Peaks close eluting

Figure 3. A Composite Peak Formed by Two Closely Eluting Peaks of Different Size...

Figure 5. Curves Relating Apparent Separation Ratio Relative to Actual Separation Ratio for Two Closely Eluting Peaks...

Considerable care must be taken when accessing closely eluting peaks. If the resolution is inadequate, measurements must be taken on the individual solutes, chromatographed separately on the column. 

FIGURE 7 A schematic diagram showing two closely eluting peaks at various resolution values from 0.6 to 2.5. Figure reprinted with permission from Reference 7. 

Desalted peak fractions were analyzed by both IP-RP-HPLC and RP-HPLC (derivatized with FMOC-Cl) to investigate the presence of pyridinoline- and lysinonorleucine type cross-links, respectively. Two fractions. 111 and V-1-1, were found to contain the cross-links HP and DHLNL, respectively. Additional analyses by cation exchange HPLC showed TV and V-2 as closely eluting peaks and VI as a mixture of lysi-noalanine and an unidentified amino acid (fig. 3a). 

Carry-over can be problematic for closely eluting peaks in stop-flow mode [46], and these are better analysed using the loop collection mode (see below). 

Automation of LC-NMR is now at a stage where the operator can inject a sample and leave the HPLC interface to detect and store peaks and the NMR spectrometer to collect one- and two-dimensional data with signal-to-noise-dependent collection. An example of automated loop collection and transfer of closely eluting peaks is shown in Figure 6.38. Structures were deduced from the aromatic peak patterns and LC-MS information. Peaks 1-5 all elute within 5 min with no carry-over present in any of the H spectra. 

The Heat of Adsorption Detector, devised by Claxton (16) in 1958 has been Investigated by a number of workers (17,18,19) but although once commercially available, has not been extensively employed as an LC detector. One reason for this is the curious and apparently unpredictable shape of the temperature-time curve that results from the detection of the usual Gaussian or Poisson concentration peak profile. The shape of the curve changes with detector geometry, the operating conditions of the chromatograph, the retention volume of the solute and for closely eluted peaks, it produces a complex curve that is extremely difficult to interpret. 

Figure 2.4 NMR spectra of closely eluting peaks (At = 0.4 min) in different working modes...

The detector time constant is the response time of the detector to the signal passing through it. A slower time constant will result in less apparent noise, but it will compromise signal and also resolution for closely eluting peaks. For closely eluting peaks, therefore, it is important to use a faster time constant. If there is plenty of resolution, a slower time constant will provide a smoother baseline. The effect of the time constant is illustrated in Figure 8.6. 

The GSC differs from a TIC in MS or a FID chromatogram in GC. The relative intensities of the chromatographic peaks are sometimes very different in IR due to differences in absorp-tivities between chemicals. For example, chemicals containing phosphorus-oxygen bonds have high absorptivities. Additionally, as the chromatographic resolution is lower in GC/FTIR, closely eluting peaks may overlap in IR, but not in MS or GC. 

Improvement of the resolution of poorly resolved analytes then could be pursued in two different ways either by increasing the efficiency or by improving the selectivity. The resolution value equal to 1.5 is usually regarded as sufficient for the baseline separation of closely eluted peaks and if we consider that typical average efficiency of modern HPLC column is equal to 10,000 theoretical plates, then we can calculate the selectivity necessary for this separation to get a resolution of 1.5. It will be also useful to compare what would be required in terms of efficiency and selectivity to improve the resolution from 1 to 1.5. Corresponding calculations are shown in the Table 1-1. 

For closely eluted peaks and relatively high efficiency of the system, these assumptions do not lead to the significant deviations of equation (2-22) from true resolution given by equation (2-19). 

Additionally, the quality of separation is evaluated by measurement of resolution, R between two closely eluting peaks. 

When the selectivity is close to 1, the peaks elute closely together, as illustrated in Fig. 11.2. They may even overlap one another, in which case we say that they are incompletely resolved. This concept of peak resolution Rs is put on a quantitative basis by ratioing twice the separation d between two closely eluting peaks (determined by the column s selectivity), to the sum of their two widths at the base, Wb, (Fig. 11.2). 

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... 

Burt et al. [38] coupled an HPLC to detect and identify several PAHs through measurement of their fluorescence decay lifetimes. Several wavelengths were monitored simultaneously to differentiate some closely eluting peaks. 

The efficiencies that can be expected at the optimum velocity fi-om columns of different lengths, packed with particles of different size, are shown in table 8.1. As chiral separations often involve closely eluting peaks, the efficiency limitations disclosed in table 8.1 can be important in chiral chromatography. 

It is clear that considerable care must be taken when assessing closely eluting peaks. In fact, if the resolution is inadequate, the measurements must be taken on the individual isomers and chromatographed separately on the column. It is important to know the value of the separation ratio above which, accurate measurements can be made on the peak maxima of the individual peaks in the envelope. In figure 10.4, the apparent peak separation ratio is shown, relative to the actud peak separation, in standard deviation units (c) for columns of different efficiency. These have been theoretically calculated. It is seen that for a low efficiency... 

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