Ultraviolet-visible fluorescence instrumentation

Optical instruments A broad term for instruments that measure absorption, emission, or fluorescence by analyte species based on ultraviolet, visible, or infrared radiation. 

Spectrometric methods Methods based on the absorption, emission, or fluorescence of electromagnetic radiation that is related to the amount of analyte in the sample. Spectrophotometer A spectrometer designed for the measurement of the absorption of ultraviolet, visible, or infrared radiation. The instrument includes a source of radiation, a monochromator, and an electrical means of measuring the ratio of the intensities of the sample and reference beams. 

Optical spectroscopic methods are based on six phenomena (1) absorption, (2) fluorescence. (. ) phosphorescence. (4) scattering. (5) emission, and (6) chemiluminescence. Although the instruments for measuring each differ sontetehat in configurai ion, nio.sl of their ha -sic components are remarkably similar f urthermore, the required properties of these components are the same regardless of whether they are applied to the ultraviolet. visible, or infrared portion of the spectrum. ... 

We use the term colorimeter to designate an instrument for absorption measurements in which the human eye serves as the detector using one or more color-comparison standards. A photometer consists of a source, a filler, and a photoelectric transducer as well as a signal processor and readout. Note that some scientists and instrument manufacturers refer to photometers as colorimeters or photoelectric colorimeters. Filter photometers are commercially available for absorption measurements in the ultraviolet, visible, and infrared regions, as well as emission and fluorescence in the first two wavelength regions. Photometers designed for fluorescence measurements arc also called fluorometers. 

The phenomenon of fluorescence has been synonymous with ultraviolet (UV) and visible spectroscopy rather than near-infrared (near-IR) spectroscopy from the beginning of the subject. This fact is evidenced in definitive texts which also provide useful background information for this volume (see, e.g., Refs. 1-6). Consequently, our understanding of the many molecular phenomena which can be studied with fluorescence techniques, e.g., excimer formation, energy transfer, diffusion, and rotation, is based on measurements made in the UV/visible. Historically, this emphasis was undoubtedly due to the spectral response of the eye and the availability of suitable sources and detectors for the UV/visible in contrast to the lack of equivalent instrumentation for the IR. Nevertheless, there are a few notable exceptions to the prevalence of UV/visible techniques in fluorescence such as the near-IR study of chlorophyll(7) and singlet oxygen,<8) which have been ongoing for some years. 

Phosphorimetric methods have been used to determine such substances as nucleic acids, amino acids, and enzymes. However, this is not a widely used method since it cannot be run at room temperature. Measurements are usually performed with liquid nitrogen to prevent degradation due to collision deactivation. Fluorometric methods are used to determine both inorganic and organic species. Instruments used for measuring fluorescence and phosphorescence are fluorometers and spectrofluorometers, respectively. These instruments are similar to ultraviolet and visible spectrometers,... 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Section I covers the more conventional equipment available for analytical scientists. I have used a unified means of illustrating the composition of instruments over the five chapters in this section. This system describes each piece of equipment in terms of five modules - source, sample, discriminator, detector and output device. I believe this system allows for easily comparing and contrasting of instruments across the various categories, as opposed to other texts where different instrument types are represented by different schematic styles. Chapter 2 in this section describes the spectroscopic techniques of visible and ultraviolet spectrophotometry, near infrared, mid-infrared and Raman spectrometry, fluorescence and phosphorescence, nuclear magnetic resonance, mass spectrometry and, finally, a section on atomic spectrometric techniques. I have used the aspirin molecule as an example all the way through this section so that the spectral data obtained from each... 

Sources that emit a few discrete lines find wide use in atomic absorption spectroscopy, atomic and molecular fluorescence spectroscopy, artd Raman spectroscopy (refractomeiry and polarimciry also u.se line sources). The familiar mercury and sodium vapor lamps provide a relatively few sharp lines in the ultraviolet and visible regions and are used in several spectroscopic instruments. I loilow-cathodc lamps and clectrodelcss discharge lamps are the most important line sourcc.s for atomic absorption and fluorescence methods. Discussion of such sources is deferred to Section 9H- . 

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