Tannic Acid HPLC Method 1

PAPER 1. Using hexane, methanol-tetrahydrofuran

verzele1983.pdf

0.53MB

Normal phase

A 25 x 0.46 cm column packed with 5-pm ROSiL (a spherical silica gel from Alltech-RSL) was used. The solvent employed were (A) hexane (Burdick & Jackson Labs.) and (B) methanol-tetrahydrofuran (75:25) (both from Burdick & Jackson Labs.). To solvent B, 0.25% of pure citric acid was added. The gradient used was 80% A and 20% B at 0 min changing over 15 min to 50% A and 50% B and changing further over the next 15 min to 35%A and 65%B. The flow-rate was 1 ml/min. Detection was at 280 nm. It is essential when using this method to use solvents of high purity.

Reversed phase

Conditions were as above but with a 15 x 0.46 cm column packed with extra demineralized 5-pm ROSiLGs-D (an octadecylated spherical silica gel from All- tech-RSL). The solvents were (A) water containing 0.5% phosphoric acid and (B) methanol (Burdick & Jackson Labs.) containing 0.5% phosphoric acid. A linear gradient of 90% A and 10% B changing over 30 min to 0% A and 100% B was used. The concentration of the internal standard was 9.99 mg per 100 ml for all measure- ments. The amount of the other compounds was varied between 1 and 10 mg for gallic acid and between 10 and 100 mg for the tannic acids. The point at which integration of the composite tannic acid peak was started is indicated on some of the chromatograms shown. At least four points on the calibration graph were deter- mined for all calibrations: the linearity and fit for all points was very good. The results are summarized in Table I.
The linearity and good fit of the points leading to the data in Table I reflect


— ANALYSIS OF TANNIC ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
M. VERZELE* and P. DELAHAYE
Laboratory of Organic Chemistry, State University of Ghent, Krijgsiaan, 281 (S.4), B-W00 Ghent
(Belgium)
(Received June 3rd, 1983)

Journal of Chromatography, 268 (1983) 469476
Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

PAPER 2. Quantitative Estimation (정량분석) — MeOH, DW 이용

quantitative-estimation-of-tannins-by-hplc.pdf

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Preparation of mobile phase
The mobile phase was prepared by mixing methanol and water in ratio of 50:50 and filtered through 0.2-μm filter, using vacuum pump and sonicated for 30 min [4, 5, 6].

PAPER 3. Acetic Acid & ACN

kjfp-26-7-808.pdf

0.51MB

HPLC 분석

추출된 상등액은 HPLC 분석을 위해 추가적인 탄닌 분리
실험을 수행하였다. 총 탄닌 분석에 사용된 20 μL를 제외한 추출된 6 mL의 상등액을 진공 회전농축기(concentrator plus, Eppendorf, Hamburg, Germany)를 이용하여 용매층을 농축하고 최종 볼륨 3 mL가 되도록 H2O를 첨가하였다. 추출물에서 지방족 화합물을 제거하기 위하여 chloroform 5 mL를 첨가하고 5분간 초음파 추출기를 이용하여 혼합한 후, 1,500 rpm에서 10분간 원심 분리하였다. 상층용매(H2O 층) 2 mL를 microtube에 옮겨 15,000 rpm에서 10분간 원심 분리하고 최종적으로 1 mL의 H2O층을 취하였다. 1 mL의 H2O층은 진공 회전농축기를 이용하여 30°C 온도에서 완전 농축하고, 500 μL의 methanol으로 재용해 후, 0.22 μm의 membrane filter(Millipore, Burlington, USA)로 여과하여 HPLC 분석에 사용하였다.

DAD(Diode Array Detector)와 Capcell Pak C18 Column (4.6×250 mm I.D., Osaka Soda, Japan)이 장착된 HPLC (Agilent 1100 series, Agilent Technology, California, USA)을 이용하여 monomer 탄닌(catechin, epicatechin, gallocatechin, epigallocatechin) 및 dimer 탄닌(procyanidin B1, procyanidin B2)을 분석하였다. 주입량은 10 μL로 하였으며, 이동상은 용매 A(0.1% acetic acid in H2O)와 용매 B(100% acetonitrile) 를 A:B 용매 혼합비율 90:10(1 min), 78:22(21 min), 50:50(23 min), 10:90(25 min), 10:90(28 min), 90:10(30 min), 90:10(33 min)의 조건으로 1 mL/min의 유속에서 분석하였다. 검출 파장은 280 nm이었으며 모든 분석은 4반복으로 이루어졌다.

— “포도 과피 및 종자의 탄닌 분석을 위한 추출 용매 조건 탐색”, 김현일.허윤영.임동준.이동훈.정성민.박서준.김수진
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