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Available online at www.sciencedirect.com

Separation and Purification Technology 61 (2008) 358–365

Supercritical carbon dioxide extraction of rice bran oiland column partition fractionation of �-oryzanols

Chao-Rui Chen a, Ling-Ya Wang a, Chih-Hung Wang a, Wai-Jane Ho b, Chieh-Ming J. Chang a,∗a Department of Chemical Engineering, National Chung Hsing University, #250, KuoKuang Road, Taichung 402, Taiwan, ROC

b Department of Bioresources, Dayen University, #112, ShanJiao Road, ChangHua 515, Taiwan, ROC

Received 8 August 2007; received in revised form 29 October 2007; accepted 1 November 2007

bstract

This work studies how pressure and temperature affect supercritical carbon dioxide extraction of rice bran oil from powdered rice bran, followedy the concentration and isolation of �-oryzanols by column partition purification. The two purest �-oryzanols (>98 wt.%) were isolated fromeagent chemical �-oryzanols by preparative reverse-phase high-pressure liquid chromatography. In searching for a suitable range of extractiononditions, supercritical extraction at 350 bar and 313 K yielded 17.5% oil and the extraction efficiency of �-oryzanols was 84.9%, using 1200 g ofarbon dioxide over 4 h. Finally, supercritical extractions at pressures from 250 to 350 bar and at temperatures from 313 to 333 K were chosen based

n the response surface methodology to determine their effects on concentration of �-oryzanols in the extracted oil. Pressure proved more significanthan temperature in increasing the concentration of �-oryzanols. A significant amount of the purest oil that contained 37.0 wt.% �-oryzanols wasbtained following a normal-phase medium-pressure column partition fractionation of the extracted oil.

2007 Elsevier B.V. All rights reserved.

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eywords: Column chromatography; Extraction; �-Oryzanols; Rice bran; Supe

. Introduction

Rice bran is comprised of 11–15% proteins, 34–62% carbo-ydrates, 7–11% crude fibers, 7–10% ashes and 15–20% lipids,hich are by-products of the rice-refining process [1]. Thisaterial contains nutritional and non-nutritional compounds that

enefit humans. Rice bran oil contains 95.6% saponifiable lipids,uch as glycolipid and phospholipids and 4.2% unsaponifiableipids, such as tocopherols, tocotrienols, �-oryzanols, sterolsnd cartenoids [2]. Lipids are comprised of mostly triglycerides.owever, a lipase can hydrolyze these triglycerides to yield free

atty acids [3].The content of �-oryzanols in the extracted rice bran oil is

pproximately 1.8–3%, according to Hu et al. [3]. Xu and God-er [4] employed a low-pressure normal-phase silica column toield an enrichment of oryzanol in the original oil. This RBO

as further partitioned using a preparative normal-phase HPLCethod. Finally, ten �-oryzanols have been identified by reverse-

hase HPLC coupled with GC-Mass chromatography. They

∗ Corresponding author. Tel.: +886 4 2285 2592; fax: +886 4 2286 0231.E-mail address: cmchang@dragon.nchu.edu.tw (C.-M.J. Chang).

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383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2007.11.003

cal carbon dioxide

oncluded that cycloartenyl ferulate, 24-methylenecycloartanylerulate and campesteryl ferulate are three major �-oryzanolsn rice bran. These �-oryzanols have several biological andhysiological effects, such as serving as anti-oxidation [5],nti-blood cholesterol [6] and anti-carcinogenic agents [7–10].he LC–MS/MS method is frequently adopted in the structural

dentification of �-oryzanols [11]. Stoggl et al. simultane-usly identified and quantified tocopherols, �-oryzanols andartenoids [12]. Aguilar-Garcia et al. also performed a bio-ogical study of a correlation between �-oryzanols content andnti-oxidant capacity [13].

Wastes of edible rice bran oil can be processed into bio-iesel by acid-catalyzed transesterification [14]. In recent years,upercritical fluid extractions of powdered rice bran haveemonstrated that the amount of extracted oil is equivalent tohat obtained by Soxhlet N-hexane extraction [15]. Deacidi-cation of rice bran oil using supercritical carbon dioxide isecognized as a very environmental friendly process. Kim etl. investigated the time-related mass transfer kinetics of oil

omponents between solid-phase (rice bran) and fluid-phasecarbon dioxide) in supercritical carbon dioxide extraction [16].hang et al. studied supercritical carbon dioxide extractioninetics and high-pressure vapor–liquid-phase equilibrium mea-

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urements between oil compounds and carbon dioxide [17,18].any multi-stage supercritical fluid deacidifications of rice bran

il have been conducted to remove free fatty acids or triglyc-rides from raffinate oil using a packed column at a middle–highressure at 333–353 K to increase concentrations of �-oryzanolsnd phytosterols in the oil [19–24].

