Absorption Coefficients of Phenolic Structures in Different Solvents Routinely Used for Experiments

Abstract

Phenolic structures are of great interest due to their antioxidant properties and various postulated benefits on human health. However, the quantification of these structures in fruits and vegetables, as well as in vivo or in vitro experiments, is demanding, as relevant concentrations are often low, causing problems in exactly weighing the respective amounts. Nevertheless, the determination of used concentrations is often a prerequisite for accurate results. A possibility to quantify polyphenol is the use of UV/vis spectroscopy. Therefore, the absorption coefficients of selected phenolic structures were determined in three different solvents relevant for polyphenol research (water/methanol (50/50, v/v), water, and phosphate buffer at pH 7.5). To confirm the values based on weight and to avoid errors due to impurities, hygroscopic effects, and inadequate balance care, the mass concentrations were additionally determined by quantitative NMR (q-NMR). The coefficients presented in this article can help to quickly and easily determine accurate concentrations in a laboratory routine without wasting the often-precious standard compounds.

Keywords: absorption coefficient; anthocyanins; polyphenols; q-NMR.

Figure 1

Overview of phenolic compounds investigated.

Figure 2

UV spectra of hydroxycinnamic acids in water/methanol (50/50, v/v, black), water (red), and phosphate buffer pH 7.5 (blue). (A), coumaric acid; (B), caffeic acid; (C), ferulic acid; (D), sinapinic acid. Concentrations are different for the four hydroxycinnamic acids but similar among the solvents.

Figure 3

UV-spectra of IRH-3-rut (A) and Q-3-glc (B) in water/methanol (50/50, v/v, black), water (red), and phosphate buffer pH 7 (blue). Concentrations are different for the two flavonoids but similar between the solvents.

Figure 4

UV spectra of CA (A), EGCG (B), and PHL (C) in water/methanol (50/50, v/v, blue), water (orange), and phosphate buffer pH 7.5 (gray).

Figure 5

¹H-NMR example spectrum of procyanidin-B2 (1868 mg/L) in methanol-d₄/D₂O (50/50, v/v). The signals in the range of 6.5–7.15 ppm (the six protons of Ring B and E) and 2.6–3.0 ppm (the two diastereomeric protons F4) were used for summary quantification (Figure 1, ¹H-NMR spectra with signal assignments for all PP are provided in the Supplemental Material Figure S2, including references).