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Bob Bickler

Can reversed-phase chromatography compound elution volume/time be predicted?

November 18, 2020 at 9:26 PM / by Bob Bickler

When developing reversed-phase flash chromatography methods it is important to understand the impact that a change in solvent ratio has on compound retention and, therefore, separation performance. Unlike normal-phase chromatography where you can optimize separations using TLC and a wide variety of solvents and solvent ratios, reversed-phase limits you to 3 to 4 solvents, including water, using either HPLC or small flash columns for method development. Those solvents include:

  • Water
  • Methanol
  • Acetonitrile
  • Tetrahydrofuran

One of the most frequently asked question I am asked is with what % organic do I begin my method development? This is a pertinent question as a 0-100% gradient is not necessarily a good method or even a good starting point.

You do need to consider sample solubility when determining the gradient starting point or % B. If your product is water or alcohol soluble, start with a low % B (e.g. 10%), if organic soluble (hexane, DCM, EtOAc, acetone), start with a high % B (e.g. 40%) and ramp 50% B over 10 CV, then adjust accordingly as outlined below.

I have posted on this previously but want to address another aspect of method development – predicting elution volume or time based on the amount of compound retention shift you see with a change in strong solvent %, e.g. % methanol or acetonitrile. Rather than just tell you, I believe it important to show you the relationship of compound retention and % B both graphically and in a table.

To illustrate the % B change impact on compound retention I set up a series of experiments with a gradient slope of 5%B/column volume (50% increase in B over 10 column volumes). Changing gradient slope is another variable in method development to be covered in a different post.

This 5% slope paradigm enabled me to run the following 10 column volume (CV) acetonitrile gradient experiments to determine the correlation.

  • 10% to 60% B
  • 20% to 70% B
  • 30% to 80% B
  • 40% to 90% B

My sample was a 1:1 mix of methyl paraben (MP) and butyl paraben (BP) in acetone. My column was a 12-gram Biotage® Sfär C18, the flash system used was a Biotage® Selekt at 30 mL/min.

The four runs provided useful data points and provided me enough information to determine their correlation. The results show a linear correlation of compound retention and % B, Table 1, which is better seen in the graph in Figure 1.

Table 1. Elution volumes for methyl paraben (MP) and butyl paraben (BP) with different water/acetonitrile gradient starting points.

Start% B     MP (mL)     BP (mL)
10                    118              176
20                    82               141
30                    49               108
40                    31                69

Retention change with % B

Figure 1. Compound retention change with increasing % B shows a nearly linear correlation for both compounds.

With correlation factors of essentially 1, we can determine that the retention vs. % B slope is essentially linear. We also now know that we can use the slopes for each of the two compounds to predict their elution volume for any other 5% slope gradient starting between 10% and 40% B.

Putting this in practice, we know the methyl paraben slope is ~30 mL/10% B change or 3 mL/1% change and the butyl paraben slope per 10% change in B is ~36 mL (3.6 mL/1% change). So, for a 25% to 75% acetonitrile gradient, the predicted elution volumes for methyl paraben and butyl paraben are 64 mL and 126 mL, respectively.

How did I get there?

Methyl paraben -

Its slope = 3 mL/%; Slope (3 mL/%) x 5% change in gradient starting % (30% B – 25% B = 5%) = 3 mL/% x 5% = 15 mL; 15 mL + 49 mL (elution volume at 30% B) = 64 mL

Butyl paraben -

Slope = 3.6 mL/%; Slope (3.6 mL/%) x 5% change in gradient starting % (30% B – 25% B = 5%) = 2.6 mL/% x 5% = 18 mL; 18 mL + 108 mL (elution volume at 30% B) = 126 mL

The results show the prediction was very close and provided actual elution volumes of 62 mL and 123 mL, Figure 2.


Parabens 25-75% acn

Figure 2. Chromatogram of methyl and butyl paraben in a 25-75% acetonitrile gradient shows good correlation to predicted elution volumes.

Methanol/water gradients will also show linearity within specific starting points as well, when gradient slope is maintained.

So, when you are developing a reversed-phase method and your compounds elute too late or too early, consider changing the starting and ending % B while maintaining the gradient slope. Run a 10-60% gradient and a 40-90% gradient. If your target compound is not eluting and separating as desired, determine the retention slope and choose a starting % B which provides the needed retention.

For more flash chromatography information download our whitepaper - Successful Flash Purification.


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Topics: Chromatography Fundamentals, Reversed-phase, Method development

Bob Bickler

Written by Bob Bickler