Advanced techniques in separation science for unraveling the complexity of natural substances: from small chiral molecules to large proteins

Natural substances are built up or transformed into each other in living organisms. Depending on the substance class, their tasks are varied and range from simple metabolism or energy production via cell components and building materials of the organism to complex control tasks. Regarding their functions, one can distinguish between primary and secondary natural products. Primary natural products include all the compounds needed for supporting and growing organism. These include, in particular, fats and biopolymers of carbohydrates and proteins. Secondary natural products are usually smaller molecules than primary products. They are formed for reasons often unknown and are subdivided into the broad classes of terpenes, aromatics, and alkaloids. Thus, the demand to study a range of increasingly complex samples of plant and animal origin, trying to solve and identify their chemical entities with greater confidence, has prompted researchers to develop innovative analytical approaches. The work showed in this thesis has the aim to investigate new analytical proficiencies to enrich separation science, especially by using cutting-edge chromatographic and detection techniques, and their applicability in secondary natural products chemistry and proteomics. The first section of this dissertation has been focused on terpenophenolic and polyphenolic secondary metabolites deriving from Cannabis sativa L. and Malus pumila Miller (cultivar Annurca) respectively. In the former case, the attention was focused on a fascinating feature of cannabinoids (C21 terpenophenolic compounds specific of Cannabis): the chirality. Chiral natural products are usually generated in optically pure form; however, occasionally both enantiomers are biosynthesized. It becomes evident, therefore, the worth of determination of the enantiomeric purity (i.e., enantiomeric excess, e.e.) in naturally occurring samples. The problem faced in the stereoselective analysis of Cannabis plant extracts was that vegetable extracts are highly enriched complex mixtures and often the minor enantiomers or the racemates, are not available as reference samples. In order to overcome this limitation, our group has previously developed a method for the identification of enantiomeric couples and accurate quantification of the minor enantiomer in trace analysis of natural products, named the ‘‘inverted chirality columns approach’’ (ICCA). The method allows determining the e.e. of (–)-Δ9-THC in medicinal marijuana. The e.e. was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not to be underestimated, and it is worth a systematic evaluation of bioactivity. In the latter case, an improved online comprehensive two‐dimensional liquid chromatography was developed. The use of a hydrophilic interaction chromatography column in the first dimension coupled to a trapping column modulation interface, and using a high retentive fully porous monodisperse reversed-phase column in the second dimension, enabled the simultaneous separation of multiple polyphenolic classes, as well as oligomeric procyanidins up to a degree of polymerization of 10. Moreover, thanks to hyphenation with an ion trap time-of-flight mass spectrometer, the tentative identification of 121 compounds has been possible. Thus, the presented system showed it could be a powerful analytical tool for the accurate profiling of complex polyphenolic-rich matrices. The topic of the second part of this dissertation concerns the study of analytical strategies applicable in the field of proteomics. In particular, the focus has been on the development of monolithic stationary phases to be used in capillary high performance liquid chromatography. Indeed polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization were used, and the HPLC experimental conditions, such as nature of the organic modifiers, content of acid additive, and column temperature, were optimized for the separation of nine standard proteins with different pI, hydrophobicities and a wide range of molecular weights. The high working flow and high efficiency of these columns have allowed employing a longer column (up to 500 and 1000 mm), and thus, to reach a peak capacity value up to 1000. In order to probe the capacities of the monolithic columns, the range of molecular weights of the proteins examined was further expanded (from 3000 Da of glucagon to 150 kDa of monoclonal antibodies), showing in both cases excellent results. Precisely, monoclonal antibodies are the protagonists of the last chapter of this thesis, where a sample treatment procedure and an analytical method are developed in order to characterize the oxidized/reduced state of disulfide bridges. Indeed, upon incubation of antibodies with the reducing agent tris(2‐carboxyethyl)phosphine, three reduced isoforms of light chain can be identified at different reaction times: light chain, partially reduced light chain+2H, with one of two disulfide bridges opened in the constant or variable region, and entirely reduced light chain+4H.