In bacteria, PG is biosynthesized from phosphatidic acid by enzymatic reactions via an intermediate cytidine diphosphate diacylglycerol . Other secondary biosynthetic pathways produce PG, e.g., the catabolism of cardiolipin or other phospholipids, catalyzed by phospholipase D, and eventually, cardiolipin synthase (ClsB) , which can partially change the stereochemistry of the second glycerol group (free glycerol). The other two isomers, i.e., S,R, and S,S, could also be present in organisms if PG biosynthesis starts with glycerol 1phosphate ((2S)-2,3-dihydroxypropyl dihydrogen phosphate), which is the enantiomer of glycerol 3-phosphate. Both enantiomers are biosynthesized by reducing dihydroxyacetone phosphate by the respective enzyme, i.e., sn-glycerol-1-phosphate dehydrogenase (EC 1.1.1.261) or sn-glycerol-3-phosphate dehydrogenase (EC 1. . So far, it has been assumed that in archaea, the starter unit is glycerol 1-phosphate while in bacteria and eukaryotic organisms it is its enantiomer glycerol 3-phosphate . However, genetic analyses of archaea and bacteria showed that both enzymes are present in both domains. For glycerol-3phosphate dehydrogenase, the bacterial vs archaeal ratio was found to be 477,032:2,634, whereas for glycerol-1-phosphate dehydrogenase it was 2,459:2,169, (sequences available in NCBI) . It is clear from these values that, in some bacteria, the presence of all four PG isomers is not excluded. The analysis or determination of individual PG isomers can be carried out in several ways. Direct measurement of optical rotation was insufficiently sensitive to distinguish between diastereomeric PG . Circular dichroism used by Batrakov et al . makes it possible to determine the absolute configuration of chiral compounds. However, its use is not simple and practical in obtaining the composition of the mixture. Therefore, only chiral chromatography of derivatized phospholipids is used. One of the most suitable derivatizing agents is 3,5-dinitrophenyl isocyanate, which reacts with free OH groups of PG to form 3,5-dinitrophenylurethane (DNPU). These derivatives of PG can be separated on a stationary column with a chiral phase (e.g., phase based on (R)-and (S)-1-(1naphthyl)ethylamine), as has already been described many times .