Preparation and Evaluation of a Safranin O-Modified Weakly Acidic Cation-Exchange Resin as an HPLC Stationary Phase for Amino Acid Separation
DOI:
https://doi.org/10.64354/byct9r71Keywords:
: Safranin O; weakly acidic cation-exchange resin; HPLC; stationary phase; amino acids; chromatographic resolution; HETP.Abstract
We prepared an ion-exchange stationary phase by modifying a weakly acidic cation-exchange resin with the cationic aromatic dye Safranin O. We evaluated it for high-performance liquid chromatography (HPLC) separation of selected amino acids. The resin was swollen in deionized water, treated with Safranin O, washed to remove loosely retained dye, and dried before packing the column. We evaluated the modification by visual color change, Fourier-transform infrared (FTIR) spectroscopy, and chemical-stability testing. The modified resin retained its red coloration after washing, and FTIR analysis showed spectral changes consistent with Safranin O incorporation into the resin matrix. No detectable Safranin O release was observed after treatment with 0.1 M HCl, 0.1 M KOH, or the organic solvents investigated. We evaluated column performance as a function of flow rate and acetonitrile content. The minimum HETP was obtained at 1.0 mL min⁻¹, which was selected as the optimum flow rate. Increasing the acetonitrile content from 5% to 30% improved the resolution of Gly/Phe from 0.1034 to 1.47 and Tyr/Lys from 0.710 to 1.68. Overall, the results indicate that Safranin O modification changes the interaction environment of the weakly acidic polymeric ion-exchange phase and improves amino-acid separation under the investigated chromatographic conditions.
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