Absorbance ratio method

Erk [20] described a spectrophotometric method for the simultaneous determination of metronidazole and miconazole nitrate in ovules. Five capsules were melted together in a steam bath, the product was cooled and weighed, and the equivalent of one capsule was dissolved to 100 mL in methanol this solution was then diluted 500-fold with methanol. In the first method, the two drugs were determined from their measure d%/dk values at 328.6 and 230.8 nm, respectively, in the first derivative spectrum. The calibration graphs were linear for 6.2—17.5 pg/mL of metronidazole and 0.7—13.5 pg/mL of miconazole nitrate. In the second (absorbance ratio) method, the absorbance was measured at 310.4 nm for metronidazole, at 272 nm for miconazole nitrate and at 280.6 nm (isoabsorptive point). The calibration graphs were linear over the same ranges as in the first method. 

Quantitation was carried out by the simultaneous equation (Method 1) and the absorbance ratio (Method 2). The wavelengths selected for Method 1 were 257.10 and 288.66 nm of both drugs, respectively. In Method 2, two wavelengths 257.10 nm of paracetamol and 284.36 mn, the isobestic point, were selected. Both the methods were validated for linearity, accuracy, and precision. 

The publications of American Cyanamid investigators included instrumental analysis of chemicals in the industrial environment, including two-component mixtures of nitrobenzene and aniline that were of value in industrial hygiene investigations. Details of this absorbance-ratio method were presented at the 4th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy held in March 1953114. 

When a spectral plot is not available, the absorbance ratio method is a satisfactory method for characterizing a real sample, using both spectrophotometric and HPLC assays. In addition, better performance needs to be achieved in carrying out advanced experiments, such as solid-phase extraction and other purification techniques coupled with HPLC quantification, in order to improve the separation properties. " ... 

R. C. Hirt, F. T. Kirg, and R. G. Schmitt, Graphical Absorbance-Ratio Method for Rapid Two-Component Spectrophotometric Analysis, Anal. Ghent. 26, 1270, 1954. 

Graphical Absorbance Ratio Method. The method of Hirt et al. quickly gives the relative concentration of two components without solving simultaneous equations. It is most convenient when the two components spectra exhibit an iso-absorptive point, a wavelength at which the spectra cross each other, showing that the compounds have equal absorptivities. 

The absorbance ratio method (and, indeed, most of the calculations involved in quantitative spectrophotometry was discussed and explained by Ish-Shalom et at. 

An absorbance ratio method permitting the use of solutions of unknown concentration was described by Perry ei aL... 

Mixture of o- and p-toluenesulfonamides determined in ultraviolet by an isoabsorptive point, absorbance ratio method... 

Factor analysis affords a method of determining the number of components in a series of mixtures. This technique has been recently applied to infrared spectroscc y (1). The absorbance ratio method already has had application in determining the component spectra (2,3). A recent refinement of the ratio method suggests a method of internal calibration which allows the quantitative determination of the amounts of each component in the spectra of the amorphous phase (4). 

An absorbance ratio method has been applied to mixtures of simple organic molecules and also to polymers (2,3) to determine the component spectra without separation or isolation. The method can also be applied to determine the relative concentrations of the components without external calibration (4). 

Both the method of continuous variations and the mole-ratio method rely on an extrapolation of absorbance data collected under conditions in which a linear relationship exists between absorbance and the relative amounts of metal and ligand. When a metal-ligand complex is very weak, a plot of absorbance versus Ay or n-J m may be curved, making it impossible to determine the stoichiometry by extrapolation. In this case the slope ratio may be used. 

The molecular absoi ption spectra, registered at a lower temperature (e.g. 700 °C for iodide or chloride of potassium or sodium), enable one to find the absorbance ratio for any pair of wavelengths in the measurement range. These ratios can be used as a correction factor for analytical signal in atomic absoi ption analysis (at atomization temperatures above 2000 °C). The proposed method was tested by determination of beforehand known silicon and iron content in potassium chloride and sodium iodide respectively. The results ai e subject to random error only. 

The absorbance ratio AnlAxi for the solute peak should be close to zero. If it is not, then this suggests that the peak is not what we think it is. For example, there may be another component that elutes at the same time, so the ratio method is a simple way of indicating the purity of the peaks. 

The carbonyl index is not a standard technique, but is a widely used convenient measurement for comparing the relative extent and rate of oxidation in series of related polymer samples. The carbonyl index is determined using mid-infrared spectroscopy. The method is based on determining the absorbance ratio of a carbonyl (vC = 0) band generated as a consequence of oxidation normalised normally to the intensity of an absorption band in the polymer spectrum that is invariant with respect to polymer oxidation. (In an analogous manner, a hydroxyl index may be determined from a determination of the absorbance intensity of a vOH band normalised against an absorbance band that is invariant to the extent of oxidation.) In the text following, two examples of multi-technique studies of polymer oxidation will be discussed briefly each includes a measure of a carbonyl index. 

A general protocol for the use of DCIA for fluorescently labeling proteins that contain sulfhydryl residues may be obtained by following the method discussed for AMCA-HPDP (previous section). After purification of the labeled protein, the F/P ratio of fluorophore incorporation may be determined by measuring its 382nm/280nm absorbance ratio. 

Two differential spectrophotometric methods were used by Chatterjee et al. for the simultaneous analysis of diloxanide furoate and metronidazole in pharmaceutical formulations [24]. The first method involved measurement of the absorbance of a methanolic solution of the two drugs at 259 and 311 nm. Since the absorbance of diloxanide furoate at 311 nm is zero, the concentration of metronidazole is directly measured, and a simple equation based on absorbance ratios is used to calculate the concentration of diloxanide furoate. The second method was a differential spectrophotometric determination based on pH-induced spectral changes, on changing from an acidic to an alkaline solution. A marked bathochromic shift was exhibited by metronidazole, while diloxanide furoate showed a slight hypsochromic shift. The wavelength of maximum absorption difference for diloxanide furoate was 267 nm, where metronidazole did not absorb. Similarly, diloxanide furoate did not interfere with metronidazole at when measured at 322 nm. 

Peak Identity Confirmation by Measurement of Absorbance Ratios. It is known that peak absorbance ratio measurements provide an alternative method of peak identity confirmation to running the sample on two different columns, fraction collection for MS and so on. If Beer s law is obeyed... 

Identification is considerably improved by the use of multiwavelength detection and absorbance ratioing. It was possible to analyse some commercial hair dyes with the method described here. However, several substances were found that could not be identified with the present standard substances. Recent results in this laboratory make a possible improvement of the chromatography probable. Future work will consider this aspect and an increased number of standard substances (17) will be examined. 

One of the major problems in studying polymers quantitatively is the absence of model compounds for the purpose of calibration. A method of obtaining spectra of the components of a mixture spectra is based on obtaining the ratio of absorbances. This method was first used by Hirschfeld 851 for mixtures of components differing in relative concentration. This approach was later generalized but is limited to a rather... 

Oxidation studies have been made for cured epoxy resins and the relative stability of the functional groups was established by following the changes in the absorbance ratios of bands associated with the particular functional group 231>. Hence the most unstable groups can be determined easily and their reactions separated from the more stable units. The irreversible 232) and reversible 2331 effects of moisture on epoxy resin systems have also been studied by FT-IR using the difference spectrum method 234). (Fig. 20) shows the interaction spectra obtained after three cycles of water sorption and redrying. The reversible nature of the interactions are clearly demonstrated. 

For general purpose tracer work, however, and particularly in polymer chemistry, the liquid scintillation counter surpasses all other instruments in its sensitivity and adaptability. There is no question on the author s mind that at the present time such an instrument would be the first choice, particularly where tritium, carbon-14 or sulphur-35 were involved. Samples for assay are dissolved in a phosphor whose major solvent usually consists of toluene, toluene-alcohol, or dioxan. Many polymers and low molecular weight compounds are readily soluble in these solvents. Prospective users should not be deterred by alleged complications due to "variable quench effects" as these effects are readily corrected for via internal or external standards or the channels ratio method (7, 46, 91). Dilution quench corrections, though valid, are tedious and unnecessary. Where samples are insoluble in phosphor they may be suspended (e.g. as gels or as paper cut from chromatograms, etc.) or they can be burnt and the combustion products absorbed in a suitable phosphor solution. A modification of the Schoniger flask combustion technique is particularly suitable for this purpose (43—45). 

The intense absorption of water over most of the infrared spectrum restricts the regions where aqueous solutions of carbohydrates can be usefully studied. Absorbance subtraction makes it possible to eliminate water absorbance and magnify the remaining spectral features to the limit of the signal-to-noise ratio. Many other data-processing techniques, such as the ratio method,4 the least-squares refinement,5 and factor analysis,6 should be of benefit in the study of carbohydrate mixtures. 

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