Why the retention factor needs to be kept in check

In chromatography, there is always the need to achieve a good balance between resolution, time and costs. The retention factor is also a part of this balancing act. There is an ideal retention factor range that helps you achieve high resolution at acceptable costs and run times. Read this post to find out what this retention factor range is and why these values are so golden.

It’s starting to get quite chilly outside, so I am busy thinking up nice indoor activities for the evenings and weekends. And I’ve decided to take up painting. I’m a colourful scientist and what is more colourful than painting? As a novice, I went to the store to pick out paints, brushes and a canvas.

At first, I was tempted to pick out a large canvas. I imagined my future landscape painting would look really nice, with beautiful resolution and detail on my wall. But then I started to think about the time it would take me to paint such a large picture. And then I had to consider the cost of how much paint it would take me to complete my vision.

So I settled for a smaller canvas size.

Actually, painting and chromatography have more in common than just colours. The need to achieve this delicate balance between high resolution, time and costs is prevalent in chromatography. Just as it takes more resources and time to paint a larger picture with better resolution, it also takes more time and solvent to achieve higher resolution during a separation.

Take the retention factor (k) as a great example of where balance is key to an optimized chromatography process.

Simply put, the retention factor is the ratio of the amount of time an analyte spends in the stationary phase to the time the analyte spends in the mobile phase. The stronger the interactions of the analyte with the surface, the longer the retention and the higher the retention factor.

There are several ways you can manipulate the retention rate:

  • Alter the mobile phase composition
  • Alter the nature of the stationary phase
  • Change the length of the column

Out of these, the first point, manipulating mobile phase composition, is the easiest way to increase the retention factor. This is frequently done by using a mobile phase that is a weaker solvent. When the mobile phase has lower solvent strength, solutes spend more time interacting with the stationary phase and need a longer time to elute.

That seems easy enough, but how can you determine the ideal value of the retention factor for your purification?

Well, the largest gain in resolution is achieved when the retention factor value is between 1 and 5. If the k value is lower than 1, analytes may be eluting with other sample components or with the solvent. K values above 5 only provide minimal increases in resolution.

This can be seen by the non-linear relationship between resolution and the retention factor:

efficiency, selectivity, retention, resolution, chromatography, prep HPLC, flash chromatography, preparative chromatography

Where we can see that resolution (Rs) as a function of k/(1+k) will increase with k, but not linearly.

The non-linear relationship between resolution and retention factor can also be displayed graphically  (courtesy of http://www.chromacademy.com/lms/sco2/Theory_Of_HPLC_Chromatographic_Parameters.pdf):

resolution, retention factor, chromatography, flash chromatography, preperative chromatography, prep HPLC

For more complex mixtures, the useful retention factor range is typically 2 < k < 10.

Why again? As shown above, once the retention factor exceeds a value of approximately 10, a further increase brings only marginal improvements in resolution. For example, if the original k value is 1, increasing its value to 10 gives an 82% increase in resolution. An increase of the retention factor to 15 provides a net improvement in resolution of only 87.5%
Theoretically, you can keep increasing the retention factor as you wish. But higher k values come at a cost, just as I need more supplies and more time to work with a higher canvas size when painting.

Whereas automation might make the cost of analysis time irrelevant, higher k values do make the separation slower. Even more, longer retention times result in broader peaks, which might be a problem for some applications.

Shorter run times, or smaller k values, will reduce the amount of solvent used. Lower solvent quantities reduce the purchase price, preparation costs and disposal costs associated with solvent use.

All things considered, a good balance of time, solvent costs and resolution is achieved when the retention factor is in the range:

retention factor range, resolution, chromatogrphy, flash chromatography, preperative chromatography, prep HPLC, liquid chromatography

If you have not achieved the desired resolution at k vales above 10, you might want to consider increasing the selectivity or efficiency of your separation instead and you can read about these factors in my previous blog posts.

I hope I’ve painted a clear picture of the retention factor and how to optimize its value for your flash or prep HPLC applications. If you want to know more about resolution in general, check out my previous blog post on the topic. Happy reading and stay colourful and warm.

Till next time,

The Signature of Bart Denoulet at Bart's Blog