My LC Blog: Selectivity (α)

Yes, this is a science blog, but no reason not to do it with a little bit of Christmas spirit. I hope the deeper meaning of the title picture will become clear with reading the text. And with Selectivity, in my opinion I saved the best for last, as in the last article for 2023. As already pointed out by Michael Dong in his comment on My LC Blog: The Resolution Equation, “One of the most useful concepts in practical method development for hplc/uv methods is the separation of critical pairs using selectivity tuning”. And today we’ll have a look at what that means “Selectivity” and what are the parameters that affect it, so that we can use them to improve an LC separation.

Selectivity (α) is defined as the difference in the relative retention of two analytes for given stationary phase and mobile phase conditions. In other words, α is a measure of peak spacing. It describes the separation of two peaks relative to each other and can be calculated as the ratio between the retention factors k2 and k1 of late- and early-eluting peaks (figure 1).

For details on retention and calculation of k, please refer to My LC Blog: Retention (k).? Retention factor (k) and selectivity (α) are both affected by parameters that determine retention. Accordingly, selectivity can be altered by changes in stationary phase chemistry, and mobile phase composition. So, what’s the difference then – you may ask. Let me try to explain by re-using a picture from an earlier blog.

In figure 2 you can see how an increase in retention may result in better resolution, but always at the cost of longer retention time, which in an isocratic run will also result in broader peaks. When you increase selectivity, you don’t have to necessarily increase retention, but rather change it, in a way that affects the different compounds to a different degree, to increase the space between them. This could mean reduce retention for one but maintain it for the second eluting compound.

In method development that would probably mean you identify a suitable combination of stationary and mobile phase in a screening experiment to gain adequate retention and some separation. From there you move to optimizing your method by looking at other parameters that affect selectivity to improve the separation. In my last article I covered the effect of changes in %organic modifier on retention, so that is one possibility to look at, and also changes in the pH of the aqueous mobile phase and column temperature will most likely result in your peaks moving around. Let me show that in some application examples.

Starting with temperature. According to the Van 't Hoff equation, if I we were to plot log k vs. 1/T (in Kelvin) this would result in a straight line, illustrating that higher temperature generally reduces retention and results in sharper analyte peaks, as it affects mobile phase viscosity. However, temperature also affects analyte solubility and ionization, as well as ionization of the chargeable functional groups of the stationary phase, which will result in changes in retention behavior of the different analytes as can be seen in the example in figure 3.

While a decrease in temperature of 5 °C and then 11 °C results in slightly increased retention for most of the analytes, the effect on compound 6 is much more pronounced, increasing the resolution of the critical pair (5, 6) to baseline separation.

The effect of changes in pH can be much less subtle, when the ionization of chargeable groups is changed due to the mobile phase conditions. In my example we’ll look at water soluble vitamins and at the movement of folic acid and p - aminobenzoic acid in particular. Structural formulas and ionizable functional groups for both compounds are given in figure 4.

At a pH equivalent to the pKa of an acidic or basic analyte 50 % of the molecules will be charged, while 50 % remain neutral, which can result in a split or broad peak for that compound. When considering mobile phase pH, it is therefore best to stay well away from the pKa (+/- 1.5), so that the analyte of interest is either fully charged or uncharged. However, when working with a range of analytes and some of them carrying multiple ionizable groups, that may not always be feasible. Figure 5 shows the separation of water soluble vitamins, including p-aminobenzoic and folic acid, where the aqueous mobile phase was adjusted to pH 2.2, 2.7 and 3.5 respectively.

Looking at p-aminobenzoic acid, we can see the peak (3) shifting to shorter retention with decreasing pH, as the carboxylic acid group (pKa = 2.7) changes from uncharged to charged, meaning more polar. Peak 4 on the other hand, with its multiple ionizable groups behaves less predictable and shows a strong increase in retention with a decrease in pH from 3.5 to 2.7. However, with a further decrease to pH 2.2, it moves to shorter retention, co-eluting with peak 5.? A more detailed explanation of the role of pH on retention and selectivity can be found in this article written by John Dolan and published in LCGC: Back to Basics: The Role of pH in Retention and Selectivity (chromatographyonline.com).

While writing this part I realized I haven’t really touched upon the choice of mobile phase with regards to ionic strength, pH or choice of buffer, so that’ll be a good start in the New Year. If you want to do some further reading on the topic of method development before that, I recommend Snyder, L. R., Kirkland, J. J. & Glajch, J. L. (2012); Practical HPLC Method Development, John Wiley & Sons, Inc.

That leaves me to wish everyone some more relaxed and joyful days as we approach the end of the year and all the best for a good start in 2024. I hope you keep reading.