Standards quantum-yield

The easiest way to esdmate the quantum yield of a fluoro-phore is by comparison with standards of known quantum yield. Some of the most used standards are listed in Table 2.4. The quantum yields of these compounds are mosdy independent of excitadon wavelength, so the standards can be used wherever they display usdid absorpdon. 

Determinadon of the quantum yield is generally accomplished by comparison of the wavelength-integrated intensity of the unknown to that of the standard. The optical density is kept below 0.0S to avoid inner filtn effects, or the optical densities of the sample and reference are matched at the excitadon wavelength. The quantum yield of the unknown is calculated using 

This pression is mosdy intuitive, ex pt for the use of die ratio of the refractive indices of the sample (/() and r eienoe (ffjt)- This ratio has Its origin in consldantion of the intensity observed from a p t source In a medium of Inactive index n,- by a detector in a medium of reactive 

The conected intensities for quinine sulfate are shown on Rguie 2.41, and these calculated values are seen to match the initial luiear portum of the curve. For precise corrections, it is preferable to prepare calibration curves using the precise compounds and conditions that will be used for the actual experimentation. Empirical corrections are typically used in most procedures to correct for san ik absev-bance.  

It is intoesting to note that for front-fii illumination, the intensities are expected to become ind ndent of 

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In most cases, the linear absorption is measured with standard spectrometers, and the fluorescence properties are obtained with commercially available spectrofluo-rometers using reference samples with well-known <1>F for calibration of the fluorescence quantum yield. In the ultraviolet and visible range, there are many well-known fluorescence quantum yield standards. Anthracene in ethanol (Cresyl Violet in methanol (commonly used reference samples for wavelengths of 350-650 nm. For wavelengths longer than 650 nm, there is a lack of fluorescence references. Recently, a photochemically stable, D-ji-D polymethine molecule has been proposed as a fluorescence standard near 800 nm [57]. This molecule, PD 2631 (chemical structure shown in Fig. 5) in ethanol, has linear absorption and fluorescence spectra of the reference PD 2631 in ethanol to... 

Fig. 5 Linear absorption (1, 2) and one-photon-excited fluorescence (1, 2 ) for the quantum yield standard Cresyl Violet (1, 1 ) and the proposed standard PD 2631 (2, 2 ) for NIR wavelengths. Molecular structures are shown to the left...

Molecular rotors are useful as reporters of their microenvironment, because their fluorescence emission allows to probe TICT formation and solvent interaction. Measurements are possible through steady-state spectroscopy and time-resolved spectroscopy. Three primary effects were identified in Sect. 2, namely, the solvent-dependent reorientation rate, the solvent-dependent quantum yield (which directly links to the reorientation rate), and the solvatochromic shift. Most commonly, molecular rotors exhibit a change in quantum yield as a consequence of nonradia-tive relaxation. Therefore, the fluorophore s quantum yield needs to be determined as accurately as possible. In steady-state spectroscopy, emission intensity can be calibrated with quantum yield standards. Alternatively, relative changes in emission intensity can be used, because the ratio of two intensities is identical to the ratio of the corresponding quantum yields if the fluid optical properties remain constant. For molecular rotors with nonradiative relaxation, the calibrated measurement of the quantum yield allows to approximately compute the rotational relaxation rate kor from the measured quantum yield [Pg.284]

Tetramethylammonium ozonide, 736 Tetramethyl-l,2-dioxetane (TMD) chemical titration, 1224 chemiluminescence, 1221, 1234 quantum yield standard, 1224, 1226 N,N, N, A -Tetramethyl-p-phenylenediamine hydrogen peroxide determination, 735, 631, 633... 

We often differentiate between the primary quantum yield, which focuses on only the first event (here the quantum yield cannot be >1), and secondary quantum yield, which focuses on the total number of molecules formed via secondary reactions (here the quantum yield can be high). The common emission quantum yield measurement involves the comparison of a very dilute solution of the studied sample with a solution of approximately equal optical density of a compound of known quantum yield (standard reference). The quantum yield of an unknown sample is related to that of a standard by equation 16.5... 

The quantum yields of complexes 44 and 45 were measured using recrystallized quinine sulfate in 1 N H2S04 and the widely referred complex Ru(bpy)3(PF6)2 as quantum yield standards. The data obtained using both standards are in excellent agreement and indeed show yields that are remarkably high, i.e., 80%. 

A standard material is selected firom a list of quantum yield standards (6. 7 and rofde T). Hie standard should have an absorption spectrum in the same range as that of the sample. The fluorescence spectra of the standard and sample should span a similar i range. 

Last but not least, quinine has also two unique properties a bitter taste and a strong fluorescence. The first is widely used in beverage and confectionary industry, which consumes ca. 25% of the world production of quinine [20], whereas fluorescence of quinine sulfate is routinely used in fluorescence spectroscopy as quantum yield standard [21, 22]. 

The reported quantum yields of the long-chain aldehydes in the luminescence reaction catalyzed by P. fischeri luciferase are 0.1 for dodecanal with the standard I (Lee, 1972) 0.13 for decanal with the standard I (McCapra and Hysert, 1973) and 0.15-0.16 for decanal, dodecanal and tetradecanal with the standard III (Shimomura et al., 1972). Thus, the quantum yield of long-chain aldehydes in the bacterial bioluminescence reaction appears to be in the range of 0.10-0.16. 

