US EPA

1451644 

Journal Article 

Purification of berry flavonol glycosides by long-bed gel permeation chromatography 

Riihinen, KR; Goedecke, T; Pauli, GF 

2012 

Yes 

Journal of Chromatography A
ISSN: 0021-9673
EISSN: 1873-3778 

1244 

20-27 

22609168 

10.1016/j.chroma.2012.04.060 

WOS:000305361400003 

While Sephadex LH-20 gel is frequently employed as a
stationary phase during pre-separations and in open column chromatography systems, its separation
power in long-bed gel permeation chromatography (GPC) applications is much less prevalent. Aimed
at the characterization of bioactive constituents, a long-bed GPC protocol was established for
lingonberry juice concentrate. The method included pre-fractionation over HP-20 resin to
eliminate sugars and organic acids as well as a major part of other predominant berry flavonoids
(anthocyanins, flavan-3-ols and proanthocyanidins), prior to the elution of the fraction
containing 10% (w/w) of quercetin glycosides (QGs). Subsequently, seven major QGs were purified
using a 10-m Sephadex LH-20 system and isocratic elution with methanol. The total mass recovery
was 99.3 +/- 1.4%, after eluting the highly-retained compounds from the employed pre-column with
70% acetone. Injecting 1070 mg per run, the yield of purified QGs ranged from 2 to 6 mg per
collected single fraction. The LC-UV/PDA purities of isolated Q-3-O-alpha-rhamnoside and Q-3-O-
beta-galactoside were 82 and 94 area% at 250 nm, while the three Q-pentosides showed purities of
59, 30, and 57 area%. By comparison, purity assessment of these isolates by quantitative H-1 NMR
(total integral and modified 100% method) led to significantly lower purities of 70 and 52% for
Q-rha and Q-gal and 38,25 and 46% for Q-pentosides, respectively. This can be explained by the
presence of hidden residual complexity (RC), which is revealed by the quantitative NMR method.
This finding has potentially broader implication as it reveals an unexpected degree of RC in GPC
fractions. Despite remarkable separation power for congeneric flavonoids, long-bed GPC on
Sephadex LH-20 produces materials, which require careful analysis of purity before interpreting
bioassay results. (C) 2012 Elsevier B.V. All rights reserved. 

Food and dietary supplement analysis; Berries; Flavonoid; Fractionation; Polyphenols; qHNMR