Retention measurements, close eluting peak

The resolution factor is usually estimated from the peak retention times and widths observed in a chromatogram of a mixture of solutes. However, in a rigorous way, a more accurate estimation requires the separate injection of the individual compounds. This is particularly true for closely eluting peaks. It has been established that the retention times measured at the peak apex, and mote so the peak widths, are different if the measurement is made on individually injected solutes or on the peaks in a mixture. This difference is more pronounced when the peak shape cannot be described simply by a Gaussian profile, and where the center of gravity of the peak does not correspond to the peak apex (8). Nevertheless, the chief drawback of the resolution factor Rs is the fact that it does not take into account the relative peak height (9) of the... 

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 ... 

The insecticides are identified on the two different types of gas chromatographic columns by comparing the relative retention times of the recorded peaks of the samples with those of standards. If a closely eluting component interferes with the identification, a small amount of the suspected insecticide can be added to the sample which is injected into the column. This injection consists of a small measured amount of the insecticide standard drawn into a microliter syringe already charged with an aliquot of the sample extract. 

Figure 16 shows the viscometer and DRI traces of another star-branched polystyrene. This sample contained about 12% of the starting linear arm precursor which eluted at retention volume ca. 52 ml. The kinetic molecular weight of the linear precursor was 260,000. The results obtained for the individual peak through the SEC/Viscosity methodology are summarized in Table 7. It is seen that the measured of the linear arm is very closed to the kinetic value. The average functionality of this star polymer is calculated to be f = 10. 

The majority of chromatographic separations as well as the theory assume that each component elutes out of the column as a narrow band or a Gaussian peak. Using the position of the maximum of the peak as a measure of retention time, the peak shape conforms closely to the equation C = Cjjjg, exp[-(t -1] ) The modelling of this process, by traditional descriptive models, has been extensively reported in the literature. 

The CD spectra of the second eluted isomers of three complexes, [Co(L-ser)3 n(6-ala)n](n=0,l and 2) are not exactly the same with but quite similar to those of the corresponding first eluted isomers, except that the CD sign is opposite. From these CD spectra, the CD spectra of A- and A-[Co(D-ser)3 n(f -ala)n] are estimated. They could not be measured directly because only a small amount of D-serine complexes was available. When eluted with Na2S0l. solution, the elution curve of /ac-[Co(D-ser)3 n(f -ala)n] is the same as that of the corresponding L-serine complexes. Thus, the configuration of the first eluted isomer in the D-serine complexes must be A. In this way, we have obtained two series of the retention volumes of diastereomeric pairs, A(major peak) and A(minor peak) for L-serine complexes and A(major peak) and A(minor peak) for D-serine complexes. When eluted with NapSOL solution, the ratio for each enantiomeric pair is always quite close to unity, which means that they are certainly enantiomeric pairs. 

A " is a measure of the munber of additional volumes of mobile phase in the system required to elute a solute following the elution of a nonsorbed component and hence is a direct measure of solute retention. In practice, often the easiest way of improving the resolution of a separation is to ensure that a system is used in which the compounds to be separated have a reasonable value of k . A value of A = 0 means that the solute is not retained at all— it elutes at the solvent front. In most cases, a useful value of A is between 1 and 10, and m attempting to resolve two close peaks, it is rarely of any value to increase retention so that A" > 20. A values of greater than 20 represent inordinately long retention times from the above equation, it can be seen that as A increases, A /A +1 tends toward 1, and further increases in A have little effect in improving resolution. 

In order to optimize a separation and produce it in the minimum time, the capacity ratios and separation ratios must be measured for a given pair of enantiomers under known conditions of mobile phase composition and temperature (this will be discussed in detail later in this chapter). Unfortunately, when two peaks are eluted close together, which frequently occurs in chiral chromatography, the positions of the peak maxima are distorted due to the immediate presence of the other peak. An example of this problem is shown in figure 10.1, where the peaks are simulated and added, and the composite envelope plotted over the envelope of each individual peak. It is seen that the actual retention difference, if taken from the maxima of the envelope, will give a value of less than 60% of the true retention difference. Unfortunately, this type of error will probably not be taken into account by most data processing software. It follows, that if such data is used in an attempt to calculate the... 

An approach that is closely related to the plate height method is the technique of peak profiling. This approach was first suggested in 1975 by Denizot and Delaage. In this method, measurements of the retention time for an analyte (tn) and the elution time of a non-retained solute (Im) are made on the same column. These elution times are then used with variances observed for the peaks of the analyte (a ) and non-retained species (dissociation rate constant (kj pply as is demonstrated in Eq. 

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