Peak Separation overlap

Resolution (R) is the measure of the chromatographic separation of two components. Ideally, mixtures should be completely separated, but in many instances spots or peaks will overlap (co-elute). For gas chromatography, where the components are isolated as Gaussian peaks, the resolution is given by ... 

Generally, kfs range from 1 to 8 for analytical separations and 4 to 12 for preparative, a s range from 1 to 2 at a = 1, peaks completely overlap, much above a = 2 and the separation can be made in a separatory funnel. For N, values may range from hundreds (poor resolutions) to tens of thousands (good resolution). 

The close coupling between the phenoxazine or phenothiazine modifier and a graphite electrode (E-electron overlapping) results in very fast charge transfer rates between the modifier and the electrode seen as small peak separations in cyclic voltammetry (83,86,87,92). The adsorption of the mediators also results in a further decrease in the E0 values with about 50 to 100 mV compared with the bulk values (83,84,92). Drastic changes in the pKg values of both the reduced and the oxidized forms of the adsorbed mediator have also been noticed (83,84,92). 

CD detection can be used advantageously when the two peaks largely overlap. Only one peak is revealed by the UV detector, while two peaks are observed when CD is used for monitoring, Figure 5(a). The information that is obtained demonstrates the extent of the enantiomeric separation, and can... 

Figure 5.7. The resolution of two peaks, defined as R, = AA /(2cr1 + 2a2), measures peak separation X relative to the zone dispersion (2 and 2

As was discussed in Chapter 1 resolution, R, is a measure of the distance between two adjacent peaks in terms of the number of average peak widths than can fit between the band (zone) centers. Assuming symmetrical (Gaussian) peaks, when R = 1, peak separation is nearly complete with only about 2% overlap. This case was shown in Chapter 1, Figure 1-4. Resolution results from the physical and chemical interactions that occur as the sample travels through the column. It should, therefore, be no surprise that resolution may also be expressed in terms of the contribution of the individual column characteristics separation factor (selectivity, a), efficiency (narrowness of peak, N), and capacity factor (residence time, k ) of the first component. The equation that describes this interrelationship is... 

Figure 2.5 shows that relative concentration (or for analytical separations chromatographic response) must also be taken into account when considering resolution. In particular it should be noted that the position of the valley between the peaks is shifted to longer times as the relative concentration of the second peak is decreased. When the peak separation is 3.2ct, then = 0.8 and no valley is detected if the ratio of the concentrations of the two peaks is 10 1. When the separation of the peak maxima is equal to 6a, then Rg = 1.5, and the degree of peak overlap is less than 1%. 

As an example of a continuous flow chromatogram Fig. 9.3.6 shows a stack of NMR spectra (right) as a function of elution time for five vitamin A acetate isomers on a cyanopropyl column in n-heptane recorded at a flow rate of 0.2 ml/min [Albl]. The first two peaks somewhat overlap in the ultraviolet (UV) detected chromatogram (left), but in the NMR spectra (right) the peaks are well separated due to different chemical shifts. Forty-eight scans were added for each trace, so that the time resolution was 62 s. 

Determining the integrated intensity becomes particularly difficult when the peaks partially overlap. A careful choice of fitting functions can somehow make it possible to properly deal with this issue and to separate the contributions of each peak. This is illustrated by Figure 3.15. 

The above example illustrate that a separation between two compounds requires two factors (1) that their retention times are different, and (2) that the resulting peaks are sufficiently narrow so that, despite their different retention times, they do not overlap appreciably. Consequently, both peak position (as defined by tr or VT) and peak width (as characterized by ay) are important for a chromatographic separation. Retention times are proportional to Np, whereas peak widths are (approximately) proportional to V(Vp. This is why, everything else being the same, larger values of Np yield better peak separations. 

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