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The selectivity (α) of an analytical system describes the ability to discriminate between sample components based on differences in chemical and physical-chemical properties.
The selectivity (α) of an analytical system describes the ability to discriminate between sample components based on differences in chemical and physical-chemical properties. In chromatographic terms, it describes the spacing between the apices of the peaks within the chromatogram (Figure 1) and is determined not only by the analyte properties, but also those of the stationary phase and eluent system.
The selectivity obtained by an analytical method is fundamentally important to chromatographic separations and, in combination with peak efficiency (crudely, peak width), will determine the resolution of peaks within the chromatogram. When selectivity changes, resolution may decrease, making peak area measurement less accurate and reproducible, as well as decreasing confidence in peak identification or spectral structural elucidation.
Selectivity changes can range from subtle shifts in relative retention of a single peak within the chromatogram to wholescale retention order swapping of every peak, and we need to be able to spot these changes and investigate the causes in a systematic and proficient manner. However, with so many variables effecting selectivity, it is sometimes difficult to know where to begin our diagnostic investigations.
Figure 2 shows an example of a subtle change in chromatographic selectivity (peaks 1 and 2) which led to difficulties with accurate peak quantitation.
As we can see from Figure 2, the selectivity change is also accompanied by a slight shift to later retention, and when retention shift occur, it’s a good idea to check the selectivity values for the peaks in question to confirm that a selectivity change has occurred. In this case it’s visually obvious that we have had a change to the chemistry of the system, and therefore we will go ahead and invoke the selectivity diagnostic checklist to investigate the issues.
It’s important to note that the checklist is in order of "ease and probably," for example, things which are simple to check are included earlier in the checklist, to make our investigations as efficient as possible.
Again, note that any volatile eluent additives may evaporate on standing and this may effect both retention and selectivity over long analytical campaigns or between batches where the eluent has been allowed to stand on the instrument.
Sometimes we are a little over ambitions when programming ballistic gradients, those which rise over a large range of eluent B in just a few minutes or even seconds (it can be argued the current method under investigation falls into this category). Unless we are using the highest efficiency, lowest dead-volume pumping systems, then the instrument may not be to able to accurately or reproducibly deliver the required gradient. Again, a detailed treatment of this topic is beyond the scope of our discussion here, but Reference 4 contains more details on this topic. Once any instrument problems have been rectified,
In our example, we followed the checklist to item number 5 and found that there was an error in the eluent preparation, which had been prepared volumetrically rather than gravimetrically. A 0.01% measure of formic acid prepared volumetrically had resulted in an aqueous solution pH of 3.22, whereas the gravimetric method resulted in a solution pH of 3.27. While this difference of 0.05 pH units may seems vanishingly small and inconsequential, when the eluent pH is so close to the pKa value of the analytes of interest, this had resulted in the change of chromatographic selectivity. The separations obtained from the freshly made eluent, compared to the last time the method was run as shown in Figure 3. The further point to be made here is that, in as far as is possible, methods should be designed with the eluent pH at least 1 (and preferably 2) pH units away from the pKa values of the analytes of interest. Undoubtedly the analyst who developed this method used fine control of pH to finesse this rather difficult separation, however, perhaps a better route would have been to have the analytes in their fully ionised or non-ionized forms and sought an alternative column chemistry and or organic modifier in order to obtain a satisfactory separation. Reference 5 contains more details on this type of alternative approach to method development.
It is also worthy of note that we also studied the gradient delivery performance for the instrument used, and the accuracy and reproducibility of the gradient profile we satisfactory, so this was ruled out as a potential issue.
In summary, changes in chromatographic selectivity can be among the most difficult to diagnose and correct. One should properly establish that selectivity has changed, and implementation of the diagnostic checklist outlined above represents a logical and time-efficient way in which to isolate and ultimately correct the problems with the chemistry of the separation. Wherever possible, chromatographic separations should be designed with robustness in mind, and where this is not possible, method specifications should be carefully written in order to highlight the key variables within the separation conditions.
Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.