Concentration difference spectrophotometry

In turbid or cloudy solutions, in which absorbance is affected by scattering of the sample, two absorbance measurements are made one at and the other at a different wavelength where the analyte does not absorb. Then, the difference being proportional to concentration of the analyte, the analyte concentration can be calculated. Difference spectrophotometry is used in the determination of constituents in tablets, complex pharmaceutical preparations, plant extracts, syrups, biological matrices such as blood and serum, injectable oil preparations, and the like. For this technique, in addition to other general requirements such as well-matched cells that are positioned accurately, the solution should be made homogeneous, and the instrumental stray radiation at the wavelength of interest should be extremely small. 

As previously discussed, all nutrients are quantified by spectrophotometry. Calibration in the context of spectrophotometric analysis means comparison of the sample absorption after chemical reaction with the absorption of a standard (Le., an artificial sample) of known concentration, which has been treated in exactly the same manner. The reliability of calibrations decreases with increasing concentration differences between sample and standard. Consequently, the best calibration and thus the best analytical results are obtained with exactly matched sample and calibration ranges. Since the methods for calibration, blank determination and calculation of results follow the same procedures for all nutrients discussed here, the different steps to be taken simultaneously are outlined in the following. 

Figure 6.4 displays the denaturation transition curve of chymotrypsinogen at 293 nm in urea. Variations of the extinction coefficient with urea concentration has also been observed for the native and for the unfolded states. These properties have been used to estimate the number of exposed or buried chromophores either by difference spectrophotometry or by the method of solvent perturbation (Herskovits and Laskowski, 1962, 1968 Herskovits, 1965,1967). 

In Figure 5.1, the fraction of iron whose concentration is being reported is identified as the total dissolved iron concentration. In practice, this fraction is operationally defined by the analytical method used in its measurement. For the data in Figure 5.1, the total dissolved iron concentration was determined by filtration to remove the solid iron, followed by colorimetric analysis to quantify the solutes. Another analytical technique, such as filtration followed by atomic absorption spectrophotometry, might yield a different total dissolved concentration, so it is important to be aware of the analytical methods used. To address this issue, marine chemists engage in intercalibration experiments to assess differences in results from various analytical methods. 

Kinetic study of this reaction usually requires sampling the polymerizing mixture and analyzing for the concentrations of the various reaction species at different polymerization times. Vofsi and Tobolsky in 1965 reported the use of radioactively tagged initiator (10), while Saegusa amd coworkers in 1968 developed a "phenoxy end-capping" method in which the oxonium ion is trapped with sodium phenoxide and the derived phenyl ether at the polymer chain end quantitatively determined by UV spectrophotometry (11). 

A technically simple method for determination of aqueous H2O2 in high concentration is by NIR spectrophotometry, taking advantage of the slight differences in the O-H... 

Mn2+ increased severalfold in concert with these changes. As in the water column, there was no significant difference in Mn2+ determined by ESR spectroscopy and total Mn determined by AA spectrophotometry after acidification. Similarly, there was no difference in Mn concentration between filtered and whole-water samples. All the Mn released into the water column was present as soluble-colloidal Mn2+ species. Although surprising when it occurred, the mobilization of Mn2+ under oxygen actually is in accord with the reports of other groups, who have observed accumulation of Mn2+ under oxic conditions. 

The GECE sensors were used for lead determination in real water samples suspected to be contaminated with lead obtained from water suppliers. The same samples were previously measured by three other methods a potentiometric FIA system with a lead ion-selective-electrode as detector (Pb-ISE) graphite furnace atomic absorption spectrophotometry (AAS) inductively coupled plasma spectroscopy (ICP). The results obtained for lead determination are presented in Table 7.1. The accumulation times are given for each measured sample in the case of DPASV. Calibration plots were used to determine the lead concentration. GEC electrode results were compared with each of the above methods by using paired -Test. The results obtained show that the differences between the results of GECE compared to other methods were not significant. The improvement of the reproducibility of the methods is one of the most important issues in the future research of these materials. 

As was stressed above, in case of direct excitation of the lanthanides, the differences in the absorption spectra between the solvated ion and the complex formed are usually considered as slight but allow nevertheless the use of spectrophotometry for speciation purposes (for a recent example, see Giroux et al. (2000)). In the case of U(VI), the absorption changes observed upon complexation have often been used to determine equilibrium constants (for the classical example of U(VI) hydrolysed species, see Dai et al. (1998), Meinrath (1998)). For curium, no information of this kind is available the low molar absorption coefficient implies the use of solutions with high total curium concentrations, which are almost impossible to handle nowadays due to safety reasons. The absorption spectra of curium are known only for aqueous solutions of HCIO4 (Carnall et al., 1958), HC1, H2SO4 and HNO3 (Pascal, 1962). 

Erk presented a ratio spectra derivative spectrophotometric method and a Vierordt s method for the simultaneous determination of hydrochlorothiazide in binary mixtures with benazepril hydrochloride, triametrene, and cilazapril [19]. A mass of powder equivalent to one tablet was dissolved in 1 1 methanol/0.1 M Hydrochloric acid (solvent A), shaken for 30 min, filtered and the residue was washed with 3x10 mL of solvent A. Combined solutions were diluted to 100 mL with solvent A, and then diluted with methanol for analysis by (i) Vierordt s method, and (ii) ratio spectra derivative spectrophotometry. For method (i), the absorbance of the solution was measured at 271.7, 238.1, 234.1, and 210.7 nm for hydrochlorothiazide, benazepril, triametrine, and cilazapril, respectively, and the corresponding concentrations were calculated using simultaneous equations. For method (ii), the absorption spectra of solutions containing different amounts of hydrochlorothiazide in a... 

The adsorption of vitamins was carried out from ethanol solution at 20°C for 2 h onto unmodified and modified fumed silica. The extent of modification by -Si(CH3)3 groups is given by the TMS loading 6 rMs = 0.10, 0.40 and 0.70. The solution of a vitamin (10 ml) with different initial concentrations Co was mixed with an adsorbent (0.05 g). After adsorption for 2 h, the equilibrium concentration of the vitamin (Ceq) was determined by UV spectrophotometry. The difference in the maximum of the absorption of the solution before and after adsorption at k = 284 nm (Vitamin E) or 250 nm (Vitamin C), was used to... 

Redox titrations involve determination of equilibrium between the enzyme and a redox agent of known redox potential. The method requires a redox agent with redox potential close to the protein of interest, to ensure reversibility. The protein is exposed to different concentrations of the redox agent, and once equilibrium is attained, the half cell potential is measured with electrodes and the oxidation-reduction state of the proteins is measured by some physical technique, usually UV-Vis spectrophotometry. The concentration of the oxidized and reduced forms is determined at isosbestic points, and thus spectral characterization of redox species (ferric enzyme,... 

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