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December 2010


mixture and volume must be injected and that the injector must be working reproducible.


Critical peak pairs are clearly seen in some of the input runs seen in Fig. 1, for example in the lower left chromatogram (tG 40°C) benzylamine tR


: 15 min and T: and procainamide peaks (tR


: 5.16 min, peak area 258) : 5.25 min, peak


: 11.46 min, peak area 317), one of the neutral components) which elutes before quinoxaline (tR


area 200) only exhibit partial separation. Phenol (tR


: 11.67 min, peak area 940) when


chromatographed with a shallower gradient (i.e. lower right chromatogram, tG 40°C) now elutes (phenol, tR area 317) after quinoxaline (tR


: 45min, T:


: 21.57 min, peak : 20.53 min, peak


Figure 2. Screenshot in DryLab® Binary gradients using as eluent B: B1 (MeOH) and tG


2010 of the resolution map obtained using the chromatographic conditions stated in Fig. 1 – –T-model. Red regions in the resolution map represent regions of


“baseline resolution” Rs, crit > 1.5, blue colour means co-elution of 2 or more peaks.


area 918). Interestingly, increasing the operating temperature from 40°C (bottom left chromatogram) to 60°C (top left chromatogram) induces the same movement of phenol with respect to quinoxaline as on increasing the gradient time. It is quite evident from Fig.1 that some peak pairs are better separated at the higher temperatures. These observations are based on the different tendency of peak movements and are definitely not the result of decomposition.


The critical peak pair was not constant between the input chromatograms, indicating the presence of several “critical moving peaks”in the complex mixture.


3.2 Generation of the tG –T models


Once a matched peak table (identified by a green colour) had been obtained, all the data were automatically transferred into DryLab®


2010, where the resolution map of the MeOH-plane of the cube was generated and optimal conditions could then be ascertained.


From the 4 basic input experiments, a tG –T


model could be created, showing a resolution map in which approximately 10,000 virtual chromatograms can be represented with extremely high precision (Fig. 2).


The accuracy of the DryLab®


Figure 3. Comparison of the experimental (in blue) and modelled chromatograms (in green) in PeakMatch® obtained using chromatographic conditions stated in Fig. 1 – (Binary gradient using MeOH-tG


-T plane) for the control of the model.


generate numbers of 2-4 digits. The legends associated with each chromatogram permit easy identification of the parameters that have been changed for each chromatogram (i.e. composition of eluent B – ratio of MeOH:AN, the temperature and gradient time). The low standard deviation (i.e. 0.7%) of the sums of the peak areas per runs. Fig.1., green table at lower right corner shows the precision of the experiments in the tabulated data to be excellent. This observation proves that peak areas which correspond to sample masses can be used in an easy and precise way for peak


assignment / tracking. Decomposition or large extinction changes on altering the chromatographic conditions due to the dilution of the sample in the column are very seldom. UV or mass spectra are often helpful in peak assignment / tracking, but their use requires more expertise and cost. The UV-spectra of related substances and impurities are often too similar for discriminatory and identification purposes and up to 30-40% of analytes may not be ionised and hence not detected by mass spectroscopy. However, the only prerequisite for successful peak tracking using peak area is, that the same sample


2010 software to


model the original input experiments is shown in Fig. 3. The original experiments are in blue and the DryLab®


2010 models are in green.


The comparison shows that all four input runs are precisely calculated by DryLab®


2010 and


hence validates the model (i.e., the model does what is expected). Fig. 3 highlights that these four input experiments of the same sample generate very different selectivities.


The above process is then repeated using the ternary mobile phase composition of (B1:B2)(MeOH:AN)(50:50 V/V) as eluent B as shown in Fig. 4.


Comparison of Figs. 1 and 4 highlights the differing elution orders (i.e. differing chromatographic selectivity) that are


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