Analysis of Spectrophotometric Data

In this chapter you ll learn how to handle calibration curves that are not straight lines, how to analyze the spectra of mixtures of components and how to deconvolute a spectrum into its individual absorption bands. 

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The dimer U2(X1), U204, was detected by its absorption at 737 m/t. Analysis of spectrophotometric data using the rate law... 

A suitable chemometric analysis of spectrophotometric data was used for simultaneous determinations of MSG, GMP, and IMP. The overlapping of absorption spectra from each flavor enhancer was resolved by multicomponent analysis using chemometric techniques. 

H. Gampp, M. Maeder, C.J. Meyer and A.D. Zuberbuhler, Calculation of equilibrium constants from multiwavelength spectroscopic data. Ill Model-free analysis of spectrophotometric and ESR titrations. Talanta, 32 (1985) 1133-1139. 

To determine the effect of acid catalysed decomposition of NADH on the electrochemical response in our experiments, the decrease in oxidation current for NADH was recorded as a function of time. The results of this experiment were compared with the decrease in NADH concentration as spectrophotometrically determined. The rates of decrease of the current and the concentration of NADH are both first-order and occur on similar timescales (Fig. 2.14). Analysis of the data for the two experiments provide first-order rate constants of 1.68 and 1.16 x 10-4 s-1 for the electrochemical and spectrophotometric measurements, respectively. The small difference between these two constants can be explained by the additional consumption of NADH by reaction at the electrode during the electrochemical measurement. This electrochemical process is also a first-order rate process, and the extent of the effect can be determined by using the treatment of Hitchman and Albery [50] for electrolysis using a rotating disc electrode. The results are consistent with the observed difference in the two rate constants. 

More recently, Albery et al. [203] carried out an improved analysis of experimental data for dilute acidic solutions which was based on an extension of McCauley and King s treatment [204] of kinetic data for the reaction of diazoacetone with water and halide ions. Albery et al. determined rate coefficients as well as product ratios, p - (moles ethyl halogenoacetate formed)/(moles ethyl glycollate formed), with the aid of UV spectrophotometric and gas—liquid chromatographic methods. In eqn. (48), the logarithms of the activity coefficient ratios were considered to be linearly dependent on the ionic strength, viz. 

Equilibrium studies involving double-stranded trihelicates have demonstrated the co-operative nature of metal ion binding. A spectrophotometric titration investigation of the stepwise binding of copper(I) to a (substituted) tris-bipyridyl ligand system of the present type in dichloromethane-acetonitrile (1 1) indicated the formation of a single, well-defined product. From analysis of the data, the self-... 

The advent of low priced powerful microcomputers has enabled easy processing of spectrophotometric data. In addition, fast scanning detectors have allowed elimination of errors resulting from poor time stability when a dual wavelength method is used with a single wavelength spectrophotometer. Modem multiwavelength analysis utilizes the reversed matrix representation of Beer-Lambert s law. This can be combined with chemometric techniques such as principal component analysis (PCA) and partial least squares (PLS). 

Comparative studies of the widely employed spectrophotometric readings at the Soret and Q bands (405 and 630 nm, respectively) and the elemental analysis of copper and nitrogen showed that the spectrophotometric assay based only on the Soret band can overestimate the purity of a preparation. Erroneous data were attributed to an increase in absorptivity at the Soret band when other colored compounds like metal-free analogs and carotenoids are present. Indeed, copper-free chlorin e6 exhibits a specific absorbance 3.6 times greater than that of its coppered counterpart. Therefore, measurements at the Q band (630 mn) and the establishment of the S Q ratio are preferred. 

Maeder, M., Gampp, H. Spectrophotometric data redudion by eigenvedor analysis for equilibrium and kinetic smdies and a new method of fitting exponentials. Anal. Chim. Acta 1980, 122, 303-313. 

Apparently similar flowstream universal buffers have been developed by Alibrandi and others [128,129] for assessing kinetic parameters, such as the pH-dependent hydrolysis of acetylsalicylic acid. The pH-time curves are not as linear as in the SGA system. Other reports of continuous flow pH gradient spectrophotometric data have been described, with application to rank-deficient resolution of solution species, where the number of components detected by rank analysis is lower than the real number of components of the system [130]. The linear pH-time gradient was established in the flowstream containing 25 mM H3PO4 by the continuous addition of 100 mM Na3P04. 

COMPOUNDING OF ERRORS. Data collected in an experiment seldom involves a single operation, a single adjustment, or a single experimental determination. For example, in studies of an enzyme-catalyzed reaction, one must separately prepare stock solutions of enzyme and substrate, one must then mix these and other components to arrive at desired assay concentrations, followed by spectrophotometric determinations of reaction rates. A Lowry determination of protein or enzyme concentration has its own error, as does the spectrophotometric determination of ATP that is based on a known molar absorptivity. All operations are subject to error, and the error for the entire set of operations performed in the course of an experiment is said to involve the compounding of errors. In some circumstances, the experimenter may want to conduct an error analysis to assess the contributions of statistical uncertainties arising in component operations to the error of the entire set of operations. Knowledge of standard deviations from component operations can also be utilized to estimate the overall experimental error. 

Four analysts obtain the following data for a spectrophotometric analysis of an injection containing the local anaesthetic bupivacaine. The stated content of the injection is 0.25% weight in volume (w/v). 

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