Fluorescence spectrophotometer

All samples were monitored using a Perkin-Elmer 650-10S Fluorescence Spectrophotometer. Fluorescence excitation and emission wavelengths for the PAHs in this study were obtained from Berlman (24). The resulting spectra were analyzed using an Apple 11+ computer by integrating peak areas to determine total changes in... 

Retzik, M. and Froehlich, P., Extending the capability of luminescence spectroscopy with a rapid-scanning fluorescence spectrophotometer, Am. Lab., March, 68, 1992. 

During the course of these studies, it was found that fluorescence intensity from the polymeric films rapidly decreased on continued excitation in a fluorescence spectrophotometer (ca. 30% loss in 1 min for L). Herein, we (1) elaborate further upon the fluorescence loss studies, (2) provide direct evidence for RET from fluorescence lifetime measurements, and (3) present preliminary findings on the photochemistry of model compounds for polymer 1. The results support the conclusion, from previous studies, that the effectiveness of added stabilizer decreases with time due to formation of a photoproduct(s) from the polymer which competes in RET, and is less able to dissipate the resulting excitation energy.1... 

In order to determine the effect of air on fluorescence loss, free films of polymer 1 (15 ym thick) were placed in a quartz cuvette, which was evacuated prior to excitation in the fluorescence spectrophotometer. Although the initial loss constant was not determined accurately, both constants (entry 4) were substantially smaller in vacuo relative to air. Fluorescence loss from correspondingly thick films in air is provided in entry 5. 

Ultraviolet absorption spectra were obtained from a Cary 118C Spectrophotometer. Luminescence measurements were obtained from a Perkin-Elmer Model MPF-3 Fluorescence Spectrophotometer equipped with Corrected Spectra, Phosphorescence and Front Surface Accessories. A Tektronix Model 510N Storage Oscilloscope was used for luminescence lifetime measurements. Fiber irradiation photolyses were carried out in a Rayonet Type RS Model RPR-208 Preparative Photochemical Reactor equipped with a MGR-100 Merry-go-Round assembly. 

Using a Perkin-Elmejj Model 204 fluorescence spectrophotometer, no fluorescence was found at either 1 or 1000 ug halcinonide per mL of methanol. No fluorescence was induced in 0.1M hydrochloric acid or 0.1M sodium hydroxide. 

Fogli et al. developed and validated an HPLC method with fluorescence detection for simultaneous routine TDM of anthracyclines and their metabolites.27 They coupled a Waters LC Module I Plus system equipped with a WISP 416 autosampler with a Model 474 scanning fluorescence spectrophotometer. The stationary phase was a Supelcosil LC-CN column (250 x 4.6 mm, 5 /um particle size) with a /iBondapak-CN guard column. The mobile phase consisted of 50mM monobasic sodium phosphate buffer and acetonitrile (65 35 v/v), adjusted to pH 4.0 with phosphoric acid. The flow rate was 1 mL/min. The fluorescence detection was set at excitation wavelengths of 233, 254, and 480 nm and at an emission wavelength of 560 nm. 

The film reacted with adipoyl chloride followed by coupling with 7-hydroxycoumarin was subjected to methanolysis at 1 N HC1 and 60°C. The regenerated coumarin was assayed at pH 10 by fluorescence spectroscopy at an excitation wavelength of 329 nm and an emission wavelength of 455 nm. A Hitachi MPF-4 Fluorescence Spectrophotometer was used for all fluorescence measurements. 

For a fluorescence detector, quinine sulfate is used as the standard compound. The flow cell is filled with a standard solution and the fluorescence intensity is measured. The value is compared with that measured by a fluorescence spectrophotometer. This standard solution is also used for fixing the wavelength and position of the flow cell. The Raman spectrum of water can also be used for this purpose. 

Total particulate matter in cigarette smoke Extract particulate matter with NaOH, buffer to a pH 4.6 HPLC-fluorescence spectrophotometer 0.3 mg/L 91% at 20-30 pg... 

Figure 2. General design features of a fluorescence spectrophotometer.

Any advanced absorbance/fluorescence spectrophotometer designed for routine acquistion of absorption or emission on the subsecond time scale. The basic goal is to obtain a series of complete UV/visible or fluorescence spectra as a function of time, usually after samples are mixed in a stopped-flow device. Such data help the investigator to infer the most likely structures of transient intermediates whose electronic spectra or fluorescence spectra can be determined by deconvoluting the spectra with appropriate reaction kinetic simulation software or by some other global analysis method (Fig. 1). 

A major contributing factor to the increased sensitivity of the improved HPLC system over that originally described (5 ) is the detector. The original method utilized a fluorescence spectrophotometer adapted for HPLC detection by use of a fabricated 40 ul flow cell. The present system utilizes a highly sensitive HPLC fluorescence detector and this contributes greatly to the improved detection limits. 

Instrumentation. The steady-state fluorescence spectra were measured with Perkin-Elmer MPF-44B fluorescence spectrophotometer. The single-photon counting instrument for fluorescence lifetime measurements was assembled in-house from components obtained from EG G ORTEC. A PRA-510B light pulser filled with gas was used as the excitation source. Instrument response function was obtained with DuPont Ludox scatter solution at the excitation wavelength. 

