Standards for fluorescence

States also began to look at new products. Massachusetts promulgated legislation requiring its state energy office to set standards for fluorescent and incandescent lamps, and introduced legislation requiring standards for electric motors. Transformers were later added to the Massachusetts list. 

Table V. Materials Used as Standards for Fluorescence Decay Time, r...

A collection of corrected excitation and emission spectra can be found in Miller J. N. (Ed.) (1981) Standards for Fluorescence Spectrometry, Chapman and Hall, London. Corrected emission spectra can also be found in Appendix 1 of Lakowicz J. R. (1999) Principles of Fluorescence Spectroscopy, Kluwer Academic/ Plenum Publishers, New York. 

Miller J. N. (Ed.) (1981) Standards for Fluorescence Spectrometry, Chapman and Hall, London. 

Figure 3.2 Difference gel electrophoresis (DIGE). Ettan DIGE workflow three-color and two-color experiments including the internal standard. For fluorescence proteins tagging, two different CyDyes techniques are available. Minimal fluors allow consideration of three different CyDyes (Cy2, Cy3 and Cy5) in a multiplexing experiment.

In the second test, a number of fluorescent compounds of relatively well known lifetimes in the nanosecond time range (8,9) were used as standards, allowing evaluation of both the instrumental and computational aspects of the measurement. Table I shows the values obtained for 2,3-diphenyloxazole (PPO), anthracene and quinine blsulphate. All chemicals were analytical grade and not further purified before use. Anthracene and PPO were dissolved in cyclohexane, quinine in O.IN 8280 solvents were not degassed. The case of quinine is of interest because of its common use as a standard for fluorescence measurements, despite its complex decay kinetics (10). In agreement with previous work (10) we found satisfactory fits of our deconvolved data to a blexponentlal rather than a single exponential model. 

Wolfbeis, O. S. Urbano, E. Syntheses of fluorescent dyes. XIV. Standards for fluorescence measurements in the near neutral pH-range. J. Heterocycl. Ghent. 1982, 19, 841-843. 

This analysis, abbreviated as FIA for Fluorescent Indicator Adsorption, is standardized as ASTM D 1319 and AFNOR M 07-024. It is limited to fractions whose final boiling points are lower than 315°C, i.e., applicable to gasolines and kerosenes. We mention it here because it is still the generally accepted method for the determination of olefins. 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

Precision When the analyte s concentration is well above the detection limit, the relative standard deviation for fluorescence is usually 0.5-2%. The limiting instrumental factor affecting precision is the stability of the excitation source. The precision for phosphorescence is often limited by reproducibility in preparing samples for analysis, with relative standard deviations of 5-10% being common. 

Samples of urine are analyzed for riboflavin before and after taking a vitamin tablet containing riboflavin. Concentrations are determined using external standards or by the method of standard additions, fluorescence is monitored at 525 nm using an excitation wavelength of 280 nm. 

The determination of cesium in minerals can be accompHshed by x-ray fluorescence spectrometry or for low ranges associated with geochemical exploration, by atomic absorption, using comparative standards. For low levels of cesium in medical research, the proton induced x-ray emission technique has been developed (40). 

While some video display screens such as liquid crystal, gas plasma or vacuum fluorescent displays do not present the same charged screen hazards as CRTs, this does not imply that they are safe for use in hazardous locations. This requires special design and certification for use with a given flammable atmosphere. Non-certified equipment used in locations classified as hazardous under Article 500 of NFPA 70 National Electrical Code require a purged or pressurized enclosure to control ignition hazards as described in NFPA 496 Standard for Purged and Pressurized Enclosures for Electrical Equipment. The screen in this case is located behind a window in the enclosure. 

Additionally, as a response to rising energy prices and uncertainty of supply, several states adopted appliance efficiency standards. At the federal level, the National Appliance Energy Consei vation Act of 1987 established the first national standards for refrigerators and freezers, furnaces, air conditioners, and other appliances. The Energy Policy Act of 1992 added national standards for incandescent and fluorescent lights, small electric motors, office equipment, and plumbing products. 

Procedure. Measure the fluorescence of each of the above solutions at 445 nm, using that containing 62.0 mL of the dilute quinine solution as standard for the fluorimeter. Use LF2 or an equivalent primary filter (/cx = 350 nm) and gelatin as the secondary filter if using a simple fluorimeter. 

Requirements of Standards. Standards used In the calibration of mlcrospectrofluorometrlc Instrumentation must meet more stringent requirements than those used In macromeasurements. The effect of Increased excitation flux as well as spatial effects under magnification make many macromeasurement standards unsatisfactory for micromeasurements. Following are some of the requirements for fluorescence standards that are strongly Influenced by a change from the macro- to the micro-environment. 

The derivatization process (5) is accomplished in aqueous media at basic pH (pH 7-10) in a matter of approximately 15 min to yield a 2-cyanobenz[f]isoindole (CBI), which is stable for 10 to 12 hr in solution. As shown in Figure 1, the absorption characteristics of the CBI adducts are also readily accessible for assay by standard fluorescence or ultraviolet detection. In addition to the absorption between 200 and 300 nm, there are two maxima in the visible spectrum at approximately 420 and 440 nm accessible for fluorescence or ultraviolet detection. A probable mechanism (5,11) for the CBI formation is illustrated in Scheme 1. 

Instrumentation for fluorescence spectroscopy has been reviewed [8]. For standards in fluorescence spectroscopy, see Miller [138]. Fluorescence detection in HPLC has recently been reviewed [137], Phosphorescence detection of polymer/additive extracts is not being practised. 

The various possible schemes for fluorescence sensing are summarized in Figure 1.1. At present, most fluorescence assays are based on the standard intensity-based methods, in which the intensity of the probe molecule changes in response to the analyte of interest. However, there has been the realization that lifetime-based methods possess intrinsic advantages for chemical sensing. (A more detailed description of... 

R. A. Lampert, L. A. Chewter, D. Phillips, D. V. O Connor, A. J. Roberts, andS. R. Meech, Standards for nanoseconds fluorescence decay time measurements, Anal. Chem. 55, 68-73 (1983). 

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