August 16, 2021
Jack Silver, Applications Specialist
Proper Equilibruim: Don't start normal phase gradients at 0% B!
Flash chromatography is often run using default gradients built into the flash chromatography system. This usually produces a reasonable purification with minimal work on method development. When running normal phase, these gradients typically start at 0% B (the strong solvent). Reverse phase gradients start at 5% to 10% B to avoid phase collapse, and so will not be discussed here. Most compounds elute well with the standard gradients. Compounds that need only a small concentration of B solvent, however, will often elute later than expected, and may elute together.
The reason for this is shown in Figure 1. On the CombiFlash NextGen 300+, no sample was run, and baseline-correction was turned off to track the solvent adsorption. In this run, the solvent trace failed to follow the gradient. The step-in absorbance occurred after the “hot band" eluted from the column.
Figure 1- Ethyl acetate gradient monitored at 230 nm.
The observed “hot band" is nothing more than the heat of adsorption of ethyl acetate onto silica. As the silica is adsorbing all of the ethyl acetate, no compound can elute before ~4.7 column volumes (CV). This is analogous to the “solvent demixing" phenomenon described by Snyder & Kirkland[i].
Proper elution is achieved by starting the run at ~2% B solution and extending the equilibration to 7 to 10 CV to allow the ethyl acetate to adsorb onto the silica. As an alternative to the long equilibration, one can use manual control to wash the column with 10% B solvent for 4 CV, and equilibrate normally while still starting the gradient at 2% B.
Figure 2- Acetophenone using a gradient starting at 0% B (top) and 2% B (bottom).
Aside from hexane, dichloromethane/methanol gradients also benefit from starting at a low B concentration. Additionally, separations using a small amount of modifier such as triethylamine also benefit from a longer equilibration for this same reason.
 Snyder, L.R.; Kirkland, J.J. Liquid-Solid Chromatograph. In
Introduction to Modern Liquid Chromatography, second edition. John Wiley and Sons, New York, 1979, 99 386
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From Gary at SFU 8-27-2021: The lower trace didn't seem to offer any advantage at all---more manual operations, same amount of time, less symmetrical and fatter peak. It would have been more convincing to show a mixture separation case in which starting from low B% gave separation while starting from 0% B didn't.
Jack Silver, Teledyne ISCO 9-9-2021: The peak width is what would be normally expected for a compound eluting during such a gradient. However, when the column is equilibrated only with the weak solvent, the peak(s) that would elute prior to the complete adsorption of the weak solvent co-elute. The peaks are narrow because of the gradient pushing the back end of the peak down the column with a much higher concentration of solvent than needed for elution.
I agree that showing a mixture would be more effective, however this example does demonstrate a compound being held up due to adsorption of the B solvent. Most of the time, compounds elute sufficiently late in the gradient that this sort of “dewetting” isn’t a problem. This blog post was prompted by a customer who had a mixture of non-polar compounds who declined to allow us to share their chromatograms. We will revisit this topic when we have a suitable experiment as you describe, but the problem is sufficiently troublesome that we posted with what data we can share.
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