There is a great interest in searching for new environmentally sustainable techniques to enhance the use of agricultural byproducts. In this work, a response surface methodology was used to study the influence of the two independent variables, temperature (25-200 °C) and extraction time (3-17 min), in the extraction of antioxidants by pressurized hot water extraction (PHWE) from industrial apple byproducts. The optimized extraction method for determination of flavonols was at 120 °C and 3 min, giving a predicted total yield of flavonols of 1.3 μmol/g dry apple byproduct. Results obtained suggest that new antioxidant compounds were formed at the higher extraction temperatures. A desirability function response surface, considering maximum antioxidant capacity and minimal formation of brown color, was calculated and gave an optimum of 125 °C and 3 min. This latter PHWE method correlates well with the obtained results for flavonols; thus, a desirability function is a simpler alternative method for finding optimal conditions.

A method was developed based on supercritical fluid chromatography for quantitative determination of carotenoids in extracts of Scenedesmus sp. By utilizing the low backpressure in supercritical fluid chromatography, a C18 column and a 2-ethyl pyridine column were coupled in series. It was concluded that even minor changes in temperature had a substantial effect on selectivity. A standard mixture of 8 carotenoids and microalgae extracts obtained through supercritical fluid extraction with and without 10% ethanol as a co-solvent were successfully separated. All of the carotenoids were separated within 10 minutes, while the total analysis time was 20 minutes. The method was validated and the carotenoids of microalgae extracts were quantified. Furthermore, the method should be seen as a more rapid and environmentally sustainable alternative to traditional high-performance liquid chromatography methods utilizing organic solvents.

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In this study, a rapid and sensitive ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) method has been developed and partially validated for the separation of carotenoids within less than 6 min. Six columns of orthogonal selectivity were examined, and the best separation was obtained by using a 1-aminoanthracene (1-AA) column. The length of polyene chain as well as the number of hydroxyl groups in the structure of the studied carotenoids determines their differences in the physiochemical properties and thus the separation that is achieved on this column. All of the investigated carotenoids were baseline separated with resolution values greater than 1.5. The effects of gradient program, back pressure, and column temperature were studied with respect to chromatographic properties such as retention and selectivity. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared in both positive and negative mode, using both direct infusion and hyphenated with UHPSFC. The ESI in positive mode provided the highest response. The coefficient of determination (R (2)) for all calibration curves were greater than 0.998. Limit of detection (LOD) was in the range of 2.6 and 25.2 ng/mL for α-carotene and astaxanthin, respectively, whereas limit of quantification (LOQ) was in the range of 7.8 and 58.0 ng/mL for α-carotene and astaxanthin, respectively. Repeatability and intermediate precision of the developed UHPSFC-MS method were determined and found to be RSD < 3 % and RSD < 6 %, respectively. The method was applied in order to determine carotenoids in supercritical fluid extracts of microalgae and rosehip. Graphical Abstract Ultra-high performance supercritical fluid chromatography-a rapid separation method for the analysis of carotenoids in rosehip and microalgae samples.

In situ instrumentation that can detect amino acids at parts-per-billion concentration levels and distinguish an enantiomeric excess of either d-or l-amino acids is vital for future robotic life-detection missions to promising targets in our solar system. In this article, a novel chiral amino acid analysis method is described, which reduces the risk of organic contamination and spurious signals from by-products by avoiding organic solvents and organic additives. Online solid-phase extraction, chiral liquid chromatography, and mass spectrometry were used for automated analysis of amino acids from solid and aqueous environmental samples. Carbonated water (pH *3, *5 wt % CO 2 achieved at 6 MPa) was used as the extraction solvent for solid samples at 150°C and as the mobile phase at ambient temperature for chiral chromatographic separation. Of 18 enantiomeric amino acids, 5 enantiomeric pairs were separated with a chromatographic resolution >1.5 and 12 pairs with a resolution >0.7. The median lower limit of detection of amino acids was 2.5 mg/L, with the lowest experimentally verified as low as 0.25 mg/L. Samples from a geyser site (Great Fountain Geyser) and a geothermal spring site (Lemon Spring) in Yellowstone National Park were analyzed to demonstrate the viability of the method for future in situ missions to Ocean Worlds.