## 2 Results and Discussion
### (\(\pm\))-Ectoine Enantiomers Separation  
To our best knowledge this is the first time that enantiomers separation of (\(\pm\))-ectoine has been performed using chiral stationary phases. In general, there are several subgroups of chiral stationary phases, and some of these subgroups were examined: polysaccharides-based chiral stationary phases (cellulose, amylose), brush-type chiral stationary phases, and stationary phases with macrocyclic selector respectively macrocyclic glycopeptide (teicolamine). Several factors must be considered when developing the method. The first is the solubility of (\(\pm\))-ectoine, which is soluble in water and MeOH, less soluble in EtOH and ACN, and insoluble in non-polar organic solvents. This physical property restricted the choice of stationary phase to reverse phases only. Furthermore, the ectoine is small organic molecule present in zwitter-ion without chromophores. Ectoine does not possess an \(\pi\)-\(\pi\) system to enable \(\pi\)-\(\pi\) interactions with suitable chiral selectors. The interactions that ectoine can form include hydrogen bonding, electrostatic interactions, dipole-dipole interaction, and charge transfer interaction. The interactions can be attractive or repulsive, they can be single or multiple, but at least one must be stereochemically dependent. Separations on brush-type ((\(R\),\(R\))-WhelkO1) and polysaccharide-type columns (Chiral-pak AD-RH, Chiralcel OD-RH, Chiralcel OJ-RH and Chiralpak AS-H and Chiralpak PST-4) gave no results (methods 1-15. Due to the nature of ectoine, there was no interactions with these stationary phases and the ectoine was eluted without retention. The exchange of solvents did not give the results. On the other hand, almost all tested methods on teicolplain-based chiral stationary phase, the Chiral-T column (Table 1, methods 16-35) separate the enantiomers to baseline (except methods 17-19 method conditions shown in Table S1). This separation of ectoine enantiomers is the result of the ability to form an interaction between ectoine and the chiral stationary phase. Chromatographic parameters such as retention time of the first and the second eluting enantiomer (\(t_{\text{R1}}\), \(t_{\text{R2}}\)), retention factor of the first and the second eluting enantiomer (\(k_{1}\), \(k_{2}\)), separation factor (\(\alpha\)), and resolution (\(R_{\text{s}}\)) are summarized in Table 1. During the development of enantioseparation method on the Chiral-T column, the following parameters were varied: mobile phase, temperature, and flow rate. Due to the solubility of ectoine, mostly water and MeOH in different ratios were used as the mobile phase. The influence of temperature was measured in range of 30-45 \({}^{\circ}\)C. The flow rate was varied from 0.5 to 1.2 mL/min. The addition of formic acid (0.1% and 0.01%), Et3N (0.01%), and ammonium formate (25 mM, pH = 3.0) was examined. The addition of formic acid in MeOH (methods 17 and 18) leads to a drastic shortening of the retention time, a deformation and broadening of the peaks and thus to a loss of resolution. On the other hand, the addition of Et\({}_{3}\)N (method 19) results in long retention time, which is caused by the formation of an ion-pair between ectoine and Et\({}_{3}\)N.  
Good results were obtained with ammonium formate (methods 33 and 34), but the best results were obtained with water and MeOH alone. Water and MeOH were also used in the scale-up method for the purpose of preparative isolation of (+)- and (\(-\))-ectoine. The effect of flow rate (method 27, 28 and 31) was investigated after the determining the optimum ratio of H\({}_{2}\)O:MeOH as 1:1 (Figure 1). According to the chromatograms (Figure 1) and the separation and resolution values (Table 1), the method 27 with flow rate at 0.5 mL/min is proved to be the best choice for the enantioseparation of racemic ectoine.