Towards an accurate method for column void volume determination using liquid chromatography-mass spectrometry

Ribar, David (author), Lukšič, Miha (author), Kralj Cigić, Irena (author)

Prediction of analyte retention times requires prior knowledge of the column void volume, the measurement of which is still highly contested within the literature and therefore experimental based prediction is often used. In this study, we investigated deuterated acetonitrile as an isotopically labelled mobile phase component to observe its elution behaviour in a binary mixture with water at 25 different mobile phase compositions (from 5 to 95 vol. % of acetonitrile), on two stationary phases (C8 and C18), and at two temperatures (30 and 40 °C) using LC-MS. The same experimental design was additionally used for three commonly used neutral void volume markers: uracil, phloroglucinol and N,N-dimethylformamide. Temperature was observed to influence the elution of acetonitrile in an inversely proportional manner with higher temperatures coinciding with lower elution times. By utilizing a three-way ANOVA, the composition of the mobile phase has been shown to have a significant effect on deuterated acetonitrile and other investigated void volume markers, demonstrating the fact that both void volume markers and acetonitrile itself exhibit retention-like behaviour. Excess adsorption isotherms for acetonitrile were calculated using deuterated acetonitrile elution data. The comparison of void volumes, obtained with conventional neutral void volume markers, revealed the former to be 24–36% lower than the void volume obtained using deuterated acetonitrile, as an isotopically labelled mobile phase component. For a water:acetonitrile mobile phase, the minor disturbance method using deuterated acetonitrile to obtain an integral average void volume (2.08 and 2.05 mL for C18 at 30 and 40 °C, respectively and 2.16 and 2.13 mL for C8 at 30 and 40 °C, respectively) was found to be the most appropriate method for determining the elusive column void volume.

• Language: English
• Keywords: reversed-phase liquid chromatography, void volume, void volume markers, retention mechanism, modelling
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2023
• Number of pages: 7 str.
• Numbering: Vol. 1706, art. 464245
• PID: 20.500.12556/RUL-148278
• UDC: 543.544.5:543.51
• ISSN on article: 0021-9673
• DOI: 10.1016/j.chroma.2023.464245
• COBISS.SI-ID: 159876099
• Publication date in RUL: 09.08.2023

Record is a part of a journal

• Title: Journal of chromatography. A
• Shortened title: J. chromatogr., A
• Publisher: Elsevier
• ISSN: 0021-9673
• COBISS.SI-ID: 6398471

Licences

• License: CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
• Link: http://creativecommons.org/licenses/by-nc-nd/4.0/
• Description: The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.

Secondary language

• Language: Slovenian
• Keywords: reverzno-fazna tekočinska kromatografija, mrtvi volumen, markerji mrtvega volumna, retencijski mehanizem, modeliranje

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P1-0153
• Name: Raziskave in razvoj analiznih metod in postopkov

• Funder: ARRS - Slovenian Research Agency
• Project number: P1-0201
• Name: Fizikalna kemija