Two-dimensional liquid chromatography as a tool for separating triacylglycerol isomers from natural matrices
Hettiarachchi, Bawanthi (2025-06-13)
Two-dimensional liquid chromatography as a tool for separating triacylglycerol isomers from natural matrices
(13.06.2025)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe2025061871600
https://urn.fi/URN:NBN:fi-fe2025061871600
Tiivistelmä
Stereospecific structural analysis of triacylglycerol (TG) in complex natural matrices is challenging. In this study, two-dimensional liquid chromatography was tested as a tool by combining reversed and chiral phases in online heart-cutting mode to facilitate isomeric separation.
The research aims to enhance the peak capacity and simultaneously separate TG isomers from complex natural samples. It is divided into three sections: familiarisation with the technology, instrument optimisation, and isomeric analysis of natural samples. Virgin olive oil was used as the natural matrix to simplify the process.
Due to the complexity of the optimisation process, first and second-dimension optimisation was conducted independently. Both dimensions were operated in online mode. The TG reference compounds were separated using a Nucleodur C18 ISIS (150 × 2.0 mm, 1.8 µm) column in the presence of an isocratic mobile phase composition of acetonitrile (55%) and isopropanol (45%) in the first-dimension. The multiple heart-cutting technique was used to sample the selected peak sections. The assigned cuts were further sent through the CHIRALPAK IF-3 (250 × 4.6 mm, 3 µm) column in the presence of acetonitrile with 0.1% ammonium acetate mobile phase to separate the cuts into enantiomers and regioisomers.
Using the optimised method, five TGs in the virgin olive oil sample were separated in the first-dimension, and the TG 16:0_18:1_18:1 peak was sampled using two cuts and further separated into its enantiomers in the second-dimension. The chosen stationary and mobile phase conditions only allowed the separation of TG 16:0_18:1_18:1 isomers present in the olive oil sample. However, by utilising cellulose-tris-(3,5-dimethylphenylcarbamate) columns in the presence of a hexane-2-propanol gradient, TG rac-18:1_16:0(sn-2)_18:2 enantiomers could be successfully separated.
This work sheds light on the qualitative analysis of complex natural matrices such as human milk fat, of which TGs could not be completely separated and studied using one-dimensional chromatography. The ability to do quantitative analysis is made possible by high-resolution sampling.
The research aims to enhance the peak capacity and simultaneously separate TG isomers from complex natural samples. It is divided into three sections: familiarisation with the technology, instrument optimisation, and isomeric analysis of natural samples. Virgin olive oil was used as the natural matrix to simplify the process.
Due to the complexity of the optimisation process, first and second-dimension optimisation was conducted independently. Both dimensions were operated in online mode. The TG reference compounds were separated using a Nucleodur C18 ISIS (150 × 2.0 mm, 1.8 µm) column in the presence of an isocratic mobile phase composition of acetonitrile (55%) and isopropanol (45%) in the first-dimension. The multiple heart-cutting technique was used to sample the selected peak sections. The assigned cuts were further sent through the CHIRALPAK IF-3 (250 × 4.6 mm, 3 µm) column in the presence of acetonitrile with 0.1% ammonium acetate mobile phase to separate the cuts into enantiomers and regioisomers.
Using the optimised method, five TGs in the virgin olive oil sample were separated in the first-dimension, and the TG 16:0_18:1_18:1 peak was sampled using two cuts and further separated into its enantiomers in the second-dimension. The chosen stationary and mobile phase conditions only allowed the separation of TG 16:0_18:1_18:1 isomers present in the olive oil sample. However, by utilising cellulose-tris-(3,5-dimethylphenylcarbamate) columns in the presence of a hexane-2-propanol gradient, TG rac-18:1_16:0(sn-2)_18:2 enantiomers could be successfully separated.
This work sheds light on the qualitative analysis of complex natural matrices such as human milk fat, of which TGs could not be completely separated and studied using one-dimensional chromatography. The ability to do quantitative analysis is made possible by high-resolution sampling.