Lambda 365 uv vis spectrophotometer

For the experimental part of this work, DMABN was purchased from Aldrich (Purity 98%, MW: 146.19 g/mol, mp: 72–75 $${}^{\circ }$$ C, bp: 318 $${}^{\circ }$$ C) and used without further purification. Solutions of DMABN in cyclohexane, tetrahydrofuran, acetonitrile, and water were prepared at room temperature. Concentrations of these solutions within the range of $${10}^{-6}$$ to $${10}^{-3}$$ M were measured. All solvents were commercially obtained from several chemical companies and used without further purification. The solvents did not show any traces of impurities in absorption and fluorescence measurements. In Table 1, a list of the solvents used in this work is given along with their physical characteristics where $$ϵ$$ is the dielectric constant and n is the refractive index of the solvent.
The absorption measurements were made using a Perkin Elmer UV/VIS Lambda 365 spectrophotometer. The fluorescence emission spectra were recorded with a Horiba Jobin-Yvon Fluorolog-3 spectrofluorometer using a Xenon lamp as a source of beam excitation at 290 nm (4.27 eV). Fluorescence spectra were collected in the signal/reference (S/R) mode to correct for the changes in the lamp output intensity. Samples for absorption and emission measurements were prepared in a 1.0 cm path length of quartz cuvettes. Solvent blanks were subtracted from both the absorption and fluorescence emission spectra before the analysis.

Absorption spectra were measured
by a PerkinElmer Lambda 365 UV–vis spectrophotometer. PL spectra
were measured by Edinburgh Instruments FLS980 spectrofluorometer with
a xenon lamp as excitation source. PL lifetimes were recorded on an
Edinburgh LifeSpec-ps spectrometer with a fixed excitation wavelength
of 404 nm.

For quantifying untreated and treated pea protein solutions, the Biuret assay based on the work of [38 (link)] was utilized. Therefore, a Biuret reagent containing 9.4 mM copper sulfate, 28.56 mM potassium sodium tartrate, and 831 mM sodium hydroxide was prepared. For the assay, 800 μL of the Biuret reagent and 200 μL of the diluted sample were mixed. Complexation was reached during 20 min of incubation at 37 °C in a shaking incubator (250 rpm). Afterward, the absorption was measured at a wavelength of 540 nm (Lambda 365 UV-VIS Spectrophotometer, Perkin Elmer, Waltham, MA, USA). Deionized water was used for dilution and as a blank. Bovine serum albumin was utilized for establishing a calibration curve in the concentration range 0.04–5 g/L.

Lipid peroxidation was assessed by measuring the malondialdehyde (MDA) content in rice leaves [79 (link)]. For this, 100 mg of rice leaves from each treatment was homogenized with 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 10 min at room temperature. Then, the supernatant was mixed with 1.5 mL of 0.5% (w/v) thiobarbituric acid (TBA) and the sample mixtures were incubated in a water bath at 95 °C for 25 min. The reaction was terminated by incubating the samples on an ice bath, and the sample solutions were again centrifuged at 10,000 rpm for 5 min. The fluorescence intensity at 532 and 600 nm was measured in the supernatant using a spectrophotometer (LAMBDA 365 UV–Vis spectrophotometer, PerkinElmer, Mumbai, India). The MDA content was calculated as follows: $\mathrm{MDA} \mathrm{equivalent} \mathrm{content} \mathrm{in} \mathrm{leaf} \mathrm{tissues}=\frac{\left(Absorbance at 523 \mathrm{nm}-Absorbance at 600 \mathrm{nm}\right)}{155} \times 1000.$
The results are expressed as nmole of MDA equivalent per gram of fresh weight (nmol MDA mL⁻¹ g FW⁻¹).