## 1 Introduction
Since 2,4-diaminobutanoic acid contains two free amino groups that have almost the same chemical properties, it is important to achieve selective acetylation of only one amino group. For obtaining ectoine, however, it is not important whether the amino group at position two or four is acetylated, because the thermal cyclization of both 2-acetamido-4-aminobutyric acid and 4-acetamido-2-aminobutyric acid yields ectoine as a product. The introduction of an acetyl group at the amino group of 2,4-diaminobutyric acid can be carried out with 4-nitrophenylacetate. [PERSON] and [PERSON] have described the dependence of selective acetylation on pH and on the equivalents of 4-nitrophenyl-acetate [24]. A more favorable method for the preparation of monoacetylated 2,4-diaminobutyric acid involves the complexation of 2,4-diaminobutyric acid with copper(II)-ions and the acetylation of the resulting complex with 4-nitrophenylacetate [25; 26; 27]. This method has not yet been used for the preparation of ectoine and its derivatives. In this work, racemic ectoine was prepared in purpose to develop an efficient chromatographic method for its enantioseparation, which can be used for the determination of the optical purity of ectoine samples, but also to test individually their osmolytic/osmoprotective activities. Moreover, this approach can lead to an alternative method for ection enantiomers production based on preparative HPLC/SMB technology. The separation of enantiomers by liquid chromatography can be performed by direct and indirect methods [28]. Indirect methods are based on the addition of chiral additives to the mobile phase, whereupon the optical isomers react with the chiral additive, and consequently, diastereomers are formed, which can then be separated on an achiral stationary phase [29]. On the other hand, direct methods separate enantiomers on a chiral stationary phase by the formation of diastereomeric labile complexes of different stability, where enantiomer that forms a more stable diastereoisomer complex remains longer on the chiral stationary phase. Chiral stationary phases can be categorized according to basic structural features and the nature of the attractive forces acting between the stationary phase and the individual enantiomers [30; 31]. The most commonly used chiral stationary phases built from synthetic polymers are polysaccharides, which consist mainly of derivatized celluloses and amyloses. These chiral stationary phases are most commonly used with non-polar mobile phases, but there are also polysaccharide-based chiral stationary phases intended for the reverse phase (Chiralpak AD-RH, Chiralpak AS-RH, Chiralcel OD-RH, Chiralcel OJ-RH, etc.). The next group of chiral stationary phases that form inclusion compounds with the analyte are cyclodextrins. Brush-type or Pirke-type chiral stationary phases contain chiral selectors with small molecular masses. Protein-based chiral stationary phases contain immobilized proteins on a silica gel support. Chiral stationary phases that have proven to be most suitable for the separation of non-derivatized amino acids are chiral stationary phases containing macrocyclic glycopeptides, crown ethers, and chiral selectors for ion exchange. Macrocyclic glycopeptides (e.g., vancomycin, teicoplanin) covalently bound to a silica gel support represent a type of chiral stationary phase that allows the use of a wide range of mobile phases, non-polar and polar, ideal for the analytical and preparative separation of neutral, polar, and ionic compounds. These chiral stationary phases interact with the analyte through six different molecular interactions, which include ionic interactions, hydrogen bonding, \(\pi\)-\(\pi\) interactions, dipole-dipole interactions, and hydrophobic and steric interactions. In addition to the mentionedmolecular interactions, macrocyclic glycopeptides contain several inclusion sites that promote selectivity with regard to the geometric shape of the analyte.