## 5 Conclusions  
In summary, we reported the synthesis of three lactate esters, after a screening of reaction conditions, to yield a selective synthetic methodology that does not cause racemization of the stereogenic center of the lactic acid. We also verified that our reaction conditions can be carried out in solvent-free conditions or in aqueous mixtures. This simple and general methodology was applied to a new and alternative analytical protocol for the measurement of the enantiomeric purity of lactic acid, even in a non-anhydrous environment. The newly synthesized lactate esters **1b**, **2b** and **3b** were analyzed by direct-phase chiral HPLC-DAD with stationary phases differing both in the chiral selector and in the methodology of immobilization. The Chiralcel OD-H and Chiralpak IA columns show similar behaviors in retention times, enantioselectivity and resolution. On the other hand, the Whelk-O2 column is low-performing in this type of separation. The resolution of the Chiralpak IA column is greater than that of the Chiralcel OD-H column. The resolution increases passing from ester **1b** to ester **2b**, in which the addition of one nitro group in the _ortho_-position is highly beneficial. On the other hand, the resolution decreases passing from ester **2b** to ester **3b**, in which the addition of a second nitro group is detrimental; in fact, retention times increased, but the overall resolution decreased. Ester **2b** is indeed the best choice for the functionalization of lactic acid (\(\alpha\) 1.13 and Rs 3.57).  
In conclusion, we define a new protocol for the measurement of the enantiomeric purity of lactic acid by direct-phase chiral HPLC-DAD. This method involves a preliminary functionalization of the starting material by means of a reaction that is potentially feasible on biological samples and analytes at low concentrations in the 2-nitrobenzyl ester. Subsequently, the measurement of the enantiomeric purity of the same ester is performed using the Chiralpak IA column.  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app15126433/s1](https://www.mdpi.com/article/10.3390/app15126433/s1)]([https://www.mdpi.com/article/10.3390/app15126433/s1](https://www.mdpi.com/article/10.3390/app15126433/s1)): Figure S1: \({}^{1}\)H-NMR (500 MHz, CDCl\({}_{3}\)) spectrum of benzyl 2-hydroxypropanoate (**1b**); Figure S2. \({}^{13}\)C-NMR (125 MHz, CDCl\({}_{3}\)) spectrum of benzyl 2-hydroxypropanoate (**1b**); Figure S3. HRMS (ESI-TOF) spectrum of benzyl 2-hydroxypropanoate (**1b**); Figure S4. FT-IR spectrum of benzyl 2-hydroxypropanoate (**1b**); Figure S5. \({}^{1}\)H-NMR (500 MHz, CDCl\({}_{3}\)) spectrum of 2-nitrobenzyl 2-hydroxypropanoate (**2b**); Figure S6. \({}^{13}\)C-NMR (125 MHz, CDCl\({}_{3}\)) spectrum of 2-nitrobenzyl 2-hydroxypropanoate (**2b**); Figure S7. HRMS (ESI-TOF) spectrum of 2-nitrobenzyl 2-hydroxypropanoate (**2b**); Figure S8. FT-IR spectrum of 2-nitrobenzyl 2-hydroxypropanoate (**2b**); Figure S9. \({}^{1}\)H-NMR (500 MHz, CDCl\({}_{3}\)) spectrum of 2,4-nitrobenzyl 2-hydroxypropanoate (**3b**); Figure S10. \({}^{13}\)C-NMR (125 MHz, CDCl\({}_{3}\)) spectrum of 2,4-dinitrobenzyl1 2-hydroxypropanoate (**3b**); Figure S11. HRMS (ESI-TOF) spectrum of 2,4-dinitrobenzyl1 2-hydroxypropanoate (**3b**); Figure S12. FT-IR spectrum of 2,4-dinitrobenzyl1 2-hydroxypropanoate (**3b**); Figure S12.