A pH dependent Raman and surface enhanced Raman spectroscopic studies of citrazinic acid aided by theoretical calculations

• Citrazinic acid, a consisting of a dihydro-pyridine ring, with different pKa values (~ 4 and ~ 11) for the two different functional groups (–− COOH and –OH groups), shows interesting range of color changes (yellow at pH ~ 14 and brown at pH ~ 2) with the variation in solution pH. The changes in color are highly reproducible and hence it is very interesting.
• In this manuscript we have presented the complete Raman spectroscopic study (both NRS and SERS) of citrazinic acid in the above pH environment. To the best of our knowledge still there is no report on vibrational analysis of this biologically significant heterocyclic compound in a varied pH environment.
• Here it is worth noting that the NRS/SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Hence different possible molecular forms are proposed to subsist in the varied pH environment and DFT calculations were performed for the molecular forms to theoretically simulate their relative abundance.
• The present report furnishes a complete picture regarding the possible molecular forms of citrazinic acid existing in solution in varied pH environment based on the spectroscopic data, ESI-MS analytical data and DFT based theoretical analysis.

A pH dependent normal Raman scattering (NRS) and surface enhanced Raman scattering (SERS) spectral patterns of citrazinic acid (CZA), a biologically important molecule, have been investigated. The acid, with different pKa values (~ 4 and ~ 11) for the two different functional groups (–COOH and –OH groups), shows interesting range of color changes (yellow at pH ~ 14 and brown at pH ~ 2) with the variation in solution pH. Thus, depending upon the pH of the medium, CZA molecule can exist in various protonated and/or deprotonated forms. Here we have prescribed the existence different possible forms of CZA at different pH (Forms “C”, “H” and “Dprot” at pH ~ 14 and Forms “A”, “D”, and “P” at pH ~ 2 respectively). The NRS spectra of these solutions and their respective SERS spectra over gold nanoparticles were recorded. The spectra clearly differ in their spectral profiles. For example the SERS spectra recorded with the CZA solution at pH ~ 2 shows blue shift for different bands compared to its NRS window e.g. 406 to 450 cm− 1, 616 to 632 cm− 1, 1332 to 1343 cm− 1 etc. Again, the most enhanced peak at ~ 1548 cm− 1 in NRS while in the SERS window this appears at ~ 1580 cm− 1. Similar observation was also made for CZA at pH ~ 14. For example, the 423 cm− 1 band in the NRS profile experience a blue shift and appears at ~ 447 cm− 1 in the SERS spectrum as well as other bands at ~ 850, ~ 1067 and ~ 1214 cm− 1 in the SERS window are markedly enhanced. It is also worth noting that the SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Therefore based on the experimental findings we propose different possible molecular forms of CZA at different pH (acidic and alkaline) conditions. For example forms ‘A’, ‘D’ and ‘P’ existing in acidic pH (pH ~ 2) and three other deprotonated forms ‘C’, ‘H’ and ‘Dprot’ in alkaline pH (pH ~ 14). The DFT calculations for these prescribed model systems were also carried out to have a plausible understanding of their equilibrium geometries and the vibrational wavenumbers. An idea about the molecular orientation of the adsorbate over nanocolloidal gold substrate is also documented.

Citrazinic acid exhibits fascinating color changes in a wide variety of solution pH. Experimental and theoretical Raman analyses relate these changes of color variation with the structural aspects of the molecule.Figure optionsDownload as PowerPoint slide