SC-2P-bonded phase was synthesized via simple solvo-thermal condensation of the pre-synthesized pregabalin-based substituted triazine ligand with the chlorinated silica. Its probable structure is shown in Fig. 2.
The organic ligand and the resultant stationary phase showed low thermal stability when temperatures higher than 150 °C were used during the synthesis or the drying steps; thus, a synthesis temperature of no more than 70 °C and a drying temperature of no more than 100 °C were suitable. This low thermal stability was observed as these materials were turning into brown color upon heating, which suspects that inter-molecular Maillard reactions are taking place . Conversely, SC-2P stationary phase showed good chemical stability when acidic (pH = 3) or basic (pH = 9) solutions were used as mobile phases, as no changes were observed in its FT-IR or UV-DRS spectra.
Characterization of SC-2P
According to the FT-IR measurements (Fig. 4), the bands observed in the range of 3000–2800 cm−1 and ~ 1600 cm−1 in both samples are for C–H and C=O/C=N stretching, respectively. The broad band in the range of 3500–2500 cm−1 which refers to the –COOH group dimers in C-2P was not observed in SC-2P; this could be due to the steric effects that prevent the formation of inter-molecular hydrogen bonds in the bonded silica. The band at 3417 cm−1 in SC-2P could be for O–H/N–H or free SiO–H stretching . The two strong bands at 1100 cm−1 and 463 cm−1 in SC-2P refer to the stretching of Si–O–Si and Si–O rocking, respectively, and the strong band at 972 cm−1 can be assigned to Si–O–Ph stretching. It is unlikely that the organic ligand binds to the silica surface via Si–N or Si–O–CO bonds as their stretching bands in the ranges of 1550–1540 cm−1 and 1770–1725 cm−1, respectively, were not observed .
UV-DRS spectra along with the Kubelka-Munk function patterns (Fig. 5) were used as a complementary characterizing method for the synthesized bonded phase. According to Kubelka-Munk equation (Eq. 1), the increase in F(R) values observed for the bonded phase in comparison to the chlorinated silica could be due to two reasons. Firstly, the presence of the organic moiety in the bonded phase leads to a significant increase in K value, and secondly, the decrease in the specific surface area which usually accompanies the grafting processes can cause a decline in S value. This pattern was not observed at wave lengths below 250 nm. This could be due to the high absorptivity of silica surface for the two samples and the low absorptivity of the organic ligand in the bonded phase sample in this range. Although the Kubelka-Munk theory of diffuse reflection has some limitations, which makes it poorly employed as a characterizing technique in the UV range, it was easily conducted and showed high repeatability between different batches of both chlorinated and bonded silica samples.
The calculated specific surface area determined in this study confirms the favorable characteristics of the synthesized SC-2P to be used as a TLC stationary phase. In addition, the applied Langmuir isotherm method showed good repeatability and flexible probability for the different types of Langmuir equation to be used.
Chromatographic evaluation of SC-2P
The properties of SC-2P as a TLC stationary phase were studied in the presence of copper ions to form ligand exchange reagent (LER). Two isomeric mixtures were used to evaluate the chromatographic characteristics of the stationary phase and its ability to solve different types of isomerism. A mixture of nitrophenol isomers was selected as a model of structural isomerism. This mixture was successfully separated on SC-2P/Cu2+ TLC plates using a mobile phase of n-hexane:DCE:n-propanol (75:20:5, v/v/v) (Fig. 6a), as three separated peaks were determined in the densitogram which were compared to those in the densitograms of control solutions. Rf values were densitometrically determined and were 0.83, 0.45, and 0.28 for o-, m-, and p-nitrophenol, respectively. The successful separation was confirmed as α-values were 6.09 for o-, m-nitrophenol, and 2.15 for m-, p-nitrophenol.
Likewise, a racemic mixture of (±)-ibuprofen was employed to study the chiral selectivity of SC-2P/Cu2+ stationary phase. Depending on the densitometric measurements of the developed spot using DCE:acetonitrile (90:10, v/v) as a mobile phase (Fig. 6b), this mixture was also separated with Rf values of 0.43, and 0.63 for (−)-, (+)-ibuprofen, respectively. The calculated α-value was 2.24.
Rf values changed within the range of ± 0.02 between plates prepared from different batches of the synthesized stationary phase, which is considered neglected and could be due to shifts in the mobile phase fractions and room temperature changes.
The same mixtures were developed on TLC plates prepared from SC-2P slurry without copper ions addition, and the same mobile phase systems were used. The resulted densitograms are shown in Fig. 7, which confirm the advantage of using copper additive to enhance the selectivity of SC-2P, as there was no resolution observed between m- and p-nitrophenol in the first structural isomeric mixture, and, similarly, the racemic mixture of (±)-ibuprofen gave one spot.
According to the literature, the selectivity of stationary phases containing LERs is mainly owing to the ability of these reagents to form (solute-Cu2+-ligand) ternary complexes. These complexes are of different stability constants due to the configuration differences and the steric effects. Our results were consistent with this proposed mechanism. For the structural isomers of nitrophenol, the steric effect of the ortho–NO2 group in o-nitrophenol led to a decrease in the capability of the –OH group of o-isomer to form ternary complexes with LERs, in addition to the simple hydrogen bonds with the free –COOH groups on SC-2P. This steric effect declines for the other two isomers which showed a closer affinity to the stationary phase, with the para-isomer exhibiting the highest capability to interfere with the surface, as it corresponded to the lowest Rf value.
The successful separation of (±)-ibuprofen stereoisomers on SC-2P/Cu2+ can be similarly illustrated, as the different spatial arrangements of atoms in the solute components will lead to the formation of diastereomeric complexes of different configurations (S-ibuprofen––Cu2+––S-ligand, and R-ibuprofen––Cu2+––S-ligand). The latter configuration is more stable than the former, which explains the difference in the observed Rf.
Moreover, both mixture components and SC-2P stationary phase are of moderate polarity and consist of a hydrophobic aromatic ring with polar hydroxyl or carboxylic acid group. Thus, other mechanisms such as π–π interactions, and H-bond formation might have a role in the separation process.
The separation of nitrophenol isomers was previously reported using RP-HPLC methods [26, 27]. However, our work is the first to describe a successful separation of the ternary mixture of nitrophenols using LER-based TLC plates.
The use of LER-based stationary phases provided good enantioselectivity to solve some racemic mixtures of NSAIDs-like drugs such as Bupropion  and Baclofen . On the other hand, the reported work that separated a racemic mixture of ibuprofen using amino acids as chiral selectors needed two-dimensional development to give an acceptable resolution . SC-2P/Cu2+ stationary phase provides successful separation of (±)-ibuprofen using LER-based stationary phase with simple one-dimensional development.
Besides, unlike most of the other previous phases which were prepared by impregnation with the chiral selector, SC-2P is based on a distinct structure of chemically stable triazine linker that binds two molecules of pregabalin with the same optical configuration to the silica surface. The stability of selector on the stationary phase could enable the employing of SC-2P in column isomeric separations using HPLC.
Therefore, the synthesized SC-2P/Cu2+ stationary phase provides a good alternative to the previously mentioned amino acid based chiral phases, as it has the advantage of being able to solve both structural and stereo-isomerism with good selectivity. However, the use of copper ions as an additive to the chiral selector was crucial to obtain acceptable separations, which is still considered a major limitation of LER-based stationary phases.