Except for cycloartenyl ferulate, no pure standard of �-ryzanols can be obtained from local chemical suppliers. Thisoal of this work was to obtain pure 24-methylenecycloartanylerulate and campesteryl ferulate from reagent chemical �-ryzanols using a semi-preparative HPLC method. Supercriticalarbon dioxide extraction of rice bran oil from powdered riceran, designed using response surface methodology, followedy the concentration of �-oryzanols in the extracted oil using aiddle-pressure column partition fractionation, was studied.

. Experimental method

.1. Reagents and materials

Ten kilograms of fresh rice bran powder were donated bylocal Da-quan rice bran producer (Taichung, Taiwan) and

ere stored in a cooler at 269 K before use. The water contentf this rice bran powder was to be under 10%, as determinedsing a moisture meter (A&D, AD-4714A, Japan). De-ionizedater was obtained using a Milli-Q reverse osmosis purifica-

aroo

Fig. 1. Schematic flow diagram of SC

tion Technology 61 (2008) 358–365 359

ion system. Analytical grade reagents—99.95% carbon dioxideToyo gas, Taiwan), 99.7% nitrogen (Toyo gas), 99.8% hydrogenToyo gas), 99% air (Toyo gas), 99.9% ethyl acetate (Mallinck-odt, USA), 99.9% N-hexane (Mallinckrodt), 99.9% methanolMallinckrodt), 99.9% dichloromethane (Mallinckrodt), 99.5%thyl ether (Mallinckrodt), 99.5% isopropanol (Mallinckrodt),9.9% acetonitrile (J.T. Baker, USA), 99.8% acetic acid (Merck,ermany) and 95% ethanol (Taiwan Sugar Co. Ltd., Taiwan),ere purchased from a local supplier and used without further

reatment. Authentic standards including 98% cycloartenyl fer-late (Wako, Japan), 95% mixed standard �-oryzanols (Wako)nd a fatty acid methyl esters standard (Supelco, USA) weresed herein only for quantification.

.2. Isolation and identification of two γ-oryzanols

A semi-preparative HPLC system using one C18 columnYMC-C18, 250 mm × 10 mm i.d.) that is connected to a UVetector (PE-785A, PerkinElmer) via a high-pressure pump (PE-10, PerkinElmer). The 1.5 mL (500 ppm) mixed standard of-oryzanols dissolved in methanol was injected into a 5 mL

oop and this sample was partitioned using a mixed solvent of

cetonitrile, dichrolomethane and acetic acid (90:6:4) at a flowate of 5 mL/min. The eluates were detected at a wavelengthf 330 nm and four isolated compounds were collected basedn their retention time. The isolated 24-methylenecycloartanyl

-CO2 extraction of rice bran oil.

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teeTcolumn (440 mm L × 37 mm i.d.) that was packed with silicagel 60 resin (LiChroprep-Si-60, 40–63 �m, Merck, Germany).The diluted solution of SC-CO2-extracted oil was pumped intothe medium-pressure column through an intelligent pump (PU-

60 C.-R. Chen et al. / Separation and Pu

erulate and campesteryl ferulate were obtained at the 10 mgevel. Their chemical structures were identified using a 400 HzH NMR spectrophotometer.

.3. Quantification of γ-oryzanols, free fatty acids andriglycerides

HPLC quantifications of four �-oryzanols (cycloartenyl fer-late, 24-methylenecycloartanyl ferulate, campesteryl ferulatend sitosteryl ferulate) and three free fatty acids (oleic acid,inoleic acid and linolenic acid) were performed using a reverse-hase analytical column (YMC-C18, 5 �m, 250 mm × 4.6 mm.d., Japan). The column was linked to a UV/vis detector (L-200H, Hitachi, Japan) by a high-pressure pump (L-7100,itachi, Japan) and the software to control the interface (D-000, Hitachi, Japan). The column temperature was maintainedt 313 K using a column oven (TCM-004657, Waters-Millipore,SA). The absorption of UV from the samples was detected

t a wavelength of 330 and 210 nm for the each of the four �-ryzanols and the three free fatty acids. The injection volume ofhe sample was 20 �L. One mobile phase of the mixed solventf 90% acetonitrile, 6% dichrolomethane and 4% acetic acidy weight fraction was used in the �-oryzanols analysis. Thether mobile phase of the mixed solvent of 85% acetonitrile,% methanol and 10% de-ionized water with 1% acetic acidas used to analyze the free fatty acids. Both R-square corre-

ation coefficients of the calibration curves of �-oryzanols andree fatty acids were at least 0.999.

