Fluorescence intensity absolute

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. 

In general, luminescence measurements are relative rather than absolute, since the Instrument characteristics and sample properties that determine the fluorescence Intensities are often not well defined. Absolute luminescence measurements are difficult to perform and require time and Instrumentation not available In most laboratories. Thus, luminescence measurements rely heavily on standards to determine Instrument responses and parameters, the chemical composition of samples, and the characteristics of chemical systems. To... 

In practice it is much simpler to determine the relative quantum yield of fluorescence than the absolute quantum yield (see Table 2.1). This is done by comparing the fluorescence intensity of a given sample to that of a compound whose fluorescence quantum yield is known. For this one must... 

The run-to-run reproducibility of the profile shape of the FOCS fluorescence-intensity signal is good however, the reproducibility of the absolute intensity values is unsatisfactory. The run-to-run variations in the fluorescence intensities are caused by the differences in resin thickness at the small area "viewed" by the optrode. In addition, substantial resin flow takes place during cure, causing the resin thickness to vary as a function of cure time. However, since this variation in resin thickness mi t be reproducible from run-to-run (if other cure parameters remain unchanged), it may be possible to develop a suitable... 

Attempts to determine absolute values for the DAN method were complicated mainly by the problem that fluorescence intensity was found to increase with decreasing particle size ( even below 80 jim). For materials of the same particle size, as in the present study, the results correlated ( r = 0.85 ) quite well with the FDNB lysine and showed good reproducibility, attributable to the unique design of the fluorometer used ( 12 ). 

From Fig. 4 it can be seen that, for finite bandpass detection, one will obtain different fluorescent intensities per emitting molecule depending on the level pumped. This can produce systematic errors in both the determination of absolute concentrations and the use of excitation scans to obtain ground state rotational temperatures (21) Also, the lack of a thermal distribution imposes restrictions on models of and data analysis in optical saturation techniques. 

The measurements are placed on an absolute scale by including a high temperature flame (H2/C>2/N2 = 4/1/2 with 1% H2S, 2350 K) which reaches thermal equilibrium rapidly. Measurement of the fluorescent intensity in the equilibrium plateau a few centimeters above the burner along with a calculation of the equilibrium concentration of each of the species at the temperature of this flame permits evaluation of the proportionality constant a (or 8) In this manner absolute concentrations can then be calculated using the relative fluorescence intensity inputs for each of the species. 

Concentration Profiles. The relative fluorescence intensity profiles for OH, S2, SH, SO, and SO2 were converted to absolute number densities according to the method already outlined. Resulting concentration profiles for a rich, sulfur bearing flame are exhibited in Figure 17. H-atom densities were calculated from the measured OH concentrations and H2 and H2O equilibrium values for each flame according to Equation 6. Similar balanced radical reactions were used to calculate H2S and S concentrations 6). Although sulfur was added as H2S to this hydrogen rich flame, the dominant sulfur product at early times in the post flame gas is S02 ... 

The concept of saturated laser fluorescence appears attractive in that the fluorescence intensity is directly related to the particular species concentration and becomes roughly independent of the laser intensity at saturation. Such a mode has been invoked already to monitor absolutely flame concentrations of Na a-4), OH (5), C2 (6,7), CH (7,8), CN (8), and MgO (4). However, during a recent study of the behavior of Na and Li in flames (9-11), we have observed evidence for laser induced chemical reactions under saturated conditions which has significant implications for the quantitative exactness of such measurements. 

As the fluorescence cross sections are known, the absolute intensity of the incoming beam can be determined readily from a measurement of the fluorescence intensity. On this basis all scattering data can be put on an absolute scale. 

Absolute measurements are not typically used in the standard HCI setting. Most HCI analysis of fluorescence intensities are based upon relative measurements, as for instance by comparing the signal intensities between different samples relative to a control. Thus, positive and negative reference controls on each assay plate are often required in order to account for slight variation of the assay handling or different power of the light source. 

Elements with low intensity fluorescence lines (e.g. Eu, Tm and Y) have been determined in aqueous solutions by depositing and drying nanoliter amounts of sample on the Ni cathode of a miniature GD source used as the atom reservoir [665], The atomic cloud thus formed was extited by a Cu-vapor laser-pumped dye laser to detect fluorescence directly. Absolute detection limits of 2 fg for Eu, 0.08 fg for Tm and 1.2 pg for Y were achieved and the total time for analysis from sample probing to data acquisition did not exceed 5 min. 

Sensitivity of the instrument, which refers to the system response to small changes in concentration, is determined by the signal to noise ratio (SNR) of the measurement, as well as, the concentration slope. The SNR of each measurement may be considerably smaller than variation between measurements made on different occasions. Measurements of concentration from absolute fluorescent intensity (O) are possible provided that the calibration of the instrument remains unchanged. Reliable fluorescent intensity measurements across different instruments or from repeated measurements with the same instrument are possible only if the above factors can either be eradicated from consideration or be controlled with an experiment independent calibration coefficient. One effective solution proposed the development of a set of standard reference materials (SRMs) for use with identical instruments [9]. Thus, for a particular instrument, the concentration of a test solution can be expressed as a ratio of the fluorescent counts for the test and SRM. Subsequently, it was recommended that the concentration of the test solution could be expressed in molecular equivalent soluble fluorophore (MESF) units. This protocol was developed with respect to the use of flow c) tometers, which can be operated close to the ideal experimental conditions. Despite this, measurements of fluorescent intensity in terms of MESF units stiU have problems, as most fluorescence-based instruments cannot be guaranteed to operate under the same conditions from day to day. 

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