Hyphenation of Supercritical Fluid Chromatography and NMR with In-Line Sample Concentration

Abstract

By coupling supercritical fluid chromatography (SFC) and nuclear magnetic resonance (NMR) in-line, a powerful analytical method arises that enables chemically specific analysis of a broad range of complex mixtures. However, during chromatography, the compounds are diluted in the mobile phase, in this case supercritical CO2 (scCO2), often resulting in concentrations that are too low to be detected by NMR spectroscopy or at least requiring excessive signal averaging. We present a hyphenated SFC-NMR setup with an integrated approach for concentrating samples in-line, which are diluted in scCO2 during chromatography. This in-line concentration is achieved by controlled in-line expansion of the scCO2. As a proof of concept four isomers of vitamin E (tocopherol) were isolated by SFC, concentrated in-line by expanding CO2 from 120 to 50 bar, and finally shuttled to the NMR spectrometer fitted with a dedicated probehead for spectroscopic characterization of microfluidic samples. The abundant isomers were readily detected, supporting the viability of SFC-NMR as a powerful analytical tool.

The blind men and the elephant: challenges in the analysis of complex natural mixtures

“…Supercritical uid extraction (SFE) coupled to both MS (DOI: 10.1039/c9fd00011a) and in-line sample concentration for NMR (DOI: 10.1039/c8fd00237a) were illustrated and discussed in terms of the selection of analytes extracted in this manner. The audience learnt that modications of the supercritical uid can readily allow for the analysis of both non-polar 14 and polar analytes with NMR spectroscopy (DOI: 10.1039/c8fd00237a): by simply adding methanol to CO 2 , one can shi the mobile phase from non-polar to be more polar. A different approach highlighted for NMR analysis was to use viscous materials such as sucrose or 1% agarose gels to enable spin-diffusion during NMR acquisitions (DOI: 10.1039/c8fd00226f), an alternative to the popular DOSY-NMR approach.…”

Characterising polar compounds using supercritical fluid chromatography–nuclear magnetic resonance spectroscopy (SFC–NMR)

“…In our previous work we have demonstrated that by expanding the sample plug in-line in a controlled fashion, the sample and co-solvent are separated from the mobile phase, CO 2 . 3 In this way, the sample is concentrated in a smaller volume, which leads to a higher concentration, which is needed for NMR detection. This method will be employed here as well.…”

“…In our group, we have recently developed a new setup, which combines supercritical uid chromatography (SFC) and NMR spectroscopy. 3 In this work we demonstrated the separation by SFC of a mixture of tocopherol (vitamin E) isomers, followed by in-line concentration and NMR detection. Although tocopherol is a nonpolar molecule and therefore very different from the mostly polar molecules present in urine, we are adapting the setup to analyse polar metabolites in urine.…”

“…This concentration is sufficient in a standard NMR setup, however in our previous SFC-NMR setup we needed samples in the mM range to be able to detect them within 50 minutes. 3 A higher signal can be gained by acquiring over a longer period of time, therefore lower concentrations may also be detectable.…”

An Optimized NMR Stripline for Sensitive Supercritical Fluid Chromatography-Nuclear Magnetic Resonance of Microliter Sample Volumes

“… 8 An interesting development is the hyphenation of supercritical fluid chromatography (SFC) with stripline NMR. 9 , 10 …”