Johnson et al. (1962) measured the quantum yield of Cypridina luciferin in the luciferase-catalyzed reaction for the first time, using a photomultiplier calibrated with two kinds of standard lamps. The measurement gave a value of 0.28 0.04 at 4°C in 50 mM sodium phosphate buffer, pH 6.5, containing 0.3 M NaCl. The quantum yield... 

Product quantum yields are much easier to measure. The number of quanta absorbed can be determined by an instrument called an actinometer, which is actually a standard photochemical system whose quantum yield is known. An example of the information that can be learned from quantum yields is the following. If the quantum yield of a product is finite and invariant with changes in experimental conditions, it is likely that the product is formed in a primary rate-determining process. Another example In some reactions, the product quantum yields are found to be well over 1 (perhaps as high as 1000). Such a finding indicates a chain reaction (see p. 895 for a discussion of chain reactions). 

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

Requirements for standards used In macro- and microspectrofluorometry differ, depending on whether they are used for Instrument calibration, standardization, or assessment of method accuracy. Specific examples are given of standards for quantum yield, number of quanta, and decay time, and for calibration of Instrument parameters. Including wavelength, spectral responslvlty (determining correction factors for luminescence spectra), stability, and linearity. Differences In requirements for macro- and micro-standards are considered, and specific materials used for each are compared. Pure compounds and matrix-matched standards are listed for standardization and assessment of method accuracy, and existing Standard Reference Materials are discussed. 

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. 

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. 

Quantum-Yield and Quantum-Counter Standards. The materials recommended as quantum-yield and quantum-counter standards are summarized In Table IV. As can be seen, several of the materials suggested as spectral responslvlty standards have also been suggested as quantum-yield and quantum-counter standards. The materials listed In Table IV cover the wavelength range from 200 to almost 800 nm, giving an excellent choice for the researcher. Quantum counters that operate further Into the red are still needed, especially for the area of... 

Table IV. Quantum Yield and Quantum Counter Standards...

Quimtua Yields of Fluorescence Measurements. All of the quantum yields of fluorescence were measured by the relative fluorescence measurement technique of Parker and Rees (24). This method compares the fluorescence of the compound of interest to the fluorescence of some known compound. All of the fluorescence quantum yields were measured using I as a reference. Compound I had previously been measured by this same method using rhodamine B as a standard. 

Repeat the above study, now using a smaller 50 x 50 = 2500 cell sample. After the sample has been allowed to come to a steady state, determine the steady-state concentrations of So, Si, and Tj, along with their standard deviations. Also determine the quantum yields (pi and p. 

There are methods available to quantify the total mass of americium in environmental samples. Mass spectrometric methods provide total mass measurements of americium isotopes (Dacheux and Aupiais 1997, 1998 Halverson 1984 Harvey et al. 1993) however, these detection methods have not gained the same popularity as is found for the radiochemical detection methods. This may relate to the higher purchase price of a MS system, the increased knowledge required to operate the equipment, and the selection by EPA of a-spectrometry for use in its standard analytical methods. Fluorimetric methods, which are commonly used to determine the total mass of uranium and curium in environmental samples, have limited utility to quantify americium, due to the low quantum yield of fluorescence for americium (Thouvenout et al. 1993). 

Table 5.6 Properties of three typical photoredox-active molecules. bpy denotes 2,2 bipyridine, TMPP is tetra N-methylpyridine porphyrin Amax is the wavelength of the absorption maximum, e is the extinction coefficient at Amax, cpT is the quantum yield of the formation of the excited triplet state, r0 is its lifetime, and E0 are standard redox potentials...

We see then that the relative fluorescence quantum yield can be determined by measuring the areas under the fluorescence bands of the sample and the fluorescent standard. However, these spectra must be corrected before their true areas can be determined. Several factors are responsible for this. The most important of these are the phototube and monochromator responses. For most phototubes the maximum response occurs within a limited wavelength range, falling off rather sharply in some cases at the short-and long-wavelength ends. This is illustrated in Figure 2.14. Similarly,... 

If the refractive indices of the solvents used for the sample and the fluorescence standard are not the same, a further correction must be made. For example, quinine sulfate in 0.1 N H2S04 (Or = 0.5) is commonly used as a fluorescence standard. If the fluorescence of the sample whose relative quantum yield is desired is determined in benzene, a correction factor of 27% must be applied in determining the relative areas under the fluorescence bands. If ethanol is used, this correction is only 5.5%. 

A ferrioxalate actinometer was used to determine the lamp light intensity (12). The quantum yield of loss (4>d) and of product formation ( p) were then calculated by standard methods (12). 

A liner containing glass wool was installed in the injection port of the GC to trap polymer residues. A solution containing a known amount of the carbamate in THF along with sulfolane as an internal standard was used to establish the concentration of carbamates in the PMMA and PPMA matrix. Quantum yields were then determined. Product ratios were calculated from UV difference spectra (taken on a Beckman DK-2A spectrometer) of films before and after photolysis. 

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