How to seperate two components by RP-HPLC having very near pKa values ie 12.98 and 13.38?
Is this prep or analytical LC? How are you detecting the compounds? If you are using a mass spectrometer or light scattering detector, you will not be able to use certain buffers.
Many reverse phase columns are silica based, and shouldn't be run above pH 7.5. However, this is a low enough pH to keep the compounds ionized as their conjugate acids and allow separation. Polymer columns don't have this limitation.
not sure what the question really is, but if you want to develop separation You can:
work with columns of different separation mechanisms a) C18 for pure vander Waals interactions, C18 modified with Phenyl to utilise PP interactions, Cyanopropyl or more polar columns to use dipoles or charges for the separation.
You can also modify Your eluent: acidic/non acidic, ion strength by modifying buffer concentration, MeOH based RP gradients versus ACCN based ones...
Mr. Mardia,
Generally, chromatographic data should be processed statistically in chemometric terms in order to evidence the reliability of the data even looking at one analyte.
In this context, please consider some results from our work in preparation [1], which show that looking at statistically representative set of chromatographic analyses even of one compound, there is unable very frequently to assign adequately even the data of the peak position depending on experimental conditions (see attachment presentation-RG.pdf.)
For instance, see data on Table A5 of the latter attachment. As can be seen, statistically the RTs of 3 peaks of 3 analytes (in total, there are 20 replicates of the samples) are significantly different from the mean value of standard samples at the studied concentration (ng.(L)-1.) The standard deviation is at the second decimal sign. However, the analyses are carried out using standard samples of analytes at the different concentrations. The chromatographic data, however, prevent adequate quantification of analytes in solution at the shown analyte concentration level; and, even qualitatively.
The shown results are part of our systematic research effort devoted to develop exact quantitative method for analysis, but based on mass spectrometry.
Please, consider, also works [2,3]. Therein, you shall find that unambiguous quantitative analysis can be carried out, currently, only when there is used mass spectrometry (not chromatography) within the framework of our own-authored (to me and my co-author according to the shown authorship below) stochastic dynamic theory and model formulas which provide exact quantification at analyte concetration levels up to pg.(mL)-1; thus, yielding to coefficients of linear correlation between analyte concentration and the instrumental measurable variable /r/ = 0.99997-1.
[1] B. Ivanova, M. Spiteller, Quantitative analytical aspects of the 3D mass spectrometric structural determination based on stochastic dynamics – analysis of steroids (part III) (2021/2022) in preparation.
[2] Stochastic Dynamic Mass Spectrometric Approach to Quantify Reserpine in Solution; Bojidarka Ivanova, Michael Spiteller, Analytical Chemistry Letters, 10 (2020) 703-721; Received 13 Oct 2020, Accepted 16 Dec 2020, Published online: 28 Jan 2021 Download citation https://doi.org/10.1080/22297928.2020.1865834
[3] Steroids, 164 (2020) 108750 Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller.
Many reverse phase columns are silica based, and shouldn't be run above pH 7.5. However, this is a low enough pH to keep the compounds ionized as their conjugate acids and allow separation. Polymer columns don't have this limitation.
Kindly visit..
https://www.researchgate.net/profile/Bojidarka-Ivanova/post/How_to_seperate_two_components_by_RP-HPLC_having_very_near_pKa_values_ie_1298_and_1338/attachment/6112a34c647f3906fc8f9673/AS%3A1055168925806596%401628583284927/download/Pr%C3%A4sentation1-RG.pdf
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