Once you have a separation you may want to optimize it. You may wish to shallow out the gradient to improve the separation, or you may wish to shorten the run time. Taking the illustration above one can see that all of the peptides are out by 40 minutes. This does not mean that we can change this 80 min run into a 40 min run, but there is room for improvement. The first step in the optimization is to determine the %B at which the last peak elutes. If you look at the blue gradient line you might guess that the last peak elutes near 40%B but this would be incorrect. All HPLC systems have a gradient delay. The gradient delay is the time between when the software tells the pumps to start pumping at a certain mobile phase composition and the time it takes for that solvent composition to reach the column and have an effect. A good guess for a gradient delay is 10 minutes. This would mean that our guess for the final mobile phase composition for the 40 min peak would be approximately 30%B. To observe the gradient delay time look at the illustration above and observe that the baseline returns to the starting conditions at 70 minutes and not at 60.1 minutes when our pumps have gone back to 2% B. One must take care to avoid having the last peak elute on the "equilibration cliff", (at 70 min. in this example). This can be avoided by ending the gradient at a %B that is slightly higher than that required by the last component.
Based on the separation shown at the top of this section one could rewrite the gradient to look like this:
This would make the gradient shallower and possibly give a better peak separation. To shorten the run time one could rewrite the gradient to look like this:
This last change would cut 30 min. from the analysis time. Shorter analysis times are always better for work efficiency. With every minute you can cut from the HPLC method without sacrificing your chromatographic goals you will be rewarded with better work efficiency. With this change the last peak would most likely still elute at 40 minutes and the peptide separation would essentially remain the same as in the initial 60/60 analysis.
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