Quantitative Analysis from Retention Measurements

There is an interesting consequence to the above discussion on composite peak envelopes. If the actual retention times of a pair of solutes are accurately known, then the measured retention time of the composite peak will be related to the relative quantities of each solute present. Consequently, an assay of the two components could be obtained from accurate retention measurements only. This method of analysis was shown to be feasible and practical by Scott and Reese [1]. Consider two solutes that are eluted so close together that a single composite peak is produced. From the Plate Theory, using the Gaussian form of the elution curve, the concentration profile of such a peak can be described by the following equation  

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Rearranging (multiplying the top and bottom of the exponent by (nA) and taking (nA ) inside the brackets), 

The variable (v) is replaced by the variable (t), the elapsed tirne, which is the parameter that can be accurately measured experimentally. 

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It follows that measurements must be made with a precision of about 0.2 second if quantitative results are to be of any value. It is seen from figure 4 that the experimental points lie very close to the line and a fairly accurate measurement of the distribution of the two isotopes can be obtained from retention time measurements. This method has very limited areas of application and is given here, more to demonstrate the effect of unresolved impurities on retention time, than to suggest it as an alternative to adequate chromatographic resolution. In some cases, however, particularly in the analysis of isotopes, it may be the only practical way to obtain a quantitative evaluation of the mixture by a liquid chromatographic method. 

The introduction of GC as an analytical technique has had a profound impact on both qualitative and quantitative analysis of organic compounds. Identification of compounds by GC can be accomplished by their retention times on the column as compared to known reference standards, by inference from sample treatment prior to chromatography, " or by the concept of retention index. " The latter method and tables of retention indices " with associated conditions have been reported. " Although qualitative data and analytical techniques for identification of compounds are well-established " and relative retention data for over 600 substances also have been published, " the main utility of GC undoubtedly lies in its powerful combination of separation and quantitative capabilities. Use in quantitative analysis involves the implementation of two techniques being performed concurrently, i.e., separation of components and subsequent quantitative measurement. 

Use the formulae below to calculate an EDL for each absent 2,3,7,8-substituted PCDD/PCDF. The background level (H ) is determined by measuring the height of the noise at the expected retention times of both the quantitation ions of the particular 2,3,7,8-substituted isomer. The expected retention time is determined from the most recent analysis of the CC3 standard on the same GC/MS system. 

Three standard solutions were prepared—one containing 1 mg. of DDT and 1 mg. of DDD per 10 ml. of solvent the second containing 1 mg. of diazinon per 10 ml. of solvent and the other containing 1 mg. of ethion per 10 ml. of solvent (benzene). The retention time (time required after injection of the sample for the component to reach its maximum peak height) was used as a qualitative measure to identify the component. Quantitative information was obtained from the direct relationship of the concentration of insecticide to the maximum peak height. Standards were analyzed periodically during the analysis of the residues. 

Five commercial walnut samples were compared by HPLC analysis to measure the presence of seven major polyphenol compounds and a dicarboxylic add derivative (1,3,12,17,22,23, 30, and 33). Each peak of HPLC profile was identified by comparing the retention time and ultraviolet-visible (UV) spectral pattern with those of standard compounds that were isolated from walnuts. Using calibration curves, each compound was analyzed quantitatively (Table 19.6). The results indicate virtually no difference in the content of the individual polyphenol components between raw and roasted walnuts. The results also revealed that polyphenol content in raw samples was higher in the U.S. walnuts than in Chinese walnuts. It was interesting to note that no polyphenols were detected in walnuts from which the pellicle (skin) was removed. This shows that polyphenols in walnut are present only in the pellicle portion, although a nonpolyphenol (33) was detected in pellicle-free walnuts. 

Therefore, to determine if oxidized lipids were formed by enzymic processes or by free radical autoxidation, a first step is to visualize the distribution of products. This step requires previous knowledge of the maximum number of oxidized products, their chromatographic behavior and ions associated with mass spectrometric detection of each product. Quantitative analyses almost always require the use of appropriate, pure standards. For samples from more complex sources where the lipids of interest are present at low concentration there may be many interfering ions. In these instances, tandem mass spectrometry can be used to select pairs of precursor ions and product ions formed by collision-induced dissociation in a procedure called selected reaction monitoring (SRM). This type of analysis usually provides a significant improvement in signal to noise so that the product can be accurately quantified. With modem instruments many, up to hundreds, of these transitions can be measured in a single analysis. In conjunction with retention time... 

The octanol-water partition coefficient scale may not be the best tool for hydrophobicity evaluation. The ability of MLC for hydrophobicity measurement and some studies on quantitative structure retention activity relationships (QSAR) are described in Chapter 9. Chapters 10 and 11 contain selected examples of applications in the analysis of a variety of samples, especially pharmaceutical preparations and physiological fluids, some of them are taken from the authors own experience. Details on the nature of the sample, stationary phase, mobile phase composition, detection wavelength, and figures of merit, are tabulated at the end of each of these... 

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