GC quantification of seven triglycerides was performed usingnon-polar capillary column (DB-5, J&W, USA) in a gas

hromatograph (GC-14B, Shimadzu, Japan). The column tem-erature was set to 443 K initially, and programmed to increase to88 K at 5 K/min, then to 496 K at 2 K/min, and finally to 503 Kt 1 K/min. The split ratio was 3.4: 1, and the flame ionizationetector temperature was set to 553 K.

.4. Classical extraction of solvents

For the Soxhlet solvent extraction, 15 g of rice bran pow-er was loaded into a 50 mL Soxhlet timber-type extractor witheflux having 16 turnover numbers and extracted using 300 mL-hexane, isopropanol and methanol individually for 4 h. All of

he extracts were collected and weighed. The total amount ofxtract, and the extraction efficiencies of �-oryzanols, free fattycids and triglycerides were then calculated.

.5. Supercritical carbon dioxide extraction of rice bran oil

Fig. 1 displays a schematic flow diagram of SC-CO2 extrac-ion from rice bran. 10 g was rice bran powder was packed inside250 mL stainless steel tubular extractor with an internal diam-ter of 2.5 cm and a height of 54 cm. A specified amount oflass wool was packed into the two ends of the extractor to pre-

ent the rice bran powder escaping from the extractor. LiquidO2 from a cylinder (1) into which was inserted a siphon-tubeassed through a cooling bath (3) at 277 K, was compressed tohe desired working pressure using a syringe pump (100DX,

Fo

tion Technology 61 (2008) 358–365

SCO, USA) (5), and was heated to supercritical conditionssing a double-pipe heat exchanger (8) and a re-boiler (10-). This carbon dioxide, maintained at a flow rate of 5 L/minSTP), flowed into the extractor (10-1); came into contact withhe rice bran powder, and was used to extract the oil. A heat-ng element equipped with a PID temperature controller (4)as thermostatically maintained at a constant temperature; twoack-pressure regulators (12-1, 12-2) located at the outlet wereanually adjusted to maintain constant extraction pressure. Fol-

owing the extraction, CO2 entered into a 130 mL separator (14)y a drop in pressure and was expanded through a spiral-typeozzle. The SC-CO2 extract then passed through a heated lineonnector (17) and entered in a separator that was maintainedt 50 bar and 303 K. The amount of CO2 used was measuredsing a wet gas meter (W-NK-Da-1B, Shinagawa, Japan) (15)nd thus returned to the ambient conditions. Precipitates (15%xtracted oil) that had washed out from the back-pressure regu-ators and the transfer lines were then mixed with the collectedil (85% oil) to prepare them for analysis. At the end of eachxperiment, the extracted solution was collected and the solventas dried out using a vacuum rotary evaporator. The residue was

hen weighed and stored at 273 K before use.

.6. Column partition fractionation of γ-oryzanols oil

Solid–liquid column purification, a form of partition extrac-ion, has been used in the isolation of bioactive compounds fromxtracted oil [25–27]. In this study, a total 15 g of SC-CO2-xtracted oil was obtained and diluted with N-hexane in a batch.he diluted solution was then loaded into a medium-pressure

ig. 2. Block diagram of solid–liquid column partition fractionation of rice branil.

