title: A rapid, ideal, and eco-friendlier protocol for quantifying proline.
abstract: Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λmaxof the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H3PO4, indicating negative impact of H3PO4on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H3PO4was achieved within 30 and 60 min, respectively. This revealed that H3PO4has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.
authors.affiliation: Department of Environmental Studies, University of Delhi, Delhi, 110007, India.
authors.forename: Nisha
authors.initials: N
authors.lastname: Shabnam
authors.affiliation: Department of Environmental Studies, University of Delhi, Delhi, 110007, India.
authors.forename: Indu
authors.initials: I
authors.lastname: Tripathi
authors.affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India.
authors.forename: P
authors.initials: P
authors.lastname: Sharmila
authors.affiliation: Department of Environmental Studies, University of Delhi, Delhi, 110007, India. ppsaradhi@gmail.com.
authors.forename: P
authors.initials: P
authors.lastname: Pardha-Saradhi
mesh_terms: D001671:Biochemistry / Q000379:methods*; D055772:Green Chemistry Technology / Q000379:methods*; D011392:Proline / Q000032:analysis*; D012015:Reference Standards; D013057:Spectrum Analysis; D013997:Time Factors
chemical_list: D011392:Proline
keywords: Ninhydrin; Phosphoric acid; Proline; Proline quantification; Toluene