SPECTROPHOTOMETRY AND OTHER OPTICAL METHODS

Also see the practice problems on enzyme assays in the chapters on Spectrophotometry and Other Optical Methods and Isotopes in Biochemistry."... 

This laboratory has examined the many techniques which are available. These range from the classic organic analytical methods of the 19th century through chemical spot tests, solution spectrophotometry, infrared, and other optical spectroscopic techniques through mass spectrometry. Thin-layer chromatography is, by itself, a separation technique which allows identification of the separated components by some appropriate technique. In many cases the patterns obtained may be sufiicient for identification, and in the hands of such workers as... 

Determination of the rare-element content in rock samples is a more difficult analytical problem than determination of the main components. The development of optical spectroscopy, X-ray fluorescence analysis, atomic absorption spectrophotometry (AAS), mass spectrometry and other analytical methods from the middle of the 20 century made careful mapping of the composition of the crust possible, even for the rarer elements. The content of each element is given in the corresponding element chapter. 

Numerous methods have been pubUshed for the determination of trace amounts of tellurium (33—42). Instmmental analytical methods (qv) used to determine trace amounts of tellurium include atomic absorption spectrometry, flame, graphite furnace, and hydride generation inductively coupled argon plasma optical emission spectrometry inductively coupled plasma mass spectrometry neutron activation analysis and spectrophotometry (see Mass spectrometry Spectroscopy, optical). Other instmmental methods include polarography, potentiometry, emission spectroscopy, x-ray diffraction, and x-ray fluorescence. 

This article provides some general remarks on detection requirements for FIA and related techniques and outlines the basic features of the most commonly used detection principles, including optical methods (namely, ultraviolet (UV)-visible spectrophotometry, spectrofluorimetry, chemiluminescence (CL), infrared (IR) spectroscopy, and atomic absorption/emission spectrometry) and electrochemical techniques such as potentiometry, amperometry, voltammetry, and stripping analysis methods. Very few flowing stream applications involve other detection techniques. In this respect, measurement of physical properties such as the refractive index, surface tension, and optical rotation, as well as the a-, //-, or y-emission of radionuclides, should be underlined. Piezoelectric quartz crystal detectors, thermal lens spectroscopy, photoacoustic spectroscopy, surface-enhanced Raman spectroscopy, and conductometric detection have also been coupled to flow systems, with notable advantages in terms of automation, precision, and sampling rate in comparison with the manual counterparts. 

As described in the article on the theory of surface plasmon resonance, surface plasmons create a surface-bound evanescent electromagnetic wave which propagates along the surface of an active medium (usually a thin metallic film), with the electric field intensity maximized at this surface and diminishing exponentially on both sides of the interface. As a consequence of this property, the phenomenon has been utilized extensively in studies of surfaces and of thin dielectric films deposited on the active medium. Although numerous other optical techniques have also been applied to such systems (e.g. ellipsometry, interferometry, spectrophotometry, and microscopy the surface plasmon resonance (SPR) method has some important advantages over all other optical techniques, as follows. The method utilizes a relatively simple optical system, it has a superior sensitivity, and the complete system of measurement is located on the side of the apparatus that is remote from the sample, and thus there is no optical interference from the bulk medium. Furthermore, the surfaces of the sample need no extra treatment to increase... 

Provided that the other precautions commonly employed in analyses by spectrophotometry are observed, and that the spectral characteristics of the compound are favourable, results of high accuracy can be expected if a constant temperature is employed for both the pH and the optical density measurements. Jacketed cell holders which can be connected to a thermostatically controlled bath are commercially available for this purpose. If a pH meter is used in the preparation of the buffer solutions, the precision of the method will be determined largely by its performance. Similarly the accuracy of the results will be a function of the accuracy with which it is possible to use a glass electrode, and the same limitations will apply to this method as to the potentiometric method (see p. 38). 

BeryUium aUoys ate usuaUy analyzed by optical emission or atomic absorption spectrophotometry. Low voltage spark emission spectrometry is used for the analysis of most copper-beryUium aUoys. Spectral interferences, other inter-element effects, metaUurgical effects, and sample inhomogeneity can degrade accuracy and precision and must be considered when constmcting a method (17). 

The pulse radiolysis method has been described in detail in some of the early papers (22, 22), in a brief review of the subject (23), and in a current comprehensive review (14). It is, in brief, a fast reaction method in which the external perturbation applied to the system is a microsecond pulse of electrons. The current is sufficiently high to produce an instantaneous concentration of transient species high enough to be observed by fast measurement of the optical absorption. Spectra may be recorded either photographically or spectrophotometrically. The kinetics are studied by fast spectrophotometry. Since a perturbing pulse as short as 0.4 /xsec. has been used, the time resolution has approached 10-7 sec. The flash photolysis method used in some of the other studies (27, 15) has been reviewed in detail (24). 

Analytical techniques used for clinical trace metal analysis include photometry, atomic absorption spectrophotometry (AAS), inductively coupled plasma optical emission (ICP-OES), and inductively coupled plasma mass spectrometry (ICP-MS). Other techniques, such as neutron activation analysis (NAA) and x-ray fluorescence (XRF), and electrochemical methods, such as anodic stripping voltammetry (ASV), are used less commonly For example. NAA requires a nuclear irradiation facility and is not readily available and ASV requires completely mineralized solutions for analysis, which is a time-consuming process. 

Similarly, bilirubin and methotrexate can be determined in serum with a Hber-optic system terminated in a 19-gauge hypodermic needle and a reflective cap at its end so to produce a small absorbance cell [50]. Although these methods usually do not yield absolute analyte concentrations owing to background absorption or fluorescence of serum, they do reflect relative concentration changes sufflciently correctly. This kind of sensor was expected to be applicable also to the photometric determination of other important clinical analytes such as drugs, toxins, and biomolecules, but the limited selectivity and sensitivity of spectrophotometry will possibly also limit the scope of the method when applied directly to serum or whole blood. 

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