Graduation Semester and Year
Spring 2026
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Dr. Morteza G. Khaledi
Second Advisor
Dr. Purnendu K Dasgupta
Third Advisor
Dr. Saiful Chowdhury
Fourth Advisor
Dr. He Dong
Abstract
Comprehensive characterization of the lipidome remains a significant analytical challenge due to the immense structural diversity, wide dynamic range, and complex physicochemical continuum of lipid species. Conventional extraction methods, such as those by Bligh–Dyer and Folch, and one-dimensional liquid chromatography (1D-LC) often fail to resolve this complexity, leading to chromatographic overlap, severe ion suppression, and limited coverage of low-abundance or highly hydrophobic species. This dissertation addresses these bottlenecks by establishing a separation-driven analytical framework that integrates novel fluoroalcohol-induced multiphase systems (FAiTPS) with orthogonal multidimensional liquid chromatography–mass spectrometry (LC×LC–MS).
The core of this research is the development and physicochemical characterization of FAiTPS—a quaternary solvent system composed of dichloromethane (DCM), hexafluoroisopropanol (HFIP), 1,3-propanediol (PD), and water. This system generates three reproducible, immiscible phases spanning a broad polarity continuum. A fundamental aspect of this work involves investigating the role of HFIP as a strong hydrogen-bond donor, which facilitates unique lipid partitioning behavior. The system enables polarity-driven fractionation, where highly hydrophobic lipids (e.g., triacylglycerols) enrich in the DCM-rich middle phase, amphiphilic lipids (e.g., phospholipids and sphingolipids) partition into the HFIP-rich bottom phase, and hydrophilic species remain in the aqueous top phase.
Key findings demonstrate that FAiTPS significantly outperforms traditional extraction protocols. When applied to complex biological matrices such as yeast and human plasma, FAiTPS improved lipidome coverage by over 150%, specifically enriching underrepresented and biologically significant classes like ether-linked phospholipids, ceramides, and hexosylceramides. Furthermore, the integration of FAiTPS with offline HILIC×RPLC–MS provides a hierarchical deconvolution of lipid complexity by separating species first by headgroup polarity and subsequently by acyl chain hydrophobicity.
A systematic evaluation of four distinct experimental workflows—varying in extraction timing and fractionation order—revealed that applying FAiTPS fractionation prior to HILIC separation (the Pre-HILIC FAiTPS workflow) yields the highest number of unique lipid identifications and the most balanced physicochemical representation. Detailed analyses based on lipophilicity (LogP), aqueous solubility (LogS), and carbon number distributions confirm that FAiTPS expands the detectable chemical space in a predictable and robust manner. By reducing ionization competition and chromatographic co-elution, this multidimensional strategy enhances detection sensitivity for low-abundance species. Ultimately, this work provides a mechanistic link between solvent-driven partitioning and orthogonal separation, establishing a versatile, sustainable, and high-performance platform for untargeted lipidomics with broad implications for biomarker discovery and translational research.
Keywords
Lipidomics, Fluoroalcohol-Induced Multiphase Systems (FAiTPS), Multidimensional Liquid Chromatography (LC×LC), Mass Spectrometry (LC–MS), Separation-Driven Analytical Framework, Polarity-Resolved Fractionation, Lipidome Coverage Enhancement
Disciplines
Analytical Chemistry | Biochemistry | Bioinformatics
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Amin, Md Al, "ADVANCING LIPIDOMICS THROUGH FLUOROALCOHOL-INDUCED MULTIPHASE FRACTIONATION AND ORTHOGONAL LC×LC–MS DETECTION" (2026). Chemistry & Biochemistry Dissertations. 4.
https://mavmatrix.uta.edu/chemistry_dissertations2/4