Tannic Acid HPLC Method 1

PAPER 1. Using hexane, methanol-tetrahydrofuran


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

Laboratory of Organic Chemistry, State University of Ghent, Krijgsiaan, 281 (S.4), B-W00 Ghent
(Received June 3rd, 1983)

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

PAPER 2. Quantitative Estimation (์ •๋Ÿ‰๋ถ„์„) โ€” MeOH, DW ์ด์šฉ


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

PAPER 3. Acetic Acid & ACN


HPLC ๋ถ„์„

์ถ”์ถœ๋œ ์ƒ๋“ฑ์•ก์€ HPLC ๋ถ„์„์„ ์œ„ํ•ด ์ถ”๊ฐ€์ ์ธ ํƒ„๋‹Œ ๋ถ„๋ฆฌ
์‹คํ—˜์„ ์ˆ˜ํ–‰ํ•˜์˜€๋‹ค. ์ด ํƒ„๋‹Œ ๋ถ„์„์— ์‚ฌ์šฉ๋œ 20 ฮผL๋ฅผ ์ œ์™ธํ•œ ์ถ”์ถœ๋œ 6 mL์˜ ์ƒ๋“ฑ์•ก์„ ์ง„๊ณต ํšŒ์ „๋†์ถ•๊ธฐ(concentrator plus, Eppendorf, Hamburg, Germany)๋ฅผ ์ด์šฉํ•˜์—ฌ ์šฉ๋งค์ธต์„ ๋†์ถ•ํ•˜๊ณ  ์ตœ์ข… ๋ณผ๋ฅจ 3 mL๊ฐ€ ๋˜๋„๋ก H2O๋ฅผ ์ฒจ๊ฐ€ํ•˜์˜€๋‹ค. ์ถ”์ถœ๋ฌผ์—์„œ ์ง€๋ฐฉ์กฑ ํ™”ํ•ฉ๋ฌผ์„ ์ œ๊ฑฐํ•˜๊ธฐ ์œ„ํ•˜์—ฌ chloroform 5 mL๋ฅผ ์ฒจ๊ฐ€ํ•˜๊ณ  5๋ถ„๊ฐ„ ์ดˆ์ŒํŒŒ ์ถ”์ถœ๊ธฐ๋ฅผ ์ด์šฉํ•˜์—ฌ ํ˜ผํ•ฉํ•œ ํ›„, 1,500 rpm์—์„œ 10๋ถ„๊ฐ„ ์›์‹ฌ ๋ถ„๋ฆฌํ•˜์˜€๋‹ค. ์ƒ์ธต์šฉ๋งค(H2O ์ธต) 2 mL๋ฅผ microtube์— ์˜ฎ๊ฒจ 15,000 rpm์—์„œ 10๋ถ„๊ฐ„ ์›์‹ฌ ๋ถ„๋ฆฌํ•˜๊ณ  ์ตœ์ข…์ ์œผ๋กœ 1 mL์˜ H2O์ธต์„ ์ทจํ•˜์˜€๋‹ค. 1 mL์˜ H2O์ธต์€ ์ง„๊ณต ํšŒ์ „๋†์ถ•๊ธฐ๋ฅผ ์ด์šฉํ•˜์—ฌ 30ยฐC ์˜จ๋„์—์„œ ์™„์ „ ๋†์ถ•ํ•˜๊ณ , 500 ฮผL์˜ methanol์œผ๋กœ ์žฌ์šฉํ•ด ํ›„, 0.22 ฮผm์˜ membrane filter(Millipore, Burlington, USA)๋กœ ์—ฌ๊ณผํ•˜์—ฌ HPLC ๋ถ„์„์— ์‚ฌ์šฉํ•˜์˜€๋‹ค.

DAD(Diode Array Detector)์™€ Capcell Pak C18 Column (4.6ร—250 mm I.D., Osaka Soda, Japan)์ด ์žฅ์ฐฉ๋œ HPLC (Agilent 1100 series, Agilent Technology, California, USA)์„ ์ด์šฉํ•˜์—ฌ monomer ํƒ„๋‹Œ(catechin, epicatechin, gallocatechin, epigallocatechin) ๋ฐ dimer ํƒ„๋‹Œ(procyanidin B1, procyanidin B2)์„ ๋ถ„์„ํ•˜์˜€๋‹ค. ์ฃผ์ž…๋Ÿ‰์€ 10 ฮผL๋กœ ํ•˜์˜€์œผ๋ฉฐ, ์ด๋™์ƒ์€ ์šฉ๋งค A(0.1% acetic acid in H2O)์™€ ์šฉ๋งค B(100% acetonitrile) ๋ฅผ A:B ์šฉ๋งค ํ˜ผํ•ฉ๋น„์œจ 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)์˜ ์กฐ๊ฑด์œผ๋กœ 1 mL/min์˜ ์œ ์†์—์„œ ๋ถ„์„ํ•˜์˜€๋‹ค. ๊ฒ€์ถœ ํŒŒ์žฅ์€ 280 nm์ด์—ˆ์œผ๋ฉฐ ๋ชจ๋“  ๋ถ„์„์€ 4๋ฐ˜๋ณต์œผ๋กœ ์ด๋ฃจ์–ด์กŒ๋‹ค.

โ€” โ€œํฌ๋„ ๊ณผํ”ผ ๋ฐ ์ข…์ž์˜ ํƒ„๋‹Œ ๋ถ„์„์„ ์œ„ํ•œ ์ถ”์ถœ ์šฉ๋งค ์กฐ๊ฑด ํƒ์ƒ‰โ€, ๊น€ํ˜„์ผ.ํ—ˆ์œค์˜.์ž„๋™์ค€.์ด๋™ํ›ˆ.์ •์„ฑ๋ฏผ.๋ฐ•์„œ์ค€.๊น€์ˆ˜์ง„
๊ตญ๋ฆฝ์›์˜ˆํŠน์ž‘๊ณผํ•™์› ๊ณผ์ˆ˜๊ณผ