Harmonization of Experimental Methods Used to Measure the True Hold-Up Volume of Liquid Chromatography Columns

Abstract

There are as many measurement values of the true column hold-up volume, V0, as techniques applied to evaluate this most important property in liquid chromatography (LC). The relative errors made on V0 measurements using conventional “non-retained” markers—such as acetone, uracil, or thiourea in reversed-phase liquid chromatography (RPLC), or benzene or acenaphthene in hydrophilic interaction chromatography (HILIC)—can be as large as ±30%. This situation is extremely confusing for LC users who wish to classify and predict the retention behavior of LC columns. In this work, along with advances in mass spectrometry (MS) instrumentation, the hold-up volume of any LC column—including, but not limited to, RPLC, HILIC, ion exchange chromatography (IEX), and mixed-mode columns—is accurately measured by injecting labeled deuterated acetonitrile (CD3CN) molecules and detecting them selectively by MS-single ion reaction (m/z = 45) using non-labeled and pure acetonitrile (CH3CN) as the eluent. This proposed harmonization of all conventional V0 measurement methods is illustrated and successfully applied to RPLC, HILIC, anion exchange (AEX), and RP-AEX mixed-mode chromatography, irrespective of the mobile phase composition selected.