Countercurrent flow of supercritical anti-solvent in the production of pure xanthophylls from Nannochloropsis oculata

Journal of Chromatography A, 1250 (2012) 85–91

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Countercurrent flow of supercritical anti-solvent in the production of pure xanthophylls from Nannochloropsis oculata Yueh-Cheng Cho a , Yuan-Chuen Wang b , Chwen-Jen Shieh c , Justin Chun-Te Lin d , Chieh-Ming J. Chang a,∗ , Esther Han e a

Department of Chemical Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, ROC Taiwan Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, ROC Taiwan c Biotechnology Center, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, ROC Taiwan d Department of BioEnvironmental Engineering, Chung Yuan Christian University, 200 Chung Pei Road, Chung Li 32023, ROC Taiwan e University of North Texas Health Science Center, Fort Worth, TX, USA b

a r t i c l e

i n f o

Article history: Available online 17 April 2012 Keywords: Zeaxanthin Column chromatography fractionation Supercritical anti-solvent precipitation Submicron-sized particulates

a b s t r a c t This study examined pilot scaled elution chromatography coupled with supercritical anti-solvent precipitation (using countercurrent flow) in generating zeaxanthin-rich particulates from a micro-algal species. Ultrasonic agitated acetone extract subjected to column fractionation successfully yielded a fraction containing 349.4 mg/g of zeaxanthin with a recovery of 85%. Subsequently, supercritical anti-solvent (SAS) precipitation of the column fraction at 150 bar and 343 K produced submicron-sized particulates with a concentration of 845.5 mg/g of zeaxanthin with a recovery of 90%. Experimental results from a twofactor response surface method SAS precipitation indicated that purity, mean size and morphology of the precipitates were significantly affected by the flow type configuration, feed flow rate and injection time. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Microalgae, have been considered a new source of nutrition that are commonly cultivated from seawater. Commercial packaging of microalgae in the food industry comes in different forms including tablets, capsules and liquids. These microalgae were also widely investigated in the fields of food supplements and drug manufacturing [1]. Carotenoids are rich in most kinds of microalgae and are essential nutrients in the human diet. They have mostly been used as natural food colorants and additives in animal feed. Nowadays, carotenoids are also used in cosmetics, particularly neutraceutics. Lutein and zeaxanthin are two major carotenoids found in the eyes of humans; they act as strong antioxidants and filter high-energy blue light [2]. Moreover, zeaxanthin acts as a macular pigment in high concentrations (approximately twice that of lutein that exists in the eyes) and plays a critical role in the pathogenesis of age-related macular degeneration (ARMD). Zeaxanthin, a type of xanthophyll, found richly in Marigold flowers and a few species of microalgae, is also a strong antioxidant. In a dose-controlled eye disease study, a high dietary intake of zeaxanthin was related to a lower risk of ARMD, and an improvement in visual performance [3]. It is estimated that the ratio of lutein to zeaxanthin in the diet ranges

∗ Corresponding author. Tel.: +886 4 2285 2592; fax: +886 4 2286 0231. E-mail address: [email protected] (C.-M.J. Chang). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.04.031

from about 4:1 to about 7:1 [4]. The fact of the matter is, humans are unable to synthesize zeaxanthin, and the intake of zeaxanthin is usually not a substantial amount in the average diet. Usually, the majority of the lutein and zeaxanthin are extracted from Marigold flowers. Zeaxanthin, however, also exists in a few species of bacteria and microalgae. The extraction of these anti-oxidative carotenoids from microalgae is important for island-type countries, such as Taiwan, who need to import lutein and zeaxanthin from foreign countries. Supercritical carbon dioxide extraction and supercritical antisolvent (SAS) precipitation have recently been adopted as an alternative for purifying thermo-labile compounds from natural materials. It is possible to completely remove the solvent or antisolvent (e.g. carbon dioxide) by reducing pressure to the gas phase. Moreover, the supercritical fluid molecule has a high mass transfer coefficient and is capable of penetrating easily into solutions to produce a super-saturation of solutes and to make precipitation occur rapidly. Solid particles (down to nano size) contain a large amount of bioactive compounds that are not easily obtained when using conventional liquid anti-solvents or by other techniques such as the jet milling or dry spraying [5]. Carotenoids become unstable in an environment full of light and oxygen. Although Sajilata et al. demonstrated an efficient supercritical carbon dioxide extraction methodology for zeaxanthin from dried biomass of Paracoccus zeaxanthinifaciens [6], relatively low solubilities of most carotenoids from natural materials in supercritical

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CO2 make CO2 a suitable anti-solvent for SAS precipitation of these thermo-labile compounds around room temperature [7,8]. In general, supercritical fluid extraction is suitable to recover non-polar low to middle molecular weight compounds from natural materials; supercritical anti-solvent precipitation is beneficial to purify high molecular weight polar compounds from solutions. Hence, SAS processes have recently been reported to be largely employed in the micronization of pharmaceutical ingredients [9,10]. In other words, supercritical carbon dioxide can highly dissolve into organic solvents; when it does, it makes an enormous expansion ratio, particularly at the critical point [11]. This causes a strong influence on the super-saturation ratio and on the shape and size of the crystals. Supercritical carbon dioxide has proven to be a very suitable anti-solvent for the production of micro-sized algal pigments [12]. Several authors have also documented the utilization of SAS precipitation for the separation of bioactive compounds from natural materials, including flavonoids, ␤-carotene, ginkgolides, lycopene and zeaxanthin [10,13–15]. However, experimental data on phase equilibriums between supercritical carbon dioxide and organic solvents are the key to understanding the SAS process and how supercritical, superheated, liquid and the co-existing phases directly influence the morphology, the size and the distribution of particles [16,17]. The SAS process has been carried out at the one-phase region with no existing interface between the solution and the anti-solvent fluid, thus precipitation was primarily caused by nucleation and smallsize particles that resulted in less agglomeration [18]. Pressures, temperatures, solution concentrations, feed flow rates and CO2 flow rates of the SAS precipitations of the column chromatographic fractions were studied in discovering an optimal condition for obtaining small particles with a narrow particle size distribution [12,15,19–22]. In order to form nanoparticles containing high amounts of xanthophylls obtained from algal extracts, a small scale column fractionation coupled with a concurrent flow nozzle in the SAS precipitation process was necessary for the preparation of these zeaxanthin-rich precipitates [15]. However, the agglomeration and the morphology of the precipitated compounds frequently changed with the nozzle configuration and the flow-pattern (i.e. concurrent versus counter-current). This study examined ultrasonic agitation extraction of xanthophyll from N. oculata coupled with pilot scaled column chromatographic fractionation in obtaining a xanthophyll-rich elution. In following, submicron-sized precipitates of the purest xanthophylls were successfully produced from a counter-current flow type nozzle in the SAS precipitation process.

2. Materials and chemicals

(Mallinckrodt, USA) and 99.5% methyl tert-butyl ether (Mallinckrodt, USA). Ultra pure water (>18 M) was obtained by using the UltrapureTM water purification system (Louton Co., Ltd. Taipei, Taiwan) and was filtered through a 0.45 ␮m membrane filter prior to use. Reagent grade of the PS100 resin (Mitsubishi, Japan) was purchased and used without further purification. 99.95% CO2 (Toyo gas, Taiwan) was used for SAS precipitation. The authentic standards of carotenