The Future is Supercritical: How SFC is Transforming Natural Product Analysis

Hello, and welcome back to the colorful researcher’s blog. Today, I’d like to move on from my last post about chromatography columns and the importance of screening to discuss specific chromatography applications. As I mentioned in my previous post, supercritical fluid chromatography (SFC) can be up to five times faster than prep HPLC. It is eco-friendly and efficient, making chemistry cleaner and greener. One area where prep SFC has shined is in the purification of natural products, opening up new pathways for sustainability and efficiency in our labs.

Why prep SFC?

The allure of prep SFC lies in its use of supercritical fluids like carbon dioxide, which, at the correct pressure, behaves both as a liquid and a gas. This dual nature allows it to penetrate materials like a gas and dissolve substances like a liquid. Most importantly, especially for natural products, it does so without the environmental and health risks associated with traditional organic solvents. The food and flavor industries are particularly interested in SFC since carbon dioxide and ethanol are foodstuffs, and the fractions can be tasted. In prep HPLC, methanol and acetonitrile are unhealthy, and the fractions cannot be tasted without further steps. Additionally, if your target compound is liquid, separations can be run with 100% CO2 without requiring solvents or alcohols. You receive your fractions pure without additional drying steps. 

What are the Challenges of Natural Product Analysis?

Complexity and variability pose significant challenges in analyzing natural products. This reminds me of the various types of Biryanis found across Mumbai, which combine a wealth of ingredients to create a complex flavor profile. A traditional Biryani comprises rice, meat or vegetables, marinade, spices, seasonings, herbs, fresh onions, nuts, dried fruits, and ghee. Leaving marinades overnight helps combine the flavors, as do specific cooking techniques, like “Dum”, where the ingredients are steamed together to allow the flavors to meld. It would be challenging to ascertain the complete list of ingredients just by tasting once they have been combined. The same applies to isolating compounds in biological matrices like plant extracts. It is estimated that the plant kingdom contains over 200,000 metabolites essential for growth and reproduction (primary metabolites) and environmental competitiveness (secondary metabolites), with certain plants hosting several thousand metabolites. This variability puts a high demand on the analytical methods used for natural product analysis. 

The vast developments in prep SFC and column technology, with breakthroughs in selectivity, sensitivity, and speed that I discussed in my last blog, have gone a long way toward tackling the challenges posed by not only natural products but also environmental analysis, bioanalysis, and pharmaceutical analysis. SFC's versatility makes it practical for a wide range of natural product compounds of interest. 

What are the prep SFC Natural Product Applications?

Natural products have numerous properties that are useful for a wealth of applications. Whether it’s lipids, flavonoids, or terpenes, prep SFC can separate them efficiently. Here is a breakdown of some of the main categories of natural products that are exploited for a range of purposes:

• -    Lipids: These are insoluble in water and include fats, oils, waxes, and hormones. They function as energy-storage molecules and chemical messengers and are widely studied for their nutritional and medicinal properties. SFC is ideal for analyzing lipid compositions because it separates these non-polar molecules without the need for harmful solvents. 
• -    Carotenoids: These are tetraterpene pigments that exhibit yellow, orange, red, and purple colors. They are widely distributed in nature and present in photosynthetic bacteria, fungi, algae, plants, and animals. They are essential for human health, acting as antioxidants and precursors to vitamin A. SFC is excellent for separating carotenoids due to its short analysis time, efficiency, and ability to handle these sensitive and easily degradable compounds. 
• -    Terpenes: These are major biosynthetic building blocks responsible for the smell and flavor of many plants. These unsaturated hydrocarbons are produced predominantly by plants, particularly conifers, and are significant to the pharmaceutical and food industries. SFC’s ability to effectively separate chiral compounds allows the mixture (racemate), + molecule, and – molecule to be isolated. These molecules smell different, which is essential for the fragrance industry, among others.
• -    Saponins: These are secondary metabolites that are heat-stable, amphiphilic, glycosidic compounds that are naturally found in leaves, flowers, and fruits. They have various health-promoting properties and are widely used by the pharmaceutical industry. SFC helps in isolating saponins effectively due to its superior selectivity and resolution.
• -    Cannabinoids: These are a class of biological compounds that bind to cannabinoid receptors and are sourced from plants of the Cannabis genus, including sativa, indica, and ruderalis. They are used for their therapeutic properties, and SFC is particularly suited for their analysis because it can fractionate closely related compounds cleanly and quickly. 
• -    Flavonoids: These are phytochemical compounds present in many plants, fruits, vegetables, and leaves and have numerous potential applications. SFC is useful here due to its ability to separate complex mixtures of flavonoids. 
• -    Phenolics: This is a term used to describe a heterogeneous group of compounds with a benzene ring and a hydroxyl group(s). These antioxidants have numerous health benefits and exhibit a variety of functions, including plant growth, development, and defense. Phenolics are precursors to other significant bioactive molecules used by the therapeutic, cosmetic, and food industries. SFC’s capability to handle a range of polarities makes it ideal for analyzing phenolic compounds in complex matrices.
• -    Alkaloids: These are a class of naturally occurring compounds that contain at least one nitrogen atom. Due to this nitrogen atom feature, some synthetic compounds of similar structure may also be termed alkaloids. Again, SFC’s precision and flexibility make it essential to separate alkaloids for their use in pharmaceuticals. 

What Does the Future Hold for prep SFC?

The environmental friendliness of SFC makes it stand out as a superior choice for natural product extraction and analysis. It is already used in large-scale production to purify fish oils by separating fatty acids according to their size and degree of unsaturation without additional solvents. Exceptionally high levels of purity can be achieved, and unlike other methods, no chemical solvents are required or found in the finished product. It is used for palm oil processing, decaffeinating coffee, and isolating cannabinoids, carotene, and terpenes. The application possibilities are endless, and as the technology and methodology evolve, its scope and applications are expected to expand, making it a cornerstone technique in modern chromatography. The Future is Supercritical!

Phir Milenge Chalte Chalte,

Padma