Gradient profile

Fig. 4.3b. Possible gradient profiles showing the blending of solvent...

Fig. 2.19. Chromatogram of carotenoid solution in THF. 1 lutein, 2 canthaxanthin, 3 /Tcryptoxan-thin, 4 lycopene, 5 /1-carotene. ODS column, 5/an, 150mm X 4.6mm. Mobile phase eluent A (methanol-acetonitrile 6 11), eluent B (THF) gradient profile 0-5min, isocratic conditions 95 per cent A 5-20min, gradient to 20 per cent A. Flow rate lml/min. Detection 450 nm. Reprinted with permission from C. Tricard el al. [45].

Fig. 3.34. Chromatogram of a dye mixture obtained by the proposed method and gradient profile. Reprinted with permission from J. J. Berzas-Nevado et al. [112].

A pH, B flow rate, C, D, and E buffer eomposition of firsf, second and third step of gradient profile... 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Fig, 25. (a) Reaction rate and (b) sensitivity gradient profiles for the most important elementary reactions involving CH3CI. 

Combinations of alkyl sulfonic acids from C5 to C8 were tested and the most efficient separations were achieved by running a gradient from 50 6 C6 plus 50 6 C7 to 100 6 C7. Use of the mobile phase compositions illustrated on Table I with the gradient shown on Figure has provided efficient separations for all of the toxins except C1 and C2. Small changes in mobile phase composition and/or gradient profile may be necessary to achieve the desired separations on any particular column or HPLC system. 

In some cases, the solution is possible in explicit form allowing direct calculations of the retention data however, for some combinations of gradient functions and retention equations iterative solntion approach is necessary, which can be applied using standard calculation software. An overview of possible solntions of Equation 5.3 for various HPLC modes and gradient profiles was pnblished earlier [4,33]. 

FIGURE 5.2 (A) Calculated gradient profile of 2-propanol in heptane on a Separon SGX silica gel column... 

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