Development and application of reversed phase liquid chromatography based techniques for automated purification of biologically active ingredients from plant extracts and for characterization of plant extracts and environmental pollutants

摘要

Automated preparative HPLC purification systems are an important and useful technology in pharmaceutical and chemical development. The systems have been applied to high-throughput purification of products from combinatorial compound synthesis for drug discovery, single compound isolation for further structure elucidation and activity screening, as well as fractionation of active compounds from plant extracts. Fraction collection in automated HPLC purification system can be triggered by less selective UV detection or by highly selective mass spectrometry (MS) for target oriented trigger or simultaneously by both detection systems. Unlike UV detection, mass spectrometry is a destructive detection technique. Therefore, a post-column split must be applied to send a small fraction of the column main flow to the mass spectrometer. A passive splitting device for an automated semi-preparative HPLC system using a column dimension of 10 mm i.d. was constructed and its design was optimized by theoretical modeling and experimental evaluation for both photometric and mass spectrometric fraction collection trigger. The challenges in the implementation of automatic triggers are proper synchronization of peak detection and peak collection as well as minimization of band dispersion in the connection tubing between detectors and fraction collector. The optimized instrumental setup was synchronized and characterized with both a microparticulate column operated at 5 mL/min and a silica-based monolithic C18-column (Monolith) of the same dimension performed at 10 mL/min for further increasing the speed of fractionation. Since the fast mode using a monolithic column can be accomplished on exactly the same instrumental setup, it represents an ideal alternative to the standard mode for high throughput purification of simple crude mixtures.Since the cost of isolated compounds depends strongly on their loadability on the column, maximizing loading capacity is of utmost importance. It is also known that he loadability may vary widely with the nature of the compound. Therefore, volume oading capacity for acetylsalicylic acid and mass loadability for some pharmaceutically relevant compounds (acetylsalicylic acid, amitriptyline, phenol and rutin) on some RP-HPLC columns in semi-preparative dimension (10 mm i.d.) compared to their analytical dimension (4.6 mm i.d.) was studied extensively.The optimized automated purification system (APS) was successfully applied to the fully automated fraction collection of pharmacologically and medicinally relevant components from plant extracts. The APS was utilized for fractionation of precious ingredients in St. John wort extracts (Hypericum perforatum), minor degradation products from active ingredients in a thermal stressed drug, and for isolation of the toxic protein gliadins which can cause a so-called coeliac desease. In the second part of this work, St. John wort extracts obtained from Accelerated Solvent Extraction (ASE) applying different extraction procedures were characterized. Moreover, asphalt samples containing tar were extracted, prepared and analyzed for polycyclic aromatic hydrocarbons (PAHs) and small phenols contents.