C.-R

.Chen

etal./Separationand

Purification

Technology61

(2008)358–365

361Table 1Experimental data on Soxhlet extractions and SC-CO2 extractions of rice bran oil

Exp# WCO2 (g) Woil (g) TY (%) WOry

(mg/goil)W∗

Ory(mg/gRB)

ROry

(%)βOry WFFA

(mg/goil)W∗

FFA(mg/gRB)

RFFA

(%)βFFA WTG

(mg/goil)W∗

TG(mg/gRB)

RTG

(%)βTG Others

(mg/goil)

Soxhleta (15 g rice bran)1 – 3.00 20.0 15.2 3.03 100.0 5.00 95.0 19.0 100.0 5.00 800 160.0 100.0 5.00 89.82 – 3.97 26.4 9.07 2.40 79.1 2.99 68.1 18.0 94.8 3.59 555 146.7 91.7 3.47 367.93 – 3.49 23.3 12.3 2.87 94.5 4.06 70.0 16.3 85.7 3.68 574 133.6 83.5 3.59 343.7

SC-CO2b (10–20 g rice bran)

4 600 1.64 16.4 13.9 2.28 75.2 4.59 101.0 16.5 87.0 5.31 793 129.8 81.1 4.95 92.35 800 1.69 16.9 13.5 2.28 75.3 4.46 102.0 17.2 90.6 5.37 793 133.9 83.7 4.96 91.56 1200 1.75 17.5 15.2 2.67 87.9 5.01 101.4 17.8 93.5 5.33 798 139.8 87.4 4.99 85.77 1200 2.65 17.6 14.6 2.57 84.8 4.81 105.6 18.6 98.0 5.56 787 138.8 86.7 4.92 92.98 1600 2.84 14.2 10.3 1.46 48.1 3.39 92.9 13.2 69.4 4.89 789 112.0 70.0 4.93 107.6

(1) 342 K N-hexane; (2) 356 K isopropanol; (3) 338 K methanol; WCO2 : consumption of carbon dioxide; Woil: weight of the extracted; TY: total oil yield = (Woil/WRB) × 100%; WRB: weight of rice bran;WOry: weight of oryzanols; W∗

Ory: yield of oryzanols; WFFA: weight of free fatty acids; W∗FFA: yield of free fatty acids; WTG: weight of triglycerides; W∗

TG: yield of triglycerides; ROry: oryzanols extractionefficiency = (WOry/WOry,Soxhlet) × 100%; RFFA: free fatty acids extraction efficiency = (WFFA/WFFA,Soxhlet) × 100%; RTG: triglycerides extraction efficiency = (WTG/WTG,Soxhlet) × 100%; βOry: concentration factor oforyzanols = ROry/TY; βFFA: concentration factor of free fatty acids = RFFA/TY; βTG: concentration factor of triglycerides = RTG/TY; others: weight of waxes, glycolipids and phospholipids; TYSoxhlet = 20.0 (%);W∗

Ory,Soxhlet = 3.03 (mg/g); W∗FFA,Soxhlet = 19.0 (mg/g); W∗

TG,Soxhlet = 160.0 (mg/g); βOry,FFA,TG,Soxhlet = 5.00.a Four hours Soxhlet extraction.b SC-CO2 extractions at 350 bar, 313 K, 10 g/min.

Table 2Response surface-designed SC-CO2 extractions of 10 g of rice bran powder

RSM# P (bar) T (K) Woil

(g)TY(%)

WOry

(mg/goil)W∗

Ory(mg/gRB)

ROry

(%)βOry WFFA

(mg/goil)W∗

FFA(mg/gRB)

RFFA

(%)βFFA WTG

(mg/goil)W∗

TG(mg/gRB)

RTG

(%)βTG Others

(mg/goil)

1(A) 250 313 0.86 8.6 4.6 0.40 13.1 1.51 170 14.7 77.3 8.96 784 67.6 42.3 4.90 41.22(F) 250 323 0.82 8.2 4.8 0.39 12.8 1.57 175 14.3 75.4 9.20 776 63.6 39.7 4.85 44.23(A) 250 333 0.82 8.2 4.7 0.38 12.6 1.53 217 17.8 93.8 11.42 721 59.3 37.1 4.51 57.04(F) 300 313 1.48 14.8 9.0 1.32 43.7 2.96 119 17.6 92.6 6.27 800 118.2 73.9 5.00 71.65(C) 300 323 1.43 14.3 6.5 0.92 30.5 2.14 121 17.2 90.6 6.36 786 112.0 70.0 4.91 86.66(F) 300 333 1.52 15.2 7.5 1.14 37.6 2.48 117 17.7 93.2 6.15 792 120.1 75.0 4.95 83.07(A) 350 313 1.75 17.5 14.7 2.57 84.9 4.86 105 18.4 96.7 5.54 798 139.3 87.1 4.99 82.28(F) 350 323 1.73 17.3 13.9 2.40 79.3 4.59 104 18.0 94.6 5.48 760 131.2 82.0 4.75 122.29(A) 350 333 1.73 17.3 14.7 2.55 84.1 4.86 104 18.0 94.8 5.47 747 129.4 80.8 4.67 134.4

