17


chromatograms, which can greatly assist in understanding peak movements.


An improved understanding of peak movements as a function of the influence of physico-chemical parameters (or factors) such us gradient time tG


, temperature, pH, ternary


composition, additive concentration or changing instrumentation (with differences in dwell volume), differences in flow rate, in column length and diameter, should permit the chromatographer to control his/her methods much more precisely and hence to reduce run failures and generate higher quality data.


The final tG –T plane using AN, an eluent,


Figure 4. Collection of the experimental chromatograms and tabulated peak assignments, retention times and areas in PeakMatch®


V/V) which corresponds to a MeOH:AN ratio of (1:1 V/V). obtained using the chromatographic conditions stated in Fig. 1 with the exception that eluent B was (B1:B2)(50:50


which is often selected initially, due to its lower viscosity, UV cut-off and associated better peak shape than MeOH, is shown in Fig. 5. The price of AN still remains high after the worldwide shortages some months ago (ca. 5-10 times higher than HPLC grade MeOH) therefore many laboratories now favour MeOH over AN. It is therefore expedient to investigate if MeOH offers any selectivity advantages over AN in method development strategies.


As expected, the elution times are somewhat shorter with AN than with MeOH. However we observe much more cases of co-elution with AN than with MeOH in eluent B.


3.3 Generation of the 3-dimensional


resolution model (the Cube) After the three tG


created, the DryLab® –T models have been 2010 software calculates


the 3-dimensional resolution model (the Cube), representing the simultaneous influence of three parameters (%MeOH in AN, tG


Figure 5. Collection of the experimental chromatograms and tabulated peak assignments, retention times and areas in PeakMatch®


Water)(65:35 V/V) – Binary gradient using AN-tG-T-plane.


achievable when (Water:MeOH:AN) ternary mobile phase compositions are compared to that of a binary composition (Water:MeOH). Peak areas represent the mass of the sample (concentration x elution volume = sample mass). As long as the flow rate is constant, it can be expected that the peak areas will be additive in overlapping peaks. In DryLab®


2010,


the area of peaks in overlapping bands can be calculated, such as the largest peak


(corresponding to co-elution of quinoxaline and phenol) in the lower left at tR: 10.15 min, originally with a peak area of 1242, is now subdivided into peak areas of 927+315. Other areas of greater co-elution can be observed (peak area 745), such as the peak in the same run at 14.14 min, corresponding to three peaks (remacemide, doxepin isomers 1 and 2) with the areas of 172+490+83. Each experiment in a tG


– T plane has three other


separations within the cube with an unparalleled precision of the retention times and chromatographic separation selectivity.


obtained using the chromatographic conditions stated in Fig. 1 with the exception that eluent B is now B2: (AN:


and T) on the chromatographic selectivity and critical resolution of the separation. The advantage of the 3-D resolution volume is the fact, that the 8 corner points and 4 intermediate points of the space are all measured and form a “cube” as a true “Design Space”, allowing us to predict > ca. 106


Fig. 6 illustrates the graphical representation of the three-dimensional resolution cube (top right side) of a Design Space (DS) with 3 factors: Gradient time (tG


) (x-axis), temperature


(T) (y-axis) and ternary composition (z-axis) (%MeOH in AN). The front page of the cube is shown on the top left and corresponds to the the tG


–T- plane with MeOH as eluent B (comparable to Fig. 2). The robustness of the method can be easily visualized as a geometrical body within the resolution space, in which the critical resolution does not fall


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