Lipids and SFC: The Perfect Match
Hello, and welcome back to the chromatography portion of the “Colorful Researchers” blog. In my previous blog, I discussed proteomics and peptidomics, the large-scale study of proteins and peptides. Today, in a similar vein, I want to discuss lipidomics, a relatively recent research field that studies cellular lipids in biological systems.
What is Lipidomics?
Lipidomics, a subset of metabolomics, is the large-scale study of pathways and networks of cellular lipids in biological systems. The lipidome refers to the complete lipid profile within a cell, tissue, or organism and encompasses thousands of lipid species that play critical roles in cellular functions.
Lipidomics has become essential in understanding metabolic diseases such as atherosclerosis, stroke, hypertension, and diabetes. By identifying and quantifying the various lipid species and their interactions, researchers can better understand the roles of lipids in cellular processes and disease mechanisms. This knowledge is crucial for developing targeted therapeutic strategies and advancing biomedical research. World Multiple Sclerosis (MS) Day just passed on the 30th of May, and the role of lipids on the underlying immunopathogenesis influences diagnosis and treatment. This rapidly expanding field complements the progress made in other areas of systems biology.
The structural diversity of lipids
Like proteins and peptides, lipids are a diverse group of compounds serving many critical biological functions. They help form the structural components of cells, provide energy storage, and participate in signaling pathways. Understanding and mapping these functions is vital for our understanding of biological systems. In my blog post about proteins and peptides, I mentioned the unknome project that lists the various proteins and highlights lesser-known proteins for further investigation. For lipids, I recommend checking out the LIPID MAPS® Structure Database (LMSD), the largest public lipid-only database in the world. I also love checking out the Lipid of the Month on their website; it’s a great way to keep up to date and learn more about the diversity of lipids.
How can you separate lipids?
For proteins and peptides, I recommended two techniques for their separation—Flash and prep HPLC. Flash as a pre-purification step to purify large amounts at a reasonable resolution and prep HPLC to achieve the highest resolution (purity). Traditionally, prep HPLC has been used to purify lipids using normal-phase (NP) or reverse-phase (RP). However, there are issues with this method that can be mitigated by using another method.
Can SFC be used to separate lipids?
When trying to analyze or purify lipids with RP-HPCL, the lipids can stick to the C18 column and will not elute. Using C18 columns in SFC is not typical; however, in the case of lipids, the stationary phase works well with supercritical CO2. There are also other benefits to using SFC to purify lipids. The complex nature of biological samples and matrices often results in very long chromatography methods with low throughput. Given that accurate statistics require many samples, the separation of lipids is expensive, time-consuming, and uses large amounts of solvents. SFC offers far shorter run times and can even provide higher resolution for specific lipid classes. SFC also offers considerable environmental benefits over HPLC and can be coupled with universal detectors (like UV or evaporative light scattering) and highly specific detectors (such as mass spectrometry).
What are the advantages and applications of SFC in Lipidomics?
Fatty Acyls
SFC is particularly advantageous for analyzing fatty acids due to its ability to use low temperatures, high flow rates, and direct sample infusion in n-hexane or n-heptane. Various fatty acids can be quickly and efficiently separated using a C18 column and a gradient mobile phase.
Glycerolipids
For triacylglycerols (TAGs), SFC offers rapid analysis without sample derivatization. Utilizing high temperatures and pure CO2, SFC can efficiently handle the polarity characteristics of TAGs, resulting in time-savings and improved separation compared to traditional methods.
Glycerophospholipids and Sphingolipids
Although the literature on using SFC for such applications is limited, SFC has been shown to provide notable advantages in isomer separation. This is crucial, given the functional and molecular complexity of these lipid classes.
Sterol Lipids
Sterol lipids consist of a steroid nucleus and are found in all eukaryotic organisms. Cholesterol is the primary sterol in vertebrates, and the ability to fine-tune SFC parameters makes it a powerful analytical technique for analyzing complex mixtures, such as sterol lipids.
Prenol Lipids and Polyketides
The prenol category of lipids includes subclasses like isoprenoids, polyprenols, quinones, and hydroquinones. The versatility of SFC is highlighted by its ability to separate various isoprenoids and polyketides, such as polymethoxylated flavones.
What about detection?
SFC is also compatible with powerful detection methods that offer several advantages regarding speed and reproducibility. Combining SFC with Mass Spectrometry (SFC-MS) is particularly effective for lipid analysis, for example, by allowing for the separation of equal carbon number triglycerides by degrees of saturation. This capability is unmatched by traditional Liquid Chromatography (LC) methods, making SFC-MS a powerful tool for lipidomics profiling.
The Perfect Match
Lipidomics is a rapidly expanding field crucial for understanding the complex roles of lipids in health and disease and the advent of advanced SFC techniques has significantly enhanced lipid analysis, providing superior efficiency, selectivity, and environmental benefits over traditional methods. SFC’s ability to handle complex lipid profiles, coupled with its compatibility with various detection methods, makes it an invaluable tool for researchers. As lipidomics continues to evolve, SFC will play a pivotal role in unraveling the complexities of lipid metabolism and paving the way for discoveries in biomedical science.
Phir Milenge Chalte Chalte,
Padma