2026 Volume 47 Issue 3 Pages 143-151
Temperature-responsive chromatography using poly(N-isopropylacrylamide) (PNIPAAm) has been extensively investigated as a functional chromatographic system. Because the retention behavior of analytes can be modulated by adjusting the column temperature, aqueous mobile phases without organic solvents were used. Thus, few studies have been conducted on the effects of adding organic solvents, such as methanol, to the mobile phase. In this study, methanol was introduced into the aqueous mobile phase to examine its influence on the elution behavior of steroid compounds. The results showed that increasing the methanol content reduced the retention times of the steroids because of their enhanced solubility in the mobile phase. However, the separation efficiency was strongly dependent on the methanol concentration. At methanol concentrations of 5 % and 10 %, shorter retention times were achieved while maintaining satisfactory steroid resolution. In contrast, methanol concentrations of 15 % and 20 % resulted in a noticeable decline in steroid resolution. These findings indicate that methanol addition can be an effective strategy for controlling analyte retention time in temperature-responsive chromatography, provided that the methanol content is maintained at 10 % or lower to preserve adequate separation performance.
