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Determination of Methanol Increment in Mobile Phase Consisting of Methanol and Water by On‐line UV Spectrometry in Reversed Phase Liquid Chromatography
Author(s) -
Geng XinDu,
Regnier Fred E.
Publication year - 2002
Publication title -
chinese journal of chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.28
H-Index - 41
eISSN - 1614-7065
pISSN - 1001-604X
DOI - 10.1002/cjoc.20020200114
Subject(s) - chemistry , methanol , elution , chromatography , analytical chemistry (journal) , phase (matter) , high performance liquid chromatography , reversed phase chromatography , organic chemistry
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 (CH 3 OH)/ V (H 2 O), a set of elution curves could be obtained at 198 nm by a diode‐array detector in the presence of 47% methanol, V (CH 3 OH)/ V (H 2 O) containing 0.03% hydrochloric add, V (CH 3 OH‐H 2 O)/ 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.