A comparison of two methods for the determination of dead time of a column in reverse-phase HPLC
Abstract
The dead times of chromatographic systems, determined using two different methods, were compared in this work. For one method, uracil was used as a dead time marker. As for the other method, the dead time was calculated by the retention of homologues – hexyl, heptyl, octyl, and nonyl esters of para-nitrobenzoic acid. The conducted studies showed that the resulting times could vary significantly. Thus, in eluents with methanol as an organic modifier of the mobile phase, the dead time determined by the retention of uracil was greater than the same parameter calculated by the retention of homologues. Meanwhile, for acetonitrile-based eluents, the ratio of the obtained parameters was the opposite: the dead time calculated by ether retention was greater than the time determined by uracil. The methylene difference, which was recognised as constant for homologues, was preserved only when using the dead time calculated by the retention of esters used in the work. Therefore, for all physico-chemical calculations, it is necessary to determine the retention mechanism of each sorbate and choose a suitable method for calculating retention factors and related parameters (for example, the enthalpies of sorbate transfer from the mobile phase to the stationary phase) in order to obtain the correct retention factors. To solve the issue of the retention mechanism, it was proposed to use two columns with stationary phases of the same brand, but with different lengths of the grafted radical. If the C18 phase was used, then its replacement, for example, with the C8 phase with the same composition of the mobile phase and temperature should lead to a reduction in retention time by more than twice with a distributive retention mechanism. The differences in the results obtained for mobile phases based on methanol on the one hand and acetonitrile on the other require additional research.
Downloads
References
Fekete S., Fogwill M., Lauber M.A. Pressure-Enhanced Liquid Chromatography, a Proof of Concept: Tuning Selectivity with Pressure Changes and Gradients. Anal. Chem. 2022; 94: 7877-7884. https://doi.org/10.1021/acs.analchem.2c00464.
Chen S.-H., Li C.-W. Thermodynamic studies of pressure-induced retention of pep-tides in reversed-phase liquid chromatog-raphy. J. Chromatogr. A. 2004; 1023: 41-47. https://doi.org/10.1016/j.chroma.2003.09.057
Martin M., Guiochon G. Effects of high pressure in liquid chromatography. J. Chro-matogr. A. 2005; 1090: 16-38. https://doi.org/10.1016/j.chroma.2005.06.005
McGuffin V.L., Chen S.-H. Theoretical and Experimental Studies of the Effect of Pressure on Solute Retention in Liquid Chromatography. Anal. Chem. 1997; 69: 930-943. ttps://doi.org/10.1021/ac960589d
Ohmacht R., Boros B. Effect of Pres-sure on Solute Capacity Factor in HPLC Us-ing a Non-Porous Stationary Phase. Chroma-togr. Suppl. 2000; 51: S-205-S-210. https://doi.org/10.1007/BF02492807
Fallas M.M., Neue U.D., Hadley M.R., McCalleya D.V. Investigation of the effect of pressure on retention of small molecules us-ing reversed-phase ultra-high-pressure liquid chromatography. J. Chromatogr. A. 2008; 1209: 195-205. https://doi.org/10.1016/j.chroma.2008.09.021
Deineka V.I., Chulkov A.N., Blinova I.P. Dependence of the solute retention on the column pressure in reversed-phase HPLC. Mendeleev Commun. 2023; 33: 436-437. https://doi.org/10.1016/j.mencom.2023.04.044
Rimmer C.A., Simmons C.R., Dorsey J.G. The measurement and meaning of void volumes in reversed-phase liquid chromatog-raphy. J. Chromatogr. A. 2002; 965: 219-232. https://doi.org/10.1016/S0021-9673(02)00730-6
Berendsen G.E., Schoenmakers P.J., de Galan L., Vigh G., Varga-puchony Z., Inczédy J. On the Determination of the Hold-Up Time in Reversed Phase Liquid Chroma-tography. J. Liq. Chromatogr. 1980; 3: 1669-1686. https://doi.org/10.1080/01483918008064759
Krstulović A.M., Colln H., Guiochon G. Comparison of Methods Used for the De-termination of Void Volume in Reversed-Phase Liquid Chromatography. Anal. Chem. 1982; 54: 2438-2443. https://doi.org/10.1021/ac00251a009
Tsopelas F., Ochsenkühn-Petropoulou M., Tsantili-Kakoulidou A. Void volume markers in reversed-phase and biomimetic liquid chromatography. J. Chromatogr. A. 2010; 1217: 2847-2854. https://doi.org/10.1016/j.chroma.2010.02.062
Perry P.R., Coym J.W. Comparison of common mobile-phase volume markers with polar-group-containing reversed-phase sta-tionary phases. J. Sep. Sci. 2010; 33: 2310-2315. https://doi.org/10.1002/jssc.201000176
Dorsey J.G. Dill K.A. The Molecular Mechanism of Retention in Reversed-Phase Liquid Chromatography. Chem. Rev. 1989; 89: 331-346. https://doi.org/10.1021/cr00092a005
Tijssen R., Schoenmakers P.J., Böh-mer M.R., Koopal L.K., Billiet H.A.H. Lat-tice models for the description of partition-ing/adsorption and retention in reversed-phase liquid chromatography, including sur-face and shape effects. J. Chromatogr. A. 1993; 656: 135-196. https://doi.org/
1016/0021-9673(93)80801-E
Deineka V.I., Deineka L.A., Saenko I.I., Chulkov A.N. A Float Mechanism of Retention in Reversed-Phase Chromatog-raphy. Russ. J. Phys. Chem. A. 2015; 89: 1300-1304. https://doi.org/10.1134/S0036024415070079
Burzhinskaya T.G., Deineka V.I., De-ineka L.A., Selemenev V.F. Chromatograph-ic determination of zeaxanthin in some varie-ties of Сapsicum annum. Sorbtsionnye i khromatograficheskie protsessy. 2022. 22(1): 12-20. https://doi.org/10.17308/sorpchrom.2022.22/9016 (in Russ.)
Sentell K.B., Dorsey J.G. On the cal-culation of the stationary phase volume in reversed phase chromatography. J. Liq. Chromatogr. 1988; 11: 1875-1885. https://doi.org/10.1080/01483918808069031
Subirats X., Casanovas L., Redón L., Rosés M. Effect of the solvent on the chro-matographic selectivity in reversed-phase and HILIC. Adv. Sample Preparat. 2023; 6: 100063. https://doi.org/10.1016/j.sampre.2023.100063
Deineka V.I., Nguyen A.V., Deineka L.A. Model of a Reversed Phase Grafted on Silica Gel. Russ. J. Phys. Chem. A. 2019; 93: 2490-2493. https://doi.org/10.1134/S0036024419120057
Bayer E., Paulus A., Peters B., Laupp G., Reiners J. Albert K. Conformational be-havior of alkyl chains of reversed phases in high-performance liquid chromatography. J. Chromatogr. 1986; 364: 25-27. https://doi.org/10.1016/S0021-9673(00)96192-2
