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A new article discusses the benefits and drawbacks of RPLC and RPLC×RPLC chromatography techniques. It provides recommendations for selecting the best approach for analyzing small pharmaceuticals and peptides.
Researchers from the University of Geneva in Switzerland and Université de Lyon in France have conducted a study comparing one-dimensional reversed-phase liquid chromatography (1D-RPLC) and comprehensive two-dimensional RPLC (RPLC × RPLC). Their goal was to provide an unbiased comparison of the two techniques through calculations and experimental verification.
One-dimensional reversed-phase liquid chromatography (1D-LC RPLC) is a separation technique used to separate molecules based on their hydrophobicity. It involves a stationary phase, usually a porous material such as silica, that is coated with a hydrophobic material. The sample is injected onto the column and then eluted using a mobile phase consisting of a mixture of solvents. The molecules in the sample will interact differently with the stationary phase and the mobile phase, leading to their separation based on their hydrophobicity. The separated components can be detected using various detectors such as UV, fluorescence, or mass spectrometry.
Reversed-phase liquid chromatography (RPLC) × RPLC, also known as two-dimensional liquid chromatography (2D-LC), is a powerful analytical technique that separates complex samples in two dimensions. In this technique, the sample is first separated in the first dimension by RPLC, and then the effluent is separated in the second dimension by another RPLC column. By using two different RPLC phases in the two dimensions, a higher peak capacity and resolution can be achieved compared to one-dimensional RPLC. RPLC × RPLC has various applications in the analysis of complex samples, such as proteomics, metabolomics, and environmental analysis.
The study evaluated various quality descriptors, including peak capacity, analysis time, dilution factor, number of runs in the second dimension, and injection volume, with the same strategy being applied to small pharmaceuticals and peptides. The results showed that for the best compromise between peak capacity and sensitivity, short columns of only 30 × 2.1 mm packed with sub-2-µm particles should be selected in both dimensions of the 2D-LC setup, regardless of the analysis time.
The peak capacity in RPLC × RPLC was found to be significantly improved for analysis times beyond 5 min. However, extra-column volume located after the second-dimension column was found to be particularly critical for peptides, and up to 50% lower peak capacity was observed with MS vs. UV detection.
Contrary to common belief, higher dilution is not always observed in RPLC × RPLC. With adequate analytical conditions, better sensitivity (in theory fivefold and in practice three- to fivefold) could be achieved in RPLC × RPLC compared to 1D-RPLC, regardless of the analysis time.
The study was published in the Analytical and Bioanalytical Chemistry journal on November 3, 2022, under the title "Theoretical and practical comparison of RPLC and RPLC × RPLC: how to consider dilution effects and sensitivity in addition to separation power" (1). The authors of the study are Davy Guillarme, Florent Rouvière, and Sabine Heinisch.
(1) Guillarme, D.; Rouvière, F.; Heinisch, S. Theoretical and practical comparison of RPLC and RPLC × RPLC: how to consider dilution effects and sensitivity in addition to separation power? Anal. Bioanal. Chem. 2023, 415, 2357–2369. DOI: https://doi.org/10.1007/s00216-022-04385-w