Analysis of unstable compounds using online SFE-SFC

Dr. Isabelle Möller Kontakt, Shimadzu Deutschland GmbH

Free radicals are short-lived molecular fragments of oxygen and are suspected of being responsible for aging processes but also for damage to cells or enzymes. Cells, however, have their own defense mechanisms to counter free radicals. Such antioxidants, like ascorbic acid or coenzyme Q10, react with free radicals and are provided by dietary intake.

UV-radiation, pollutants in the air or chemicals lead to the formation of highly reactive oxygen species, socalled free radicals in the human body. If they accumulate, they can cause cell damage and other signs of aging, for instance the formation of wrinkles. Free radicals are even associated with a number of diseases - including cardiovascular disease and cancer.

In addition to the body's own antioxidants mentioned above, substances provided by dietary intake can exhibit similar effects. The bestknown examples are vitamins or their precursors. This also includes coenzyme Q10 and its reduced form, which is structurally related to vitamin E and K.
Additionally to its antioxidant properties, coenzyme Q10 is also involved in important biochemical processes such as cellular respiration due to its easy oxidizability. So, how can such a sensitive and easily converted substance be carefully analyzed?

Supercritical fluids for gentle extraction

For substances that are sensitive to oxidation, analysis using supercritical carbon dioxide (CO2) offers a gentle alternative to conventional solvent extraction. Supercritical fluids combine the properties of gases and liquids: they are low in viscosity and exhibit high diffusivities similar to gases, but they are also readily soluble like liquids.

CO2 (at or above its critical temperature and critical pressure) is the most commonly used supercritical fluid for chromatographic purposes. In addition to its suitable physicochemical properties and availability, it is highly inert, non-toxic and inexpensive.

So far however, techniques like SFC (super critical fluid chromatography) and SFE (supercritical fluid extraction) could only be used as individual steps in an analysis. The newest generation of instruments now combines the entire analysis, integrating sample pretreatment, chromatographic separation and detection into a single system. With the online coupling of SFE and SFC, manual extraction and transfer steps are now replaced by a fully automated process. This does not only reduce time and personnel expenditure but also eliminates manual errors that can occur, e.g. during decanting or pipetting.

An additional advantage of the closed online SFE-SFC system is the gentle sample handling. After being placed in the instrument, the samples can be analyzed virtually without decomposition, as the entire analytical process takes place without exposure to light or air and free from humidity. Therefore, the system is ideal for the analysis of sensitive samples, for instance lightsensitive, easily oxidized or readily hydrolyzed substances.

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