###### Abstract  
Lactic acid (LA) is a natural organic acid that can be used in a wide variety of industries. Recently, the production of lactic acid by fermentation protocols has gained relevance. However, these biotechnological processes often encounter challenges in the production of enantiopure D- or L-lactic acid. Thus, the measurement and control of the enantiopurity of lactic acid and its derivatives is a crucial step in these productions, especially when monomers have to be used to synthesize polymers, such as polylactic acid (PLA). In the present work, we propose a measurement of the enantiopurity of lactic acid mixtures with HPLC-DAD by using direct-phase conditions, which are mild, and a large set of different employable chiral columns. To this end, we report the synthesis of two new LA derivatives (2-nitrobenzyl 2-hydroxypropanoate and 2,4-dinitrobenzyl 2-hydroxypropanoate) and benzyl 2-hydroxypropanoate, with a selective and non-racemizing chemical functionalization. Then, three commercially available HPLC direct-phase chiral columns are tested to achieve a method for measuring the enantiomeric purity of lactic acid derivatives. The 2-nitrobenzyl lactate was chosen as the best lactic acid derivative and the Chiralpak IA column was chosen as the most effective chiral column to achieve a new and efficient protocol (Rs = 3.57 and \(\alpha\) = 1.13) for the measurement of the enantiopurity of lactic acid. enantiomeric purity; lactic acid; HPLC-DAD; chiral direct phase; PLA Article  
acid produced by biotechnological processes has become a crucial step to validate the production process of LA through fermentation and to check the quality of the monomer leading to polylactic acid. The development of chromatographic methods to determine the enantiopurity of lactic acid and lactic acid derivatives has been studied for about 30 years [10]. High-performance liquid chromatography (HPLC) is the most widely used chromatographic analytical technique for this purpose. However, several techniques of chromatographic analysis have also been explored. One of these is based on chiral derivatization combined with gas chromatography and mass spectrometry (GC-MS). L-Menthal and acetyl chloride were used to derivatize LA enantiomers that were subsequently separated on a DB-5 MS capillary column. This method was also successfully applied to the analysis of mouse plasma samples [11]. In 1999, two HPLC methods for the determination of lactic acid and its enantiomers in calf serum were described for the first time [12]. In 2007, [PERSON] et al. [13] developed a novel method for the separation and simultaneous determination of urinary D- and L-lactic acid enantiomers based on high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The chiral separation was optimized utilizing the Chirobiotic TAG column, chosen because the polar ionic mobile phase (17% H\({}_{2}\)O, 83% EtOH/0.12% HOAc/0.30% TEA delivered isocartically at 0.2 mL/min) was well suited for the separation of small polar molecules, such as lactic acid. Some years later, the same method was exploited by [PERSON] et al. [14]. Urinary D- and L-lactic acid enantiomers were separated at 4 \({}^{\circ}\)C on an Astec Chirobiotic(tm R) chiral HPLC column. The mobile phase consisted of 15% (\(v/v\)) 33.3 mmol/L ammonium acetate and 85% (\(v/v\)) acetonitrile. Elution was performed at a flow rate of 0.7 mL/min. [PERSON] et al. [15] reported a protocol based upon the use of a reverse-phase chiral column with a \"Pirkle\"-type stationary phase, using 1 mM copper (II) sulfate in water as the mobile phase. The addition of copper sulfate to the mobile phase is needed to generate a complex that can be detected spectrophotometrically by a diode array detector (DAD), avoiding a preliminary chemical derivatization step. The HPLC-DAD approach was also used to test various types of macrocyclic glycopeptides-based chiral stationary phases for chiral HPLC separation of lactic acid in reversed-phase mode.