Gradient run time

Let us now compare columns of different lengths packed with different particle sizes as a function of the gradient run time. We also use a fixed column diameter, since we want to work with different flow rates. This allows us to see how the expansion and contraction of the gradient with different flow rates affects the peak capacity. In all the following studies, we will use a column diameter of 4.6mm. As seen above in our isocratic examples, the highest pressure will be 25 MPa (250 bar, 4000 psi). 

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. 

For very long gradient run times, for example 6h, we can reach a peak capacity of around 250 with this column. The flow rate for this gradient would be around O.hmlVmin. For more realistic conditions, for example a 45-min gradient, a peak capacity of 194 can be achieved at a flow rate of 1.4mL/min. Even for a 45-min gradient, the flow rate is... 

Calculation of In k0 and S values The retention times tgl and tg2 for a solute separated under conditions of two different gradient run times (rG1 and tG2, where tG1 < tG2) can be given by the following equations 33,40... 

Optimization of the gradient time, tG, over the entire gradient range The retention factor k is a linear function of the gradient run time tG if A(p is kept constant. Hence,... 

The optimized gradient run time /grrm can be obtained from the RRM or alternatively, from the plot of Rs versus tG, and yields for each analyte the new values of knev by /grrm being multiplied with C ... 

Experimental design methodology was applied for the optimization of the elution program for the RP-HPLC resolution of a mixture of nine phenols, with a ternary solvent system (water-AcOH-MeCN). Important factors were the initial isocratic elution, the gradient running time and the gradient curvature . ... 

The common gradient for peptide separation consists of a binary solvent system containing in solvent A 0.1% formic acid and in solvent B 0.1% formic acid and 84% acetonitrile. If no high-sensitive MS detection is required, formic acid can be replaced by TFA. For solvent B, the amount of TFA has to be reduced to 0.08% to prevent baseline shift during the gradient run time. 

For the sake of simplicity, we assume that we are running a gradient separation with a run time r,. In this case, all peaks can be assumed to have the same width w, measured in the same units as the gradient run time. If we have n number of compounds in the sample matrix, and if they arc randomly distributed over the chromatogram, the number of compounds n that interfere with the quantitation of our analyte is simply... 

If more than 25% of the gradient run time is occupied with peaks, a gradient separation is better or even the only possibility however, the %B span can be less than from 10 to 100%. A hypothetical peaks pattern is shown in the upper half of Fig. 18.2. 

Fig. 18.14 Optimization of gradient run time and temperature. (After J. W. Dolan et al., J. Chromatogr. A 803, 1 (1998).) Conditions sample, algal pigments column, 25cm x 3.2 mm i.d. stationary phase, Vydac 201tp53 Ci8, 5iim mobile phase, 0.65mlmin gradient from 70 to 100% methanol in 28 mM tetrabutylammonium acetate buffer pH 7.1. Top computer simulation of a separation at 57°C and 80 min gradient run time middle computer simulation with three fused peak pairs at 55°C and 54 min bottom experimental chromatogram under these conditions.

Figure 18.14 represents an example of the computer-assisted optimization of linear gradient run time and temperature. It was necessary to perform four preliminary experiments which included two different temperatures (50 and 60 °C) and two gradient run times (17 and 51 min). From the data of these four runs the optimum conditions were calculated by the computer to be 57 °C and 80 min. The simulated chromatogram of this proposal is shown on top of Fig. 18.14. Some peak pairs are not resolved better than with R = 0.7. Since not all peaks are of equal scientific importance, it was decided that peaks 8, 9, 12, 13, 15, and 16 did not need to be resolved. With this facilitation the computer proposed 55 °C and 54 nun (nuddle). The real chromatogram is almost identical with the simulated one (bottom).

This gradient system has been adapted for the analysis of coastal tind interstitial waters where compounds derived from amino acid degradation such as P-alaavae, taurine or amino butyric acids may occur in addition to the standard amino acids given in Table 26-1. For less complex samples such as, e.g., hydrolysates the gradient run time may be abbreviated and a linear gradient employed. It should, however, be noted that under these conditions glycine and threonine are usually not separated. 

Fig. 1. Simulated representation of the effects that the factors gradient run time and column temperature have on the quality of a chromatographic separation. Separation is insufficient in the blue zones, which is an indication of co-elution of peaks all peaks are sufficiently well separated in the red zones. On the left-hand-side of the figure, there is a color scale for correlating the worst separated...

Therefore, it was considered how more than two chromatographic factors, e.g. stationary phase, column temperature, gradient run time, solvent, gradient rise, etc., could be tested systematically in one experiment. With column thermostats, which were specifically developed for this purpose (Model H ELIOS, AnaConDa), a maximum of twelve columns and an arbitrary number of temperatures can be... 

Fig. 5. Chromatograms of a mixture of steroid contaminants obtained with the reversed phase SynergiPOLAR RP. From top to bottom column temperature 15 °C, gradient run time 30 min and 100 min column temperature 45 °C, gradient run time 30 min and 100 min.

Column no. 1 (SynergiPOLAR RP) and column no. 5 (XTerra RP18) come to the fore when compared with the others (Pig. 8). The dependence of the separation quality on the column temperature and gradient run time for column no. 1 is shown in Pig. 9. Pigure 10 presents an example of a stationary phase on which the separation does not improve by varying the two parameters of column temperature and gradient run time. 

Fig. 9. Representation of the resolution with SynergiPOLAR RP as a function of column temperature and gradient run time, x-axis column temperature y-axis gradient run time ...

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