Comparative Study of Absorption and Fluorescence Spectra of Glycitein and Glycitin†

doi:10.7503/cjcu20180421

Abstract

Abstract:

The absorption and fluorescence spectra of glycitein and glycitin in aqueous solutions with different pH values were investigated in detail, and the reasons why the two presented different spectral characteristics were explained in the viewpoint of molecular structure. The molecular form of glycitein is essentially no fluorescence. Under weak alkaline condition, the 7-OH proton ionization causes a redshift of the absorbance peak at 320 nm to 348 nm. The proton ionization constant is measured to be pK_a1=7.08±0.04, by a pH-photometric method. The univalent-anion form of glycitein exhibit a fairly strong fluorescence with maximum excitation and emission wavelengths(λ_ex/λ_em) of 334 nm/464 nm, and the fluorescence quantum yield is measured to be 0.049. In alkaline solutions, the ionization of 4'-OH proton of glycitein causes a redshift of the absorbance peak at 254 nm to 260 nm, the ionization constant is pK_a2=9.96±0.01. The molecular form of glycitin has almost no fluorescence. Under alkaline conditions, the ionization of 4'-OH proton causes a redshift of the absorbance peak at 256 nm to 280 nm, with pK_a=9.81±0.03. The anion form of glycitin has almost no fluorescence, but the cleavage reaction of γ-pyrone ketone ring occurs under hot and alkaline conditions and produces a fairly strong fluorescence, with λ_ex/λ_em of 288 nm/388 nm, and the fluorescence quantum yield of the cleavage reaction product is 0.056. Although the relationship between glycitin and glycitein is as that of glycoside and aglycone, but glycitin cannot be converted to glycitein by means of hydrolysis of glycoside under hot alkaline conditions. The fluorescence enhancement mechanisms of these two are essentially different.

Key words: Isoflavone, Glycitein, Glycitin, Cleavage reaction, Fluorescence enhancement

TrendMD:

LI Wenhong,WANG Danyang,CAO Jinjin,WEI Yongju. Comparative Study of Absorption and Fluorescence Spectra of Glycitein and Glycitin^†[J]. Chem. J. Chinese Universities, 2019, 40(1): 47.

Figures/Tables 14

Fig.1 Molecular structures of glycitein(A) and glycitin(B)

Fig.2 Absorption spectra of glycitein aqueous solutions at different pH values(A, C) and the relationship between absorbance and pH value(B, D)

Scheme 1 Proton dissociation reaction of glycitein Volume fraction of MeOH: 10%. (B) 6.56 μg/mL, 254 nm; (D) 6.76 μg/mL, 238 nm.

Table 1 Determination of pKa1 of glycitein*

pH	A	pK_a1	Average(pK_a1)
6.85	0.2824	7.14	7.08±0.04
6.94	0.3206	7.10	7.08±0.04
7.00	0.3475	7.08	7.08±0.04
7.04	0.3856	7.01	7.08±0.04

Table 2 Determination of pKa2 of glycitein*

pH	A	pK_a2	Average(pK_a2)
9.74	0.5511	9.95	9.96±0.01
9.86	0.5423	9.96	9.96±0.01
10.02	0.5311	9.97	9.96±0.01
10.21	0.5161	9.96	9.96±0.01

Fig.3 Fluorescence spectra of glycitein aqueous solutions at different pH values(A) and relationship between fluorescence intensity and pH value(B) c: 1.56 μg/mL, volume fraction of MeOH: 10%, λex/λem: 347 nm/456 nm.

Scheme 9 Cleavage reaction of glycitein

Fig.4 Absorption spectra of glycitin aqueous solutions at different pH values(A) and relationship between absorbance and pH value(B) c: 8.20 μg/mL, volume fraction of MeOH: 10%, λ=280 nm.

Scheme 3 Proton dissociation reaction of glycitin

Table 3 Determination of pKa of glycitin*

pH	A	pK_a	Average(pK_a)
9.63	0.3114	9.85	9.81±0.03
9.79	0.3342	9.87	9.81±0.03
9.90	0.3633	9.80	9.81±0.03
10.12	0.3946	9.81	9.81±0.03

Fig.5 Fluorescence spectra of glycitin alkaline solutions at different pH values(A) and influence of pH on fluorescence intensity(B) c: 1.68 μg/mL, volume fraction of MeOH: 10%, heat for 0.5 h at 100 ℃, λex/λem: 307 nm/415 nm.

Scheme 4 Cleavage and hydrolysis reaction of glycitin

Fig.6 Influence of methanol on fluorescence intensity of glyciteinc: 1.56 μg/mL, pH=8.8, λex/λem: 347 nm/456 nm.

Fig.7 Relationship between fluorescence intensity and heating temperaturec: 1.68 μg/mL, volume fraction of MeOH: 10%, heat for 0.5 h, pH=13.2, λex/λem: 307 nm/415 nm.

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