Gradient duration

A reduced peak capacity in one domain may be counterbalanced by an increased peak capacity in another domain. If we know the average peak width of a chromatographic separation and the gradient duration, we can calculate the maximum number of peaks that can be separated. (Note peak capacity does not mean that this number of compounds in a sample will be separated they may still co-elute). That means we can operate between two limits (1) a peak capacity of zero representing a flow injection analysis and (2) a minimal required peak capacity that defines the peak capacity to separate all compounds in a given mixture. Unfortunately, especially in the early stages of drug... 

FIGURE 4 Peak capacity in gradient separations, (a) Peak capacity as a function of gradient duration at constant flow rate, (b) Peak capacity as a function of flow rate at constant gradient run time. 

FIGURE 5 3-D plot of peak capacity as a function of flow rate and gradient duration. 

Cohen et al. [430] have demonstrated the validity of eqn.(4.67a) in practice for cases in which a is constant. However, they have also shown that the equation is no longer valid if a varies with composition under isocratic conditions. Nevertheless, eqn.(4.67a) may serve as a good rule of thumb for the optimization of gradient duration times (see chapter 6). 

Fig. 3. Strategy to achieve fast, faster, and ultrafast analysis. Both the particle size and the gradient duration are scaled down in proportion to the column length. This ultrafast analysis demonstrates that the smallest particle size (2.5 pm) in the shortest column length (20 mm), together with the shortest gradient duration time (tg=0.7 min), resulted in a baseline resolution of a wide range of analytes in a 9 s window. The size of the detector cell was 2.6 pi. Reprinted with permission from U. Neue [48]...

Fig. 48. Scanning of isoelectrofocusing pattern of cytochrome c (horse heart) in temperature gradient (—). Duration of focusing, 24 hours scanning rate, 0.6 cm min-1.

The gradient duration is modified to 2 min that cuts down the run time. The gradient duration may be increased to 2.5 min or more as needed when multiplex SBE is performed. 

Texture Contaminant Electrodes Processing fluid Electric gradient Duration Material Number/ position ... 

Figure 4 Fourier transform GSE spectra recorded for various values of the pulsed gradient duration 6. The sample is a 12-component liquid mixture, and the assignment of the various components are given on top. (Adopted from Ref 12.)...

At = retention factor of a component in the middle of the column, tg = gradient duration,... 

Finally, the legitimate question "All well and good, should 1 now opt for a longer gradient or for a higher flow The simplified answer is is the ultimate resolution in the foreground and does the run time play a secondary role In addition to this, does one have a small difference in %B When yes, then in this case you should increase the gradient duration. Is the separation problem not so difficult ... 

Here you should increase the flow, the possible minimal decrease in resolution is likely to have little impact, however you are finished quicker. Think in this context of the following analogy with a gradient, the gradient duration corresponds to the aqueous fraction with an isocratic separation. In both cases, an increase leads to small, broad peaks, longer retention times, better overall resolution (sum of the resolution between all peaks). 

Case 1 The Gradient Duration Remains Constant and the Fiow is Increased... 

Case 2 The Gradient Duration is Haived and the Fiow increased by a Factor of 2... 

The gradient duration is not as important as generally assumed. For 6-8 peaks a gradient longer than about 5-7min is rarely necessary, see Figure 3.4. 

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