50 February / March 2021


example [3]. If instead of relying on the r2


value, the accuracy of the


predicted retention time versus the actual retention time is evaluated, the results are shown in table 1, then an interesting observation can be made. What was previously shown, as robust reliable data where the reciprocal of the retention time varying as a linear function of the flow rate, can be seen not to be so robust since the accuracy at low flow rates can be seen to drop quite dramatically.


Figure 3. Log D plot for ketoprofen with the pKa chemicalize.org.


highlighted calculated from www.


One of the rules of thumb that separation scientists are taught early in their career is to always work 2 pH units away from the pKa


. In this case


Figure 2. Flow rate versus retention time, showing a linear response for both compounds.


Table 1. Accuracy of the predicted retention time as a function of the flow rate demonstrating that the relationship between flow rate and inverse retention time for ketoprofen is not linear.


Flow rate / mLs/min Accuracy Uracil 0.1 0.2 0.3 0.5 0.5 0.5 0.7 0.9 1.1 1.3 1.5 1.7 2


93% 99%


100% 101% 99%


100% 101% 101% 100% 100% 99%


100% 100%


Accuracy Ketoprofen 29% 86% 98%


105% 101% 104% 104% 103% 101% 101% 99%


100% 99%


Based on the accuracy data, the flow rate is affecting the retention factor for ketoprofen and in turn this implies that it is affecting the retention mechanism. After some searching of the academic literature it was noted that several authors had noticed that the pressure could


affect the retention times of compounds that are close to their pKa [4-9]. In this case increasing the flow rate was also increasing the pressure, and so closer inspection of the physiochemical properties of both analytes was undertaken. The log D plot for ketoprofen is shown in figure 3, with the pH for the pKa


the equilibrium between the charged and the uncharged forms of the molecule. Uracil has a pKa


pressure and hence flow rate in this case.


, in this case results in the molecule being present in its charged form which explains the reduction in the retention factor. Other artifacts can be also observed when analysing analytes close to the pKa


this would mean 3.9±2, which would mean that TFA would almost fit that rule, but formic acid would break it. The use of a pH that is within 2 pH units of the pKa


including peak distortion and irreproducible retention times. It should also be noted that changing the flow rate can also affect many other parameters that may be considered to be constant [24, including;


• physical dimensions of the column, • particle size and porosity, • phase ratio,


• column hold-up volume, • mobile-phase density and viscosity, • diffusion coefficients, • equilibrium constants, • and efficiency parameters.


Although pressure can affect all of these parameters the scale at which they are affected is very dependent on the exact physical configuration that is being used, and this will relate to the compound, the mobile phases and also the nature of the column housing. In this case it is the proximity of the mobile phase pH to the pKa


that is causing the shift in retention factor. Conclusion highlighted. Changing from


0.1% TFA to 0.1% formic acid changes the pH from 2.0 to 2.7, which is much closer to the pKa


and as a result changes in pressure will affect at a pH of 9 and so is not affected by


It is easy to make an assumption that data that does match the pre-conceived answer has been obtained from a poorly performed experiment. As scientists it is important that we are able to interrogate the data in an appropriate fashion and ensure that we promote the truth as opposed to making an assumption that the process is wrong, resulting in wrong data. Science is based on observation and some of the greatest scientific discoveries have been based on what might be termed failed experiments. In these cases, it was the determination of the scientist to uncover the truth that eventually delivered the real answer. These lessons can be readily applied outside the scientific


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