An experimental study on the chromatographic separation of maltopentaose from a mixture, including glucose, maltose, maltotriose, and maltopentaose, was carried out in a nonionic polymeric sorbent column while varying the operating conditions, such as the solution pH, buffer contents, and isopropyl alcohol (1PA) concentration. Unlike the pH and buffer contents, the IPA concentration had a Significant impact on the single component chromatograms for maltopentaose. The retention times of the maltooligosaccharides with the nonionic polymeric sorbent Sp207 were in the following order: glucose < maltose < maltotriose < maltopentaose. From the experimental binary, ternary, and quaternary chromatograms, gradient chromatographic separation with a changing IPA concentration as a function of time was required to obtain high-purity maltopentaose and reduce the elution time