Reductive amination of glutaraldehyde 2,4-dinitrophenylhydrazone using 2-picoline borane and high-performance liquid chromatographic analysis

Abstract

A typical method for the measurement of glutaraldehyde (GLA) employs 2,4-dinitrophenylhydrazine (DNPH) to form GLA–DNPhydrazone derivatives. However, this method is subject to analytical errors because GLA–DNPhydrazone is a quaternary bis-derivative and forms three geometric isomers (E–E, E–Z and Z–Z) as a result of the two CN double bonds. To overcome this issue, a method for transforming the CN double bond into a C–N single bond, using reductive amination of DNPhydrazone derivatives, has been applied. The amination reaction of GLA–DNPhydrazones with 2-picoline borane is accelerated with catalytic amounts of acid and is completed within 10 minutes in the presence of 100 mmol L⁻¹ phosphoric acid. Reduction of GLA–DNPhydrazone by 2-picoline borane is unique and results in the formation of N-(2,4-dinitrophenyl)-1-piperidinamine (DNPPA). NMR and LC-APCI-MS data confirmed the product identification. DNPPA is very stable and did not change when stored for at least four weeks at room temperature. DNPPA has excellent solubility of 14.6 g L⁻¹ at 20 °C in acetonitrile. The absorption maximum wavelength and the molar absorptivity of DNPPA were 351 nm and 4.2 × 10⁴ L mol⁻¹ cm⁻¹ respectively. Complete separation between the reduced forms of C1–C10 aldehyde DNPhydrazones, including DNPPA, can be achieved by operating the reversed-phase high-performance liquid chromatograph at 351 nm in gradient mode using a C18 amide column. The reductive amination method for GLA overcomes analytical errors caused by E–E, E–Z and Z–Z geometrical isomers.