8.1 An image of an odor plume taken using planar, laser-induced fluorescence. This image reveals the instantaneous scalar structure of the plume. The image was captured from the outer layer of the momentum boundary layer of the plume. It is a horizontal image spanning a lateral and streamwise range it reveals the spatial patterns at a given vertical location. The color scale indicates the concentration of the odor in the plume concentrations are normalized by the source concentration Co and color coded as shown in the legend. From Grimaldi et al.. Journal of Turbulence, 2002, The relationship between mean and instantaneous structure in turbulent passive scalar plumes, vol. 3, pp. 1-24. Reproduced with the permission of the authors and Taylor and Francis Ltd. (www.tandf.co.uk/ioumals).

At higher concentrations the relationship between F and c deviates from linearity. The plot of F vs. c rolls over as seen in Fig. 5.39. It can be seen that at higher concentrations the fluorescence intensity actually decreases because the molecules in the outer part of the sample absorb the fluorescence generated by those in the inner part of the... 

LIF is a well-established method for concentration measurement. In fact, STED is also a LIF-based method. Therefore, it is straightforward to utilize STED to measure concentration profile in nanochannels. As aforementioned, LIFPA itself measures the real concentration of fluorescent dye. The principle of measuring proton concentration in a nanochannel is that the fluorescence intensity increases with the increase of pH, which is related to H [46]. Through measurement of the calibration relationship between fluorescence intensity and pH, one can measure H by simply measuring fluorescence intensity, similar to LIFPA. 

Fig. 2 (A) Relationship between qE and internal proton concentration in the presence (closed circles) and absence (open circles) of 0.5 uM antimycin A, (B) Relationship between qE and ApH (in absence of ant.A). Conditions as for Fig.l internal proton concentration and LpH were calculated from extent of quenching of 9-aminoacridine fluorescence.

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