P: pressure; T: temperature; Woil: weight of the extracted; TY: total oil yield = (Woil/WRB) × 100%; WRB: weight of rice bran; WOry: weight of oryzanols; W∗Ory: yield of oryzanols; WFFA: weight of free

fatty acids; W∗FFA: yield of free fatty acids; WTG: weight of triglycerides; W∗

TG: yield of triglycerides; ROry: oryzanols extraction efficiency = (WOry/WOry,Soxhlet) × 100%; RFFA: free fatty acids extraction effi-ciency = (WFFA/WFFA,Soxhlet) × 100%; RTG: triglycerides extraction efficiency = (WTG/WTG,Soxhlet) × 100%; βOry: concentration factor of oryzanols = ROry/TY; βFFA: concentration factor of free fatty acids = RFFA/TY;βTG: concentration factor of triglycerides = RTG/TY; others: weight of waxes, glycolipids and phospholipids; TYSoxhlet = 20.0 (%); W∗

Ory,Soxhlet = 3.03 (mg/g); W∗FFA,Soxhlet = 19.0 (mg/g); W∗

TG,Soxhlet = 160.0 (mg/g);βOry,FFA,TG,Soxhlet = 5.00.

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3.3. Experimental designed SC-CO2 extraction

A two-factor parameter (pressure and temperature) responsesurface methodology (RSM), with four axial points, four factor

62 C.-R. Chen et al. / Separation and Pu

580, Jasco, Japan). Chromatography was then employed toractionate the �-oryzanols with an affinity for fat from otherpecies in the oil. The eluting solvent consisted of N-hexane (H)nd ethyl acetate (EA).

Fig. 2 presents an information block diagram. In thesetepwise elution processes, most triglycerides and free fattycids were initially removed by the eluent but the �-oryzanolsemained adsorbed and were later eluted. The flow rate to the col-mn was 2 mL/min. Five eluting solvent mixtures of N-hexaneith ethyl acetate in volume ratios of 100:0, 98:2, 95:5, 90:10

nd 0:100 were used in this fractionation study. After the solventad been dried out, each fraction was concentrated and analyzed.

. Results and discussion

.1. Two purified components of γ-oryzanols

The 24-methylenecycloartanyl ferulate chemical shiftsattern of 1H NMR in CDCl3, δ was 7.60 (d, J = 15.8 Hz), 7.08dd, J = 1.8, 8.1 Hz), 7.04 (d, J = 1.8 Hz), 6.92 (d, J = 8.4 Hz),.30 (d, J = 16.1 Hz), 5.85 (br s), 4.72 (br s), 4.71 (m), 4.67br s), 3.94 (s), 2.24 (sept., J = 7.0 Hz), 1.04 (d, J = 7.0 Hz),.03 (d, J = 6.6 Hz), 0.98 (s), 0.92 (s), 0.9 (d, J = 5.5 Hz), 0.9s), 0.60 (d, J = 4.0 Hz, endo), 0.37 (d, J = 4.0 Hz, exo). Theampesteryl ferulate chemical shifts pattern of 1H NMR inDCl3, δ was 7.60 (d, J = 15.8 Hz), 7.07 (dd, J = 1.8, 8.4 Hz),.03 (d, J = 1.8 Hz), 6.91 (d, J = 8.4 Hz), 6.28 (d, J = 16.1 Hz),.84 (br s), 5.40 (m), 4.71 (m), 3.92 (s), 2.41 (br s), 2.39 (br), 1.05 (s), 0.92 (d, J = 6.6 Hz), 0.85 (d, J = 6.2 Hz), 0.81 (d,= 6.8 Hz), 0.78 (d, J = 6.6 Hz), 0.69 (s). Yasukawa et al. hadlready described these spectra patterns [7].

