Absorbance ratios

As noted in Sec. 7.7, these different wavelengths correspond to absorbance by sequences of different lengths. Compare the appropriate absorbance ratios with the theoretical sequence length ratios calculated above and comment briefly on the results. 

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. 

Fig. 17. Kinetic plots showing a linear dependence on irradiation time (305 nm) of the absorbance ratios and AgJ " (O) and a linear dependence on...

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. 

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. 

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. 

Then, we calculated the < > values for the transition moments of the major bands of the 11-monolayer LB film of C12AzoC5-Ba by Eq. (2) from the observed absorbance ratios between the transmission and RA spectra. The results are shown in Table 1. The angles (a and P ) of the transition moments of the antisymmetric and symmetric CH2 stretching vibrations are 72° and 70°, and those of the antisymmetric and symmetric COO stretching vibrations are 85° and 29°, respectively. Furthermore, those of the... 

The out-of-plane orientation of chromophores can be more easily controlled in LB films as compared with the in-plane orientation. Many chromophores are known to show anisotropic orientation in the surface normal direction. The molecular structure of chromophores and their position in amphiphile molecules, the surface pressure, the subphase conditions are among those affect their out-of-plane orientation. The out-of-plane orientation has been studied by dichroic ratio at 45° incidence, absorbance ratio at normal and 45° incidence, and incident angle dependence of p-polarized absorption [3,4,27,33-41]. The evaluation of the out-of-plane orientation in LB films is given below using amphipathic porphyrin (AMP) as an example [5,10,12]. 

Figure 2. Change in surface tension(O) and absorbance ratio( ) as a function of the degree of photoisomerization(A /Aq) of 1a in water. [1] = 5-2x10 5 M Aq and A are absorbance of la at 502 nm at time 0 and t, respectively.

Procedure A standard calibration curve is plotted by thoroughly mixing together about 10% (w/w) of the analyte with the KBr-KSCN mixture and then grinding the same intimately. Now, the ratio of the thiocyanate absorption at 2125 cm-1 to a selected band absorption of the analyte is plotted against the percent concentration of the sample. Likewise, an identical disc is prepared with the unknown sample and the same KBr-KSCN mixture. Finally, its absorbance ratio is determined and the concentration (of unknown sample) is read off directly from the standard calibration curve. 

Figures la and lb show the OH and NH infrared bands of the oligomer as a function of temperature in uncatalyzed and catalyzed formulations. The uncatalyzed urethane modified epoxy oligomer shows only small changes in the OH/NH absorbance ratio at temperatures below 165°C only about a 60% conversion of the blocked isocyanate was observed. In contrast, sample of the oligomer catalyzed with 0.5% dlbutyl tin dilaurate shows nearly complete chain extension at temperatures as low as 130°C.

Absorbance ratios used peaks at 1365 cnfl and 830 cm-l as internal s tandards. 

Fig. 23 Solvent denaturation curves of 45 measured by a the UV absorbance ratio A303M287 and b the circular dichroism (g bs at 316 nm) showing the need for higher vol% acetonitrile to bring about twist sense bias than that needed for folding...

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... 

All the diamines have a UV-maximum in the 290-310 nm region and therefore 290 nm was the second detection wavelength chosen. The ratios e235/e29o were calculated for each standard substance and these values are found in Table I. The variation of the absorbance ratio with concentration is also included in the standard deviation shown in the table. 

Table II shows the substances identified and amounts determined in the various dye samples. Resorcinol and 2,5-TDA were found in most samples. The identification of a-naphthol in sample (V) is somewhat uncertain since it was found that the absorbance ratios were not independent of the concentration for this substance. In general, few peaks could be seen on chromatographing all the samples and the same simple pattern showed up with several dyes.

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. 

The AAA thus has two photometers in series. Since for every eluting amino acid both signals are recorded, the so-called 570/440 absorbance ratio may be of help in the identification process. As an example the simple primary amino acids have a 570/440 ratio of approximately 6, whereas the sulfur amino acids (cystine, sulfocysteine) have much lower ratios, approaching a value of 1. Small peptides and glycyl-amino acids will react with ninhydrin, an important fact for the diagnosis of prolidase deficiency and aspartylglycosaminuria. 

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