Ultraviolet/visible spectrophotometr

P. W. Alexander and A. Thalib, Nonsegmented Rapid-Flow Analysis with Ultraviolet/Visible Spectrophotometric Determination for Short Sampling Times. AnaL Chem., 55 (1983) 497. 

This section provides correlation charts and operational information for the design and interpretation of ultraviolet-visible spectrophotometric (UV-Vis) measurements. While UV-Vis is perhaps not as information-rich as infrared or nuclear magnetic resonance, it nonetheless has value in structure determination and sample identification. Moreover, it is extremely valuable in quantitative work. Typical UV-Vis instruments cover not only the UV and visible spectrum, but the near-infrared as well. Although there is overlap among the ranges, the approximate breakdown is ... 

Although considered a basic technique, ultraviolet-visible (UV-vis) is perhaps the most widely used spectrophotometric technique for the quantitative analysis of pure chemical substances such as APIs in pharmaceutical analysis. For pharmaceutical dosage forms that do not present significant matrix interference, quantitative UV-vis measurements may also be made directly.114,115 It is estimated that UV-vis-based methods account for 10% of pharmacopoeia assays of drug substances and formulated products.116... 

The parameter can change in a vessel being part of the analytical instrument, for example, an ultraviolet-visible (UV-Vis) spectrophotometric cell [39,41,45,14,47, 48], an infrared (IR) cell [42, 46], or a fluorometer cell [45, 51], or a polarimetric tube [27, 49]. It can change in a reactor vessel where the analytical signal can be read in some way, for example using an optical fiber cell for spectrophotometry [52-54] or a conductometric cell [16,34,40]. Another possibility is to transport the solution from the reaction vessel to the analytical instrument by a peristaltic pump [38]. When altenative ways are not practicable, samples can be taken at suitable time intervals and analyzed apart [29,31,35,39,43,50]. 

Although three early papers briefly discussed reactions between methylcobalamin and mercury compounds (30-32), the most systematic investigation has come from Wood and co-workers (33). They proposed the mechanism shown in Fig. 1, with values for the various rate constants presented in Table I. Species 2 and 3, in which the benzimidazole nitrogen no longer bonds to the cobalt atom, are termed base-off compounds, whereas 1 is base-on methylcobalamin and 4 is aquocob(III)alamin, the usual product of aqueous transmethylation by 1. Each one of these species has a unique ultraviolet-visible spectrum, which allows quantitative studies by spectrophotometric techniques to be made (28, 32, 33). The mercuric acetate-1 exchange is so rapid that it must be studied using stopped-flow kinetic techniques (33). 

Ultraviolet-visible (UV-Vis) spectrophotometric detectors are used to monitor chromatographic separations. However, this type of detection offers very little specificity. Element specific detectors are much more useful and important. Atomic absorption spectrometry (AAS), inductively coupled plasma-atomic emission spectroscopy (ICPAES) and inductively coupled plasma-mass spectrometry (ICP-MS) are often used in current studies. The highest sensitivity is achieved by graphite furnace-AAS and ICP-MS. The former is used off-line while the latter is coupled to the chromatographic column and is used on-line . 

Spectrophotometric Techniques Based on Molecular Absorption Radiation Ultraviolet-Visible Spectrophotometry... 

Spectrophotometric methods [22-26], where applicable, have the advantage of allowing a sample to be studied under reaction conditions. Ultraviolet, visible, or infrared spectra can be taken at elevated temperatures and pressures [27]. If a reaction is so fast that sampling is not practical, a pump-around loop with spectrophotometric cell can be hooked to the reactor or, even better, a probe can... 

Spectrophotometry (the measurement of light absorption or transmission), is one of the most valuable analytical techniques available to biochemists. Unknown compounds may be identified by their characteristic absorption spectra in the ultraviolet, visible, or infrared. Concentrations of known compounds in solutions may be determined by measuring the light absorption at one or more wavelengths. Enzyme-catalyzed reactions frequently can be followed by measuring spectrophotometrically the appearance of a product or disappearance of a substrate. 

A few more papers should be mentioned as regards electronic spectra of aromatic nitro compounds nitrobenzene, dinitrobenzene and trinitrobenzene [20] and the work of Barth [21 ] who subjected mono-, di- and tri-nitrobenzenes and toluenes to detailed spectrophotometric ultraviolet-visible studies and developed a quantitative analysis of TNT in the presence of cyclonite, octogene and waxes. 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Ultraviolet-visible spectrophotometry has also been applied to titrimetry. In this case the variation in the absorbance of the analyte with addition of titrant is used to obtain a spectrophotometric profile from which titration end points and/or equilibrium constants, etc., can be determined. This has been applied to the whole range of titrations in which a chromophore is generated. These include acid-base, redox, and complexometric titrations. 

R. W. Burnett, Errors in ultraviolet and visible spectrophotometric measurements caused by multiple reflections in the cell. Anal. Chem, 45 (1973) 383. 

Products were characterized by Fourier transform infrared spectrophotometry-attenuated total reflectance (FTIR-ATR), ultraviolet visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), and broadband dielectric/impedance spectroscopy (BDS). New absorption bands were observed corresponding to the conjugated pol5mieric units by FTIR-ATR and UV-Vis spectrophotometric analysis. The influence of concentration of PEDOT-PSS and PEDOT on the composite electrospun nanofibers was studied by EIS. Morphologies of electrospun nanofibers were also investigated by SEM. 

A spectrophotometric UV-Vis detector is selective, yet its selectivity can be changed simply by changing the wavelength monitored by the detector. Versatility of the detector can be increased by adding a color-forming reagent to the eluent or the column effluent. The fundamental law under which ultraviolet-visible (UV-VIS) detectors operate is the Lambert-Beer law. It can be stated in the following form ... 

Fluorimetric methods have not been used as widely as ultraviolet-visible (UV-Vis) spectrophotometric ones, although they are much more sensitive detection requires only 1% of the amount of pigment necessary in the latter method [70,71]. With this technique, chlorophylls or their derivatives have been detected quantitatively in picomole amounts [72-77]. Very low amounts of chlorophylls a and b can also be determined from their mixtures, as the fluorescence of one is independent of the other [78]. 

Dichromate visible spectrophotometric determination in saturated solutions containing chromates Various inorganic ions ultraviolet spectra of aqueous solu-tions[ ]. 

Spectrophotometric methods may often be applied directly to the solvent extract utilising the absorption of the extracted species in the ultraviolet or visible region. A typical example is the extraction and determination of nickel as dimethylglyoximate in chloroform by measuring the absorption of the complex at 366 nm. Direct measurement of absorbance may also be made with appropriate ion association complexes, e.g. the ferroin anionic detergent system, but improved results can sometimes be obtained by developing a chelate complex after extraction. An example is the extraction of uranyl nitrate from nitric acid into tributyl phosphate and the subsequent addition of dibenzoylmethane to the solvent to form a soluble coloured chelate. 

Discussion. The procedure is based on the formation of yellow tetraiodo-antimonate(III) acid (HSbI4) when antimony(III) in sulphuric acid solution is treated with excess of potassium iodide solution. Spectrophotometric measurements may be made at 425 nm in the visible region or, more precisely, at 330 nm in the ultraviolet region. Appreciable amounts of bismuth, copper, lead, nickel, tin, tungstate, and molybdate interfere. 

A common technique for measuring the values has been to employ species that produce anions with useful ultraviolet (UV) or visible (vis) absorbances and then determine the concentrations of these species spectropho tome trie ally. Alternatively, NMR measurements could be employed, but generally they require higher concentrations than the spectrophotometric methods. A hidden assumption in Eq. 5 is that the carbanion is fully dissociated in solution to give a free anion. Of course, most simple salts do fully dissociate in aqueous solution, but this is not necessarily true in the less polar solvents that are typical employed with carbanion salts. For example, dissociation is commonly observed for potassium salts of carbanions in DMSO because the solvent has an exceptionally large dielectric constant (s = 46.7) and solvates cations very well, whereas dissociation occurs to a small extent in common solvents such as DME and THE (dielectric constants of 7.2 and 7.6, respectively). In these situations, the counterion, M+, plays a role in the measurements because it is the relative stability of the ion pairs that determines the position of the equilibrium constant (Eq. 6). 

---