Separation of Calendula officinalis L lipophilic compounds using ultra-high efficiency / low-pressure supercritical fluid chromatography (UHE-LP/SFC)

As a result of the decreased usage of organic solvents, and to great chromatographic efficiencies, supercritical fluid chromatography (SFC) successfully replaced in recent years non-aqueous reverse phase liquid chromatography (NARP-LC) for the separation of non-polar compounds. High relative amounts of various triterpenoid esters are present in the Calendula officinalis flowers, offering interesting bioactive properties. Because these triterpenoids present subtle structural differences on the aglycone moiety, their separation is challenging. This paper presents a method development to separate these compounds using ultra-high-performance low pressure supercritical chromatography (UHP/LP-SFC). The lipophilic fractions were obtained by supercritical fluid extraction (SFE), in accordance to the safety required from the cosmetic industry. Five coupled C18 columns were used, and an experimental design was applied to optimize their separation. The effects of temperature and mobile phase composition, i.e. the modifier percentage and the modifier composition (methanol/acetonitrile mixtures) were studied. Five criteria were considered: analysis duration, two discrimination factors of major triterpenoid esters, peak number and fully resolved peaks. The modelling from the experimental correlated to the studied analytical factors only shows significant results for the analysis duration. Therefore, derringer functions were employed to the criteria to select an optimal separation. Resulting in an optimized separation; in less than 20 min at 20 °C, with 30 % of a mixture MeOH/ACN 75/25 as modifier mixed with carbon dioxide. Centrifugal partitioning chromatography (CPC) fractionation and NMR analysis of fractions collected by CPC allowed to confirm identification of the two main peaks as faradiol myristate and faradiol palmitate. Other minor triterpenoid esters are well separated and based on NMR analysis and retention order an attempt of identification and proportion was done.