## 2 Results and Discussion
The polarity sequence of these eluents gave good recovery with a fair distribution of the major components in the five fractions. Interestingly, primary metabolites (mainly, sugars, amino acids, sterols, fats, phospholipids, triglycerides) showed a predictable chromatographic behavior using TLC analysis (see Experimental Section 3.3), being selectively eluted as follows: saccharides and amino acids in fraction A, nucleosides in B, polar lipids including glycolipids and most phospholipids in C, sterols and free fatty acids in D and, finally, triglycerides and other neutral lipids in E. Analogously, the different classes of secondary metabolites occurring in the selected organisms also distributed widely among the five fractions. Thus, _D. membrananosa_ afforded a unique fraction (fraction D) containing 9,11-dihydroracilin A (**1**), whereas the polyhydroxylated metabolites of _A. carterae_ (Amphidinols **4** and **5**) were concentrated in fraction C. Fractionation of _R. sarai_ extracts gave more complex results. The sponge alkaloids were nicely distributed in two fractions according to the heterogeneous chemistry of these typical marine metabolites. Thus, caged zwitterionic compounds of the series of sarains A-C (e.g., **2**) were eluted by CH\({}_{3}\)CN/H\({}_{2}\)O 70:30 whereas the less complex sarains 1-3 (e.g., **3**) and the corresponding _iso_-sarains1-3 were collected by MeOH/H\({}_{2}\)O 50:50. The LC-MS analysis of these fractions also revealed the presence of a number of minor analogs that have been not reported before. The characterization of these molecules is beyond the scope of the present work but their occurrence underlines the potential of the SPE method here presented. In fact, purification of sarains as accomplished by [PERSON] and coworkers [17,18] entailed a complex and time-demanding chromatographic procedure that was not free of material loss. In line with our working hypothesis, the SPE method overcame most of these issues and furthermore offered the advantages of removing the interfering components and revealing the presence of minor compounds. The weights of the combined fractions, were used to calculate total sample recovery from both sponge and dinoflagellate extracts. Including the salt from the wash step, the procedure gave above 80% recovery over three replicates of each sample (87% \(\pm\) 6% with _D. membrananosa_; 83% \(\pm\) 3% with _R. sarai_; 84% \(\pm\) 5% with _A. carterae_). Once the reliability of the elution protocol was established, we transferred the method onto a completely automated fractionation system in order to validate the procedure for high-throughput studies (Figure 1). The use of a branded analytical platform for loading and eluting the sample on pre-packed cartridges required only an adjustment of the solvent volumes but did not entail changes in the elution order (Table 2). Considering the desired application of the method for drug-discovery, the first aim was to establish the amount for each fraction to achieve both the first steps of a bioassay-guided screening and a preliminary chemical characterization of the products by LC-MS and NMR. Different amounts of extracts were submitted to the analytical procedure in a range between 10 and 50 mg since for each organic matrix the optimal quantity of sample has to be established based on the salt content. As expected, automation gave the possibility to quickly process large numbers of samples in a short time thus simplifying the preparation of a natural products library. This was proven by application of the protocol for screening 47 samples including 27 microalgae assayed for immunomodulatory activity and 20 samples of microalgae and Antarctic sponges assessed for antimicrobial properties. Each sample (20 mg for microalgae and 40 mg for sponges) was processed in less than one hour and the whole batch of samples required almost two days of continuous work.