Direct Spectrophotometric Measurement

Aromatic acids absorb well and methods for detecting these anions are powerful. The usefulness of direct UV detection can be considered to be limited because sulfate is not detected by UV, and chloride, phosphate and others are difficult to detect. Sulfate is probably the most widely analyzed anion by ion chromatography so this is a serious limitation. On the other hand, anions that are difficult to detect make ideal eluent anions. 

The absorbance of metal chloride complexes in the ultraviolet spectrsil region has been used extensively to automatically detect metal ions in liquid chromatography [24-27], The absorption wavelength maxima of the metal chloride complexes are shown in Table 4.3. Metal EDTA complexes also absorb quite well. 

Alkali metal ions are not detected by UV. However, many anions do absorb at lower wavelengths. A list of anions and their detection wavelengths is shown in Table 4.2. 

Imanari et al. have reported a spectrophotometric detection of many inorganic anions using a post-column reactor [48]. A stream of ferric perchlorate, which is 

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Iyer, K. S. and Klee, W. A. 1973. Direct spectrophotometric measurement of the rate of reduction of disulfide bonds. Reactivity of the disulfide bonds of bovine a-lactalbumin. J. Biol. Chem. 248, 707-710. 

Biochemical research often requires the quantitative measurement of protein concentrations in solutions. Several techniques have been developed however, most have limitations because either they are not sensitive enough or they are based on reactions with specific amino acids in the protein. Since the amino acid content varies from protein to protein, no single assay will be suitable for all proteins. In this section we discuss five assays three older, classical methods that are occasionally used today and two newer methods that are widely used. In four of the methods, chemical reagents are added to protein solutions to develop a color whose intensity is measured in a spectrophotometer. A standard protein of known concentration is also treated with the same reagents and a calibration curve is constructed. The other assay relies on a direct spectrophotometric measurement. None of the methods is perfect because each is dependent on the amino acid content of the protein. However, each will provide a satisfactory result if the proper experimental conditions are used and/or a suitable standard protein is chosen. Other important factors in method selection include the sensitivity and accuracy desired, the presence of interfering substances, and the time available for the assay. The various methods are compared in Table 2.3. 

A survey of normal and derivative spectra of numerous vitamins, including vitamin Bg, has been reported. The second derivative spectra of these vitamins are especially suitable for their quantitative determinations (132). Quantitative determination by derivative spectroscopy is superior to direct spectrophotometric measurement in many problem situations. 

The fact that proteins contain chromophoric residues that absorb in the near and far UV can be used to determine protein concentrations by direct spectrophotometric measurements (Manchester 1996). The advantage of this method is that it does not destroy the protein, however the procedure is not very sensitive in the near-UV (280 nm), and is very subject to interference in the far-UV (205 nm) furthermore, exact determination of concentrations from near-UV data do require knowledge of the amino add composition. 

Analysis of the Decomposition Products of Hydroperoxides. Some authors have monitored formation of some of the decomposition products of the lipid hydroperoxides. Direct spectrophotometric measurements of the formation of oxo-octadecadienoic acids at 280 nm are possible , as are measurements of secondary oxidation products like a-diketones and unsaturated ketones at 268 nm. The formation of various aldehyde products of lipid peroxide decomposition can be monitored by reacting them with 2,4-dinitrophenylhydrazine and, after HPLC separation, measuring at 360-380 mn the DNPH derivatives formed , althongh the sensitivity of this particular technique makes it very susceptible to interference. 

These systems have been extensively studied, particularly the last. They have considerable attractions for fundamental studies, because the more or less transparent and mobile systems permit spectroscopic studies. Thus catalytic activity may be easily followed by direct spectrophotometric measurement of substrate or product... 

The tocopherols can be determined planimetrically after having been clearly separated on the plate from contaminants and visualised with molybdophosphoric acid [118]. Direct spectrophotometric measurement of remission of the unsprayed spots seems also possible now. 

H. Nagatani and H. Watarai, Direct spectrophotometric measurement of demetallation kinetics of tetraphenylporphyrinato zinc(II) at the liquid-liquid interface by a centrifugal liquid membrane method. Anal. Chem., 70, 2860-2865 (1998). 

The content of strychnine hydrochloride can be determined either by a direct spectrophotometric measurement at 254 mix on a 1 in 10 dilution in N hydrochloric acid, E(1 per cent, 1 cm) — 310 or by the colorimetric method of Rasmussen given above after extraction of the alkaloid from 20 ml, as given under Injection of Strychnine, and diluting to 10 ml before using the general method. 

In flame spectrophotometric measurements we are concerned with solutions having very small concentrations of the element to be determined. It follows that the standard solutions which will be required for the analyses must also contain very small concentrations of the relevant elements, and it is rarely practicable to prepare the standard solutions by weighing out directly the required reference substance. The usual practice therefore is to prepare stock solutions which contain about 1000 ig mL 1 of the required element, and then the working standard solutions are prepared by suitable dilution of the stock solutions. Solutions which contain less than 10 igmL 1 are often found to deteriorate on standing owing to adsorption of the solute on to the walls of glass vessels. Consequently, standard solutions in which the solute concentration is of this order should not be stored for more than 1 to 2 days. 