Emission spectra were recorded on a fluorescence spectrophotometer (Hitachi, MPF-2A) with the excitation wavelength = 337 nm. 

HPLC injector with a 50-pl sample loop fluorescence spectrophotometer, operating at an emission wavelength of 460 nm with an excitation wavelength of 300 nm. 

Dry Oil Oxidation Fluorescence may be measured within or over an oil or within or over an emulsion of lipid in water providing the pH is below 5.5. When plate fluorescence is to be measured, solid sample fluorescence spectrophotometry is necessary (23). A Hitachi Solid Sample Holder Attachment for Model MPF-2A Hitachi-Perkin Elmer Fluorescence Spectrophotometer (Figure 4) was... 

Soil screening for metal contaminants with an x-ray fluorescence spectrophotometer is a technique used in EPA Method 6200. The method is applicable for 26 analytes listed with their detection limits in an interference-free matrix in Table 3.10. Light elements, such as lithium, sodium, aluminum, silicon, magnesium, beryllium, phosphorus, cannot be detected with XRF. 

A Perkin-Elmer MPF-2A Fluorescence Spectrophotometer was used to determine the excitation and emission wavelengths required for achieving maximum fluorescence intensity for the pesticides studied. The MPF-2A contained a 150 watt xenon arc and an excitation monochromator with a grating blazed at 300 nm as the excitation unit a Hamamatsu R 777 photomultiplier tube (sensitivity range 185 - 850 nm) and an emission monochromator grating blazed at 300 nm as the emission detection unit. A DuPont Model 848 Liquid Chromatograph was used for HPLC (Figure 2). The accessory injection device included a Rheodyne Model 70-10 six-port sample injection valve fitted with a 20 y liter sample loop. A Whatman HPLC column 4.6 mm x 25 cm that contained Partisil PXS 1025 PAC (a bonded cyano-amino polar phase unspecified by the manufacturer) was used with various mobile phases at ambient temperature and a flowrate of 1.25 ml/minute. 

Measure enhancement at 450 nm in a fluorescence spectrophotometer with an excitation of 360 nm (slit width 20 nm). 

Auxiliary Instruments. Auxiliary instruments can be used on the fly as special detectors, or analytes can be trapped and taken to other instruments. Instruments that have been used with chromatography include the mass spectrometer (MS), the infrared spectrometer (IR), the nuclear magnetic resonance spectrometer (NMR), the polarograph, the fluorescence spectrophotometer, and the Raman spectrometer, among others. The two most popular ones are MS and IR, and they will be discussed in more detail in Chapter 11. In the beginning of this chapter we noted the utility iof GC/MS and LC/MS. 

Fig. 14.4. Effect of UV exposure on de novo amyloid fibril formation of prion protein, P2-microglobulin and a-synuclein. Amyloid fibril formation of (a) prion protein (b) p2-microglobulin and (c) a-synuclein. fn each panel, (filled square) represents proteins that are not exposed to UV light, and (filled circle) the UV-exposed protein. The fibril formation was monitored by ThT fluorescence. An aliquot of the sample was withdrawn at different time points and added to 0.5 ml of 10 pM ThT in 50 mM glycine-NaOH buffer (pH 8.5), and the fluorescence intensity at 485 nm with excitation wavelength set at 445 nm was measured using a Fluorolog FL3-22 fluorescence spectrophotometer. The UV-exposed proteins failed to form amyloid fibril de novo...

Fluorescence polarisation immunoassay, 154 Fluorescence spectrophotometers, 233 standardisation, 234... 

Fig. 26.4 Components of a fluorimeter (fluorescence spectrophotometer). Note that sample cells for fluorimetry must have clear sides all round.

The principal components of a fluorescence spectrophotometer (fluorimeter) are shown in Fig. 26.4. The instrument contains two monochromators, one to select the excitation wavelength and the other to monitor the light emitted, usually at 90° to the incident beam (though light is actually emitted in aU directions). As an example, the wavelengths used to measure the highly fluorescent compound naphthalene are 270 nm (excitation) and 340 nm (emission). Some examples of molecules with intrinsic fluorescence are given in Table 26.1. 

Steady-state emission spectra were recorded with a fluorescence spectrophotometer (Model 850, Hitachi Ltd.). Emission lifetime measurements were carried out using laser excitation pulses and a time-correlated single-photon counting system as described elsewhere. 

To promote good lipid accumulation in the cells, the culture was transferred to a nitrogen-absent medium. The rate of depletion of soluble potassium nitrate in culture medium was determined with Hitachi HPLC L6000. And the fluorescent intensity of the Nile Red-dyed microalgal cells was measured by Hitachi fluorescent spectrophotometer UV-2000. Stainability with Nile Red was determined as fluorescent intensity at 575 nm per cell density. After the cultivation, cells were harvested by centrifugation, washed twice with distilled water and lyophilized. Cellular lipid was extracted by the method of Bligh and Dyer [5] and determined gravimetrically. 

Hoechst 33258 binds to the minor groove of DNA. When 365 nm light (long UV) excites this dye, fluorescence results and can be measured by mini-fluorometer (as described here), fluorescence spectrophotometer (14) or fluorescence microtiter plate-reader. We have also used a microtiter (96-well) based assay 10 pL sample or standard (range 0-50 pg/mL DNA) and 100 pL Hoechst (1 pg/mL) per well. 

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