The isolation of compounds from crude extracts represent a key step common to all natural product investigations. Obtaining pure natural products at sub-milligram scale is crucial for assessment of their bioactivity profile and their full de novo structure determination when they cannot be dereplicated with LC-MS profiling methods. Ideally, this process should be performed in one-step directed from the crude extracts. For this semi-preparative chromatographic resolution should be improved and perfectly match the performance obtained by analytical HPLC . One of the limits of this method is the amount of extract that can be purified. Usually the sample is solubilized in organic solvent and injected on column through rheodyne-loop. Since the solubility of extracts is limited, high solvent volume are needed significantly compromising the resolution of separation. Moreover, when large amounts of the extract are injected there is a risk of system over pressure. A new approach was developed in order to overcome these issues. The procedure involve an initial treatment of the extract to eliminate compounds from the primary metabolism such as sugars. The separation is first optimized on an HPLC analytical column, and the conditions are transferred by software to a semi-preparative column packed with the same stationary phase to ensure similar selectivity. The sample is then injected by dry load on a dedicated cell that affords loading of hundreds of milligrams of crude extract without loss of resolution or overpressure. ELSD detection provide a semi-quantitive view of the fractions content and post-column 1H-NMR and UHPLC-HRMS profiling of all fractions gathered in 2D matrices gave a precise view of the separation and possible co-elution issues. The approach was successfully used for the fractionation of several complex natural extracts from plant and microbial origin. In all cases, an importa nt number of pure compounds was obtained in one single step.
