• Natural Product Sciences
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• v.24 no.4
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• pp.288-292
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• 2018

High-performance countercurrent chromatography (HPCCC) coupled with reversed-phase highperformance liquid chromatography (RP-HPLC) method was developed to isolate dihydrophaseic acid 3'-O-${\beta}$-D-glucopyranoside (DHPAG) from the extract of Nelumbo nucifera seeds. Enriched DHPAG sample (2.3 g) was separated by HPCCC using ethyl acetate/n-butanol/water system (6:4:10, v/v/v, normal-phase mode, flow rate: 4.0 mL/min) to give 23.1 mg of DHPAG with purity of 88.7%. Further preparative RP-HPLC experiment gave pure DHPAG (16.3 mg, purity > 98%). The current study demonstrates that utilization of CCC method maximizes the isolation efficiency compared with that of solid-based conventional column chromatography.

• Natural Product Sciences
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• v.21 no.1
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• pp.30-33
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• 2015

Anthocyanins are water soluble plant pigments which are responsible for the blue, red, pink, violet colors in several plant organs such as flowers, fruits, leaves and roots. In recent years, anthocyanin-rich foods have been favored as dietary supplements and health care products due to diverse biological activities of anthocyanins including antioxidant, anti-allergic, anti-diabetic, anti-microbial, anti-cancer and preventing cardiovascular disease. High-performance countercurrent chromatography (HPCCC) coupled with reversed-phase medium pressure liquid chromatography (RP MPLC) method was applied for the rapid and efficient isolation of cyanidin 3-glucoside (C3G) and peonidin 3-glucoside (P3G) from black rice (Oryza sativa L., Poaceae). The crude black rice extract (500 mg) was subjected to HPCCC using two-phase solvent system composed of tert-butyl methyl ether/n-butanol/ acetonitrile/0.01% trifluoroacetic acid (TBME/B/A/0.01% TFA, 1 : 3 : 1 : 5, v/v, flow rate - 4.5 mL/min, reversed phase mode) to give enriched anthocyanin extract (37.4 mg), and enriched anthocyanin extract was sequentially chromatographed on RP-MPLC to yield C3G (16.5 mg) and P3G (8.7 mg). The recovery rate and purity of isolated C3G were 76.0% and 98.2%, respectively, and those of P3G were 58.3% and 96.3%, respectively. The present study indicates that HPCCC coupled with RP-MPLC method is more rapid and efficient than multi-step conventional column chromatography for the separation of anthocyanins.

• Natural Product Sciences
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• v.21 no.1
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• pp.20-24
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• 2015

Cyanidin-3-glucoside (C3G) and cyanidin-3-rutinoside (C3R) were isolated by high-performance countercurrent chromatography (HPCCC) using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water/trifluoroacetic acid (1 : 3 : 1 : 5 : 0.01, v/v) to give pure C3G (34.1 mg) and C3R (14.3 mg) from 1.5 g crude mulberry fruit extract. Using the pure C3G and C3R, a reliable high-performance liquid chromatography (HPLC) method was developed and validated to determine the C3G and C3R contents in mulberry fruit. C3G and C3R were separated simultaneously using an Eclipse XDB-C18 column ($4.6{\times}250mm$ I.D., $5{\mu}m$) coupled with a photodiode array detector (PDA). The gradient elution of the mobile phase consisting of acetonitrile (0.5% formic acid) and water (0.5% formic acid) was applied (1.0 mL/min), and the detection wavelength was 520 nm. The calibration curves of C3G and C3R showed good linearity (both with $r^2=0.9996$) in the concentration range $15.625-500{\mu}g/mL$, and the relative standard deviations (RSD%) of intra- and inter-day variability were in the ranges 2.1 - 8.2% and 4.1 - 17.1%, respectively. The accuracies were ranged 96.5 - 102.6% for C3G and C3R, respectively. The developed HPLC method was used to determine the contents of C3G and C3R in newly harvested mulberry from eight different provinces of Korea.

• KSBB Journal
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• v.25 no.3
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• pp.205-214
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• 2010

Many successive liquid-liquid extractions occur enabling purification of the crude material to occur. In high performance counter-current chromatography (HPCCC), crude material is partitioned between two immiscible layers of solvent phases. The stationary phase (SP) is retained by hydrodynamic force field effect and the mobile phase (MP) is pumped through the column. Purification occurs because of the different solubility of the components in the liquid mobile and stationary phases. There are many key benefits of liquid stationary phases such as high mass and volume injection loadings, total sample recovery, and easy scale-up. Many researchers showed that predictable scale-up from simple test is feasible with knowledge of the stationary phase retention for the planned process scale run. In this review we review the recent advances in HPCCC research and also describe the key applications such as natural products and synthetics (small or large molecules).