.2. Extractions of rice bran oil

Table 1 presents experimental data on a few Soxhlet and SC-O2 extractions of rice bran oil. The maximum concentrationf �-oryzanols in the N-hexane-extracted oil was 15.2 mg/goil,hich is the �-oryzanols content in rice bran powder. A concen-

ration of 3.03 mg/gpowder was obtained in 4 hr from a 300 mLoxhlet N-hexane extraction of 15 g rice bran powder. This rep-esents 100% recovery of �-oryzanols from the rice bran powder.he maximal amount of this N-hexane-extracted oil was 3 g,hich was highest among all of these organic solvent extrac-

ions. The amount of SC-CO2-extracted oil is less than that ofoxhlet N-hexane-extracted oil. These two constituents reached0%, as indicated in Table 1.

Parameters of SC-CO2 extraction that potentially influencehe extraction efficiency include solids in the feed, consumptionf carbon dioxide, the temperature and the pressure. A set ofreliminary experimental tests were performed amounts of riceran powder ranged from 10 to 20 g; amounts of CO2 consumedanged from 600 to 1600 g. The experimental data obtained inhese tests revealed that the amount of solids and the consump-

ion of carbon dioxide markedly affect the extraction efficiencyf rice bran oil. The extracted amount of oil and the extrac-ion efficiency of �-oryzanols increased with the carbon dioxideonsumption; 17.5% of oil and 87.9% recovery of �-oryzanols

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S

tion Technology 61 (2008) 358–365

ere obtained at a CO2 to solids ratio of 120. The amount of oilxtracted declined as the amount of solids increased (datum# 8n Table 1).

ig. 3. Effects of pressure and temperature on SC-CO2 extractions of rice branil, (1) total oil yield; (2) extraction efficiency of oryzanols; (3) concentration fac-or of oryzanols in 3D. (F-testing: R2

(1) = 0.9979, S.D.(1) = 0.30; R2(1) = 0.9943,

.D.(2) = 3.83; R2(3) = 0.9874, S.D.(3) = 0.27).

rification Technology 61 (2008) 358–365 363

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C.-R. Chen et al. / Separation and Pu

oints and one center point was designed for the SC-CO2xtraction of rice bran oil from the 10 g of rice bran powder.he flow rate of CO2 was 10 g/min and the amount of CO2onsumed was 1200 g, as determined from the results in Table 1.able 2 presents experimental data on RSM SC-CO2 extractiont pressures from 250 to 350 bar and temperatures from 313 to33 K.

Fig. 3 shows that the total oil yield (TY), the extraction effi-iency (ROry) and the concentration factor (βOry) reaches theaxima of 17.5%, 84.9% and 4.86, respectively, at 350 bar

nd 313 K (datum# 7 in Table 2). The pressure effect seemso be more significant than the temperature effect. The totalil yield, extraction efficiency of �-oryzanols (ROry) and con-entration factor of �-oryzanols (βOry) increased with pressure,ut decreased as the temperature increased. Fig. 4 reveals that

he extraction efficiency of free fatty acids (RFFA) and the con-entration factor (βFFA) reached minima of 10.4% and 5.47,espectively, at 350 bar and 333 K (datum# 9 in Table 2). Themount of free fatty acids in extracted oil and the concentra-

ig. 4. Effects of pressure and temperature on SC-CO2 extractions of rice branil, (1) extraction efficiency of free fatty acids; (2) concentration factor ofree fatty acids in 3D. (F-testing: R2

(1) = 0.9705, S.D.(1) = 1.14; R2(2) = 0.9703,

.D.(2) = 0.60).

Fig. 5. Effects of pressure and temperature on SC-CO2 extractions of ricebtS

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ran oil, (1) extraction efficiency of triglycerides; (2) concentration factor ofriglycerides in 3D. (F-testing: R2

(1) = 0.9930, S.D.(1) = 2.72; R2(2) = 0.9830,

.D.(2) = 0.07).

ion of free fatty acids both decreased as the pressure increased,nd the temperature did not influence either. Fig. 5 shows thehree-dimensional extraction efficiency of triglycerides, whicheached a maximum of 87.1% at 350 bar and 313 K extractiondatum# 7 in Table 2). The concentration factor of triglyceridesβTG) reaches 5.0 at 300 bar and 313 K extraction (datum# 4 inable 2). The extraction efficiency (RTG) and the concentrationf triglycerides also increased with pressure.

