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

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