Determination of Methanol Increment in Mobile Phase Consisting of Methanol and Water by On—line UV Spectrometry in Reversed Phase Liquid Chromatography

ＩｎｔｒｏｄｕｃｔｉｏｎＦｒｏｎｔａｌｃｈｒｏｍａｔｏｇｒａｐｈｙ (ＦＣ) ,ａｎｏｌｄｂｒａｎｃｈｉｎｌｉｑｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ (ＬＣ) ,1ｈａｓｂｅｅｎｓｕｃｃｅｓｓｆｕｌｌｙｅｍ ｐｌｏｙｅｄｉｎｔｈｅｓｏｌｕｔｉｏｎｏｆｍａｎｙｔｈｅｏｒｅｔｉｃａｌａｓｗｅｌｌａｓａｐ ｐｌｉｅｄｐｒｏｂｌｅｍｓ—ｓｕｃｈａｓｍｅａｓｕｒｉｎｇｂｉｎｄｉｎｇｃｏｎｓｔａｎｔｂｅ ｔｗｅｅｎｃｏｍｐｏｎｅｎｔｓ2 ａｎｄｋｉｎｅｔｉｃｐａｒａｍｅｔｅｒｓｏｆｃｈｅｍｉｃａｌｒｅａｃｔｉｏｎｓ ,3 ａｄｓ…

Determination of Methanol Increment in Mobile Phase Consisting of Methanol and Water by On-line UV Spectrometry in Reversed Phase Liquid Chromatography

An on‐line UV spectrometric method for the quantitative determination of methanol increment of methanol‐water in the mobile phase (i.e., of greater concentration than that of the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography (RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%, V(CH₃OH)/V(H₂O), a set of elution curves could be obtained at 198 nm by a diode‐array detector in the presence of 47% methanol, V(CH₃OH)/V(H₂O) containing 0.03% hydrochloric add, V(CH₃OH‐H₂O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanol increment was found to be proportional to the methanol increment in the mobile phase. The methanol increment could be determined on a quantitative basis. When the method was used to investigate the elution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detected before the usual insulin plateau of each elution curve. In this case the methanol increment was found to vary with insulin concentration in the mobile phase. When that concentration was between 0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to 0.19% with a deviation of ± 0.02% and a relative deviation of ± 10%. A nuclear magnetic resonance spectrometer (NMR) was also employed to confirm the obtained result. A methodology with a very rigorous experimental procedure for obtaining results of such accuracy is also included. The presented result may be used to prove that a displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.