Chiral and achiral separation of ten flavanones using supercritical fluid chromatography. Application to bee pollen analysis.

The separation of ten flavanones (flavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, naringenin, naringin, hesperetin, pinostrobin, and taxifolin) using supercritical fluid chromatography and considering achiral and chiral approaches has been studied in this work. For this purpose, different stationary phases and organic modifiers have been checked. Considering the achiral separation, the best results were obtained with the Lichrospher 100 Diol column at 35 °C, 3 mL/min, 150 bar and a gradient of 2-propanol from 5% to 50%. The baseline separation of the ten compounds was achieved in 18 min. Using the chiral column Chiralpak AD, the separation of the ten pairs of enantiomers was obtained in 32 min. In this case, the chromatographic conditions were 30 °C, 3 mL/min, 150 bar and the organic modifier was a mixture ethanol/methanol (80:20) containing 0.1% of trifluoroacetic acid applied in an elution gradient from 15% to 50%. The applicability of the proposed chiral method was assessed by analysing bee pollen samples and 2S-pinostrobin was determined in some of them.

Polymeric stationary phases based on poly(butylene terephthalate) and poly(4-vinylpirydine) in the analysis of polyphenols using supercritical fluid chromatography. Application to bee pollen.

Two new polymer-based stationary phases; DCpak PBT (poly(butylene terephthalate)) and DCpak P4VP (poly(4-vinylpirydine)) were evaluated for the analysis of polyphenols using supercritical fluid chromatography (SFC). The compounds studied included phenolic acids and flavonoids. The different variables that influence the chromatographic separation, such as type and percentage of organic modifier, additive, pressure and temperature were examined. Using the DCpak P4VP column the retention was exceptionally high, obtaining better results with the DCpak PBT column. The separation of nine polyphenols was achieved using a gradient of modifier (methanol with 0.1% trifluoroacetic acid) from 5 to 50%, a pressure of 150 bar, a temperature of 35 °C and a flow-rate of 2 mL/min. The use of additives was necessary in order to obtain good peak shapes and efficiencies, achieving the best results with trifluoroacetic acid. LODs and LOQs values were lower than 5 µg/mL in all the cases; meanwhile, the %RSD values for method repeatability and inter-day reproducibility were lower than 3% and 10% respectively. Finally, the proposed method was successfully applied to the analysis of polyphenols in commercial bee pollen; four compounds, namely cinnamic acid, p-coumaric acid, catechin and quercetin were identified and quantified.

Comprehensive two-dimensional gas chromatography with capillary flow modulation to separate FAME isomers.

A method to separate FAME and the linoleic and linolenic acids isomers by GCxGC using an apparatus equipped with a capillary flow technology (CFT) based modulator and a FID detector has been developed. Four different column combinations (one conventional and three inverted phase sets) were used in these experiments. The conventional set first involved a DB5-MS non-polar column followed by a highly polar HP-INNOWax column in the second dimension. The inverted phase set comprised of a highly polar BPX-70 column in the first dimension and a non-polar ZB5-MS column for the second dimension. Furthermore, the influence of the length of the second dimension column on FAME isomer separation was studied in the inverted phase sets, along with other parameters like the modulation time and column flow. The best results in terms of the time required for the analysis and number of FAME identified with the inverted set were achieved with the shorter second dimension column. After supercritical fluid extraction, the method was applied to identify FAMEs in broccoli leaves from three different cultivars (Naxos, Nubia and Viola).