Complex formation constants could also be determined directly from UV spectrophotometric measurements. Addition of tert.-butyl hydroperoxide to a solution of nitroxide I in heptane at RT causes a shift of the characteristic absorption band of NO at 460 nm to lower wavelengths (Fig. 9). This displacement allows calculation of a complex equilibrium constant of 5 1 1/Mol. Addition of amine II to the same solution causes reverse shift of theC NO" absorption band. From this one can estimate a complex formation constant for amine II and +00H of 12 5 1/Mol (23 2 1/Mol was obtained for tert.-butyl hydroperoxide and 2,2,6,6-tetramethylpipe-ridine in ref. 64b). Further confirmation for an interaction between hindered amines and hydroperoxides is supplied by NMR measurements. Figure 10a shows part of the +00H spectrum in toluene-dg (concentration 0.2 Mol/1) with the signal for the hydroperoxy proton at 6.7 ppm. Addition of as little as 0.002 Mol/1 of tetra-methylpiperidine to the same solution results in a displacement and marked broadening of the band (Fig. 10b). 

Direct spectrophotometric methods have been proposed for both particulate and dissolved lignosulfonic materials. Kloster [436] used the Folin Ciocal-teumethod, which actually measures hydroxylated aromatic compounds. A general review of spectrophotometric methods was published by Bilikova [437]. 

The enzymatic activity in soil is mainly of microbial origin, being derived from intracellular, cell-associated or free enzymes. Only enzymatic activity of ecto-enzymes and free enzymes is used for determination of the diversity of enzyme patterns in soil extracts. Enzymes are the direct mediators for biological catabolism of soil organic and mineral components. Thus, these catalysts provide a meaningful assessment of reaction rates for important soil processes. Enzyme activities can be measured as in situ substrate transformation rates or as potential rates if the focus is more qualitative. Enzyme activities are usually determined by a dye reaction followed by a spectrophotometric measurement. 

In actual practice, where a test or an assay recommends the usage of a Reference Substance, the spectrophotometric measurements are always performed first with the solution prepared from the Reference Substance by the directions provided in the specific monograph and then with the corresponding solution prepared from the substance under examination. Nevertheless, the second measurement must be done immediately after the first, by employing the same cell and the same instrumental parameters. 

The level of quinine in a soft drink can be measured either by determination of the absorbance of an extract of the soft drink after making the product alkaline with ammonia or by detecting it directly spectrophotometrically or fluori-metrically (Egan et al., 1990e). 

Titration calorimetry has been successfully employed in the determination of thermodynamic parameters for complexation (Siimer et al., 1987 Tong et al., 1991a). The technique has the advantage of employing direct calorimetric measurements and has been proposed as the most reliable method (Szejtli, 1982). It should be noted that the information derived from multistep series reactions is macroscopic in nature. In contrast to spectrophotometric methods that provide information concerning only the equilibrium constant(s), titration calorimetry also provides information about the reaction enthalpy that is important in explaining the mechanism involved in the inclusion process. 

The ratio /spectrophotometric measurement, and the value of a is then calculated from eq. (3.5) to yield the desired absorption constant. The numerous absorption constants found in the literature arise from the choice of quantities incorporated in the constant b. Some of the terms most commonly used to express absorption in minerals are summarized in table 3.2. Note that optical densities (O.D.), representing the direct output from many spectrophotometers, lack specificity about sample thickness and element concentrations. Absorption coefficients (a) indicate that sample thicknesses have been measured or estimated. Molar extinction coefficients (e) require chemical analytical data as well as knowledge of sample thicknesses. 

The C-17 side-chain of corticosteroids does not contain a chromophoric group suitable for spectrophotometric measurement, nor does oxidation of the chain lead directly to spectrophotometrically active derivatives. However, the 20-keto group of the 17 a-ketol side-chain, as a chirally perturbed chromophoric group, has an optically active absorbance band in the interval 270-300 nm that is characteristic of the n-7T electronic transition for saturated ketones. An intense positive Cotton effect is observed in the CD spectra see the CD spectra for hydrocortisone and cortisone in Figures 8 and 9. 

Electron transport in cytochromes occurs by direct electron transfer between Fe2+ and Cu+ in cytochromes a and a3. These changes in metal-ion oxidation state lead to changes in the visible absorption spectra of the cytochromes spectrophotometric measurement of these changes allows quantification of the electron flow. 

Figure 8. Renaturation of low molecular weight urokinase observed in samples collected from continuous flow refolding. UK was injected in 9.3 M urea into 2.0 M urea, 20 mM Bis-Tris, pH 7.8 buffer with a gradient from 2.5 mM reduced glut-athione (GSH) to 2.5 mM oxidized glutathione (GSSG). Upper panel Data for GSSG concen-tration are from direct absorbance measurements at the secondary UV detector data for GSH were determined by DTNB titration of collected fractions. Lower panel urokinase activity in collected fractions measured by spectrophotometric assay using S-2444 (12) in a Molecular Devices titerplate reader.

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