Spectrophotometry instrument components

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). 

The principal components of a UV/visible spectrophotometer are shown in Fig. 26.2. High-intensity tungsten bulbs are used as the light source in basic instruments, capable of operating in the visible region (i.e. 400-700 nm). Deuterium lamps are used for UV spectrophotometry (200-400 nm) these... 

Spectrophotometry [ISV] (1881) n. An analytical instrumental technique for measuring color values by the relative intensity of the component spectrum colors. Broadly, a technique which, by measuring the absorption (or reflection) of electromagnetic... 

I n this chapter, we describe the components of a spectrophotometer, some of the physical processes that take place when light is absorbed by molecules, and a few important applications of spectrophotometry in analytical chemistry. New analytical instruments and procedures for medicine and biology, such as the RNA array, are being developed by combining sensitive optical methods with biologically specific recognition elements. 

Despite the fact that direct analysis methods exclude a cost-intensive separation step overall analysis cost may still be high, namely by the need for more sophisticated instrumentation (allowing for a physical rather than chemical separation of components) or extensive application of chemometric techniques. The wide variety of additives that are commercially available and employed complicate spectroscopic data analysis. For multicomponent analysis some kind of physical separation of additive signals is often quite helpful, e.g. based on mobility (as in LR-NMR or NMRI), diffusion coefficient (as in DOSY NMR), thermal behaviour (as in a thermal analysis and pyrolysis techniques) or mass (as in tandem mass spectrometry). The power of signal processing techniques (such as multi-wavelength techniques, derivative spectrophotometry) is also used to the fullest extent. 

Reflectance Spectrophotometry. Spectrophotometric measurement has been sparsely used in the past to quantify skin color for two reasons the existence of many different pigments in the skin and the inconvenience of the equipment as compared to skin colorimeter, which is much smaller and transportable. However, recent developments and improvements in the skin spectrophotometer have made them reliable and sensitive instruments to evaluate skin color, with even higher sensitivity than laser Doppler flow-metry [86]. The method offers the advantage of being able to discriminate between arterial and venous components of the inflammatory process, the oxy- and deoxyhemoglobin giving different signals. 

For determination of the elements, mainly spectrometric techniques are used here. Depending on the kind of element and the expected concentration level, the following methods are applied flame atomic emission spectrometry (flame AES), flame atomic absorption spectrometry (flame AAS), inductively coupled plasma optical emission spectrometry (ICP-OES), electrothermal atomisation (graphite furnace) atomic absorption spectrometry (ETA-AAS), inductively coupled plasma mass spectrometry (ICP-MS), spectrophotometry and segmented flow analysis (SFA). Besides, potentiometry (ion selective electrodes (ISE)) and coulometry will be encountered. In many cases, more than one method is described to determine a component. This provides a reference, as well as an alternative in case of instrumental or analytical problems. 

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