.4. Deacidification of rice bran oil using column partitionractionation

A medium-pressure normal-phase column partition fraction-tion was adopted herein to purify �-oryzanols from the Soxhletnd SC-CO2-extracted oil. Table 3 presents the amount of �-

ryzanols, triglycerides, free fatty acids and total fatty acids inach fraction. After the sixth fraction of N-hexane oil (datum#6 in Table 3) was eluted using the 90:10 solution of N-hexanend ethyl acetate, 37.0 wt.% of �-oryzanols was obtained. The

364 C.-R. Chen et al. / Separation and Purification Technology 61 (2008) 358–365

Table 3Experimental data on column partition fractionation of rice bran oil

Sample Eluent Woil (g) Ory (%) TG (%) FFA (%) FA (%) Others (%)

Soxhlet N-hexane extracted oilFeed F 20.2 2.1 53.9 40.0 93.9 4.0

F1 100:0 0.91 0 42.9 0 42.9 57.1F2 200:1 6.61 0 61.5 30.5 92.0 8.0F3 98:2 2.94 0 43.6 52.3 95.9 4.1F4 95:5 2.19 0.6 30.8 64.3 95.1 4.3F5 90:10 0.72 4.4 28.9 65.0 93.9 1.7F6 90:10 0.92 37.0 22.5 32.5 55.0 8.0F7 0:100 1.90 0.2 63.0 25.1 88.1 11.7

SC-CO2 extracted oilFeed J 14.7 1.3 83.6 12.0 95.6 3.1

J1 100:0 8.99 0 96.9 0 96.9 3.1J2 98:2 0.30 0 93.4 0 93.4 6.6J3 95:5 1.63 0.3 71.2 23.2 94.4 5.3J4 90:10 0.55 0.6 45.6 51.4 97.0 2.4J5 90:10 0.84 18.9 48.1 23.6 71.7 9.4J6 0:100 1.41 0.6 81.4 11.6 93.0 6.4

Eluent: N-hexane + ethyl acetate; Ory: percentage of oryzanols = (WOry/Woil) × 100%; TG: percentage of triglycerides = (WTG/Woil) × 100%; FFA: percentageof free fatty acids = (WFFA/Woil) × 100%; FA: percentage of fatty acids = (WFA/Woil) × 100%; Others: percentage of waxes, glycolipids and phospho-l ry: weaB

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ipids = (Woil − WOry − WFA)/Woil × 100%; Woil: weight of the extracted; WO

cids; WFA: weight of fatty acids = WTG + WFFA.old values represent the best values.

fth fraction that contained 18.9 wt.% of �-oryzanols of the SC-O2-extracted oil was obtained using the same mobile solvent.he concentration of free fatty acids in the N-hexane-extractedil from rice bran powder containing 40% FFA was higher thanhat in the SC-CO2-extracted oil from the powder with 10% FFA,o the purity of �-oryzanols was lower. In these column parti-ion fractionations, most triglycerides and free fatty acids hadeen removed by preceding solvent elutions of large fractions of-hexane. However, the remaining elutions retained a few free

atty acids in the purified �-oryzanols fraction, as revealed byata# F6 and J5 in Table 3.

. Conclusions

This study investigated the supercritical carbon dioxidextraction of �-oryzanols-containing rice bran oil from pow-ered rice bran using a design based on the response surfaceethodology and studied the fractionation of �-oryzanols

sing medium-pressure column partition chromatography. Twourest �-oryzanols were successfully isolated from a mix-ure of �-oryzanols using preparative C18 high-pressure liquidhromatography. Experimental supercritical carbon dioxidextraction indicated that the pressure dominated the increase inhe concentration of �-oryzanols in the extracted oil. The con-entration of �-oryzanols in the SC-CO2 oil was of the samerder of magnitude as that in the Soxhlet N-hexane oil. How-ver, the light-yellowish color, odor and simple post-removingf paraffins from the SC-CO2 extracted oil is superior to thatbtained by Soxhlet N-hexane extraction. Again, the SC-CO2

xtraction has been recognized as an environmental friendlyethod to produce edible oil. A purified fraction that con-

ained 18.9% of �-oryzanols was obtained using a normal-phaseedium-pressure column partition fractionation of the SC-CO2-

ight of oryzanols; WTG: weight of triglycerides; WFFA: weight of free fatty

xtracted oil. Separating free fatty acids and triglycerides fromhe purified �-oryzanols fraction remains difficult, and requiresurther study.

cknowledgements

The authors would like to thank the National Science Councilf the Republic of China, Taiwan, for financially supporting thisesearch under Contract No. NSC95-2221-E005-133. This works also supported in part by the ministry of education, Taiwan,OC under the ATU plan.

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