The disappearance of OH band from C-28 position of Betulinic acid and emergence of new NH band at 3331 cm-1 and 3334 cm-1 verified the bonding of 2-and 3-nitroaniline derivatives of Betulinic acid, respectively.
Tri and diphenyltin complexes (2-and 3-nitroaniline derivatives) were confirmed by the proton signals at 7.30-7.33 and 7.29-7.31 ppm for phenyl hydrogens.
Structures of Ligands (L1/L2) were confirmed by the formation of amide bond between Betulinic acid and 2 and 3-nitroaniline. It was further confirmed by presence of aniline (2 and 3-nitroaniline) carbon peaks (C-33 to C-38).
In FT-IR spectral analysis appearance of new NH bands at 3331 cm-1 and 3334 cm-1 verified the bonding of 2 and 3-nitroaniline to Betulinic acid and formation of ligands (L1/L2).
To study the Pi-Pi interactions between a chiral selector and the enantiomers of an analyte, a Pi- acidic or Pi-basic group would be contained in the molecules of the derivatives of hydantoin-5-acetic acid, so the aromatic amines such as phenylamine, 4methylaniline, 3,5-dimethylaniline and 3-nitroaniline were chosen as derivatization reagents.
The resolution of the 3-nitroaniline derivative of hydantoin-5-acetic acid on the CHIDMB column was the best, but the retention time was too long.
1: the 3-nitroaniline derivative of hydantoin-5-acetic acid, 2: the phenylamine derivative of hydantoin-5-acetic acid, 3: the 4-methylaniline derivative of hydantoin-5-acetic acid, 4: the 3,5-dimethylaniline derivative of hydantoin-5-acetic acid; mobile phase composition on the CHI-DMB column: n-hexane 80%; mobile phase composition on the Chiralcel OD-H column: n-hexane 85%; t 1 : retention time of the first enantiomer; t 2 : retention time of the second enantiomer; Alpha: separation factor; R s : resolution; flow rate: 1.0 ml min -1 ; column temperature: 25 deg C; UV detection wavelength: 254 nm.
The type and concentration of organic modifier were found to influence the retention and resolution of the phenylamine and 3-nitroaniline derivatives of hydantoin-5-acetic acid on the CHIDMB column dramatically.
As a compromise between resolution and retention time, 20% isopropanol in n-hexane was found to be an optimum mobile phase for the phenylamine derivative of hydantoin-5-acetic acid, and 40% isopropanol in nhexane was found to be an optimum mobile phase for the 3-nitroaniline derivative of hydantoin-5-acetic acid.
Table-2: Effect of mobile phase composition on selectivity and resolution of the 3-nitroaniline and phenylamine derivatives of hydantoin-5-acetic acid enantiomers.