Baseline method, quantitative

Ideally, the intensity (/o) of infrared radiation incident upon a sample cell is reduced (to f) by the absorption of the samples. Actually, some of the incident energy is scattered by the sample and this scattered energy makes the Beer-Lambert law inaccurate, especially at high values of absorbance [4]. The baseline method for quantitative analysis is an empirical method used to establish a calibration curve of log (/q//) versus concentration. Infrared absorption bands may overlap neighboring bands or may appear on a sloping background, so transmittance is measured in practice as shown in Figure 8.13. The absorbance. A, is determined from measurements of / and 7o, then a calibration curve of absorbance versus concentration is plotted. 

Jart (1960) has recorded spectra of many different fatty acids and fatty acid esters in carbon disulfide solution. Stress was laid on the study of the trans absorption at 962cm and absorptivities of the pure substances at the trans maximum were calculated. Beer s law applied for concentrations up to 2(X) g of substance per liter of solution. This worker used a baseline method for the quantitative determination of trans monoene fatty acids in mixtures with saturated acids and cis forms of unsaturated fatty acids and fatty acid esters. Unlike the cis compounds and the saturated compounds, trans compounds display a considerable absorption at 962 cm (CH bending about the trans C=C group). 

Chromatographic analysis of folate compounds including methotrexate and other antifolates has been reviewed by McCormack and Newman (32). Process impurities in the reduced folate compound leucovorin calcium have been monitored using a TLC method with fluorescence detection (33). An overpressure layer HPTLC procedure has been used to improve the separation of folic acid from other water-soluble vitamins with good recovery and resolution. The method uses silica gel layers developed in butan-l-ol-pyridine-water (50 35 15) at a rate of 0.25 ml/min for baseline separation. Quantitation was achieved without derivatization (11). 

The baseline method of quantitative analysis involves selecting an absorption band of the substance under analysis that does not fall too close to the bands of other matrix components. The value of the incident radiant energy Pq is obtained by drawing a straight line tangent to the spectral absorption curve at the position of the sample s absorption band. The transmittance P is measured at the point of maximum absorption. The value of log P(/P) is then plotted against eoncentration. 

After two decades of quantitative l.c. analyses, it has been established that, when proper precautions are taken, these methods can provide accurate and reproducible results.Quantitative l.c. measurements are usually as accurate as, and often more precise than, those obtained by spectrophotometric, " paper-chromatographic, and gas-liquid-chro-matographic methods. Both external and internal standardization have been used to translate peak height or areas into quantitative, solute-concentration values. Because peak heights are easy to measure, many methods use this parameter, and, when slightly overlapping peaks or unsteady baselines are encountered, it is the method of choice. With introduction of... 

The IR methods have progressed from hand-drawn baselines and peak height or area for quantitation, to spectral subtraction, to leastsquares methods. Least-squares analysis eliminates the reliance on single peaks for quantitation and the subjectivity of spectral subtraction. However, negative concentration coefficients are a problem with least-squares analysis, since they have no physical meaning. Negative components can be omitted according to some criterion and the least-squares process iterated until only... 

Another area of the use of graphics is baseline display for improved quantitation. Every chromatographer has wondered how his data system was drawing the baselines. With this system, the baselines can be visualized even to the point of extreme magnification, as in Figure 5, so the analyst is convinced that the baseline is correct. If it is incorrect, the method can be modified, the data re-integrated, and the chromatogram re-display-ed. This interactive process can be repeated in a matter of minutes. 

In the absence of definitive human data, risk assessment may have to depend on the results of cancer bioassays in laboratory animals, short-term tests, or other experimental methods. Hence the following issues must be addressed under such circumstances the ability of the test system to predict risks for man (quantitatively as well as qualitatively) the reproducibility of test results the influence of species differences in pharmacokinetics, metabolism, homeostasis, repair rates, life span, organ sensitivity, and baseline cancer rates extrapolation across dose and dose rates, and routes of exposure the significance of benign tumors fitting models to the data in order to characterize dose-incidence relationships and the significance of negative results. 

The motivation behind the considerable effort that was exerted in the development of DCV [42, 49, 50, 69] was based on the need to make CV and LSV quantitative tools for the study of electrode kinetics. At that time, there were three major problems that had to be overcome. These were (a) the precision in the measurement of Ep and AEp, (b) the problem with accurately defining the baseline for the reverse sweep and (c) the problem as to how to handle Rn in a practical manner. The development of DCV did indeed provide suitable solutions to all three of these problems, although the methods developed to handle the Ru problem [41, 42] only involve the derivative of the response in terms of precision necessary for the measurements. More recent work [55, 57] is indicative that the precision in Ep/2, Ep) and AEP measurements can be as high as that observed during DCV (see Sect. 3.4). Also, a recent study in which rate constants were evaluated using CV, DCV, and double potential step chronoamperometry for a particular electrode reaction showed that the precision to be expected frcm the three techniques are comparable when the CV baseline, after subtracting out the charging... 

High resolution l3C NMR is also used in the determination of the composition of the dispersed phase in cured rubber modified epoxies in order to analyze the chemical structure of the mobile segments 152). In this case quantitative analysis is possible because the areas under each peak are approximately equal to the number of carbons contributing to the peak, and the intensities of the broad lines from the rigid phase are very low, almost indistinguishable from the baseline noise. The structure of crosslinked networks based on poly(3,4-pyrrolidinediethylene), synthesized by different methods, was determined from gels swollen in water and chloroform 153). 

The limit of quantitation is the minimum injected amount that gives precise measurements, in chromatography typically requiring peak heights 10 to 20 times higher than baseline noise (Fig. 3). If the required precision of the method at the limit of quantitation has been specified, the Eurachem [2] approach can be used. A number of samples with decreasing amounts of the analyte are injected six times. The calculated RSD of the precision is plotted against the analyte amount. The amount that corresponds to the previously defined required precision is equal to the limit of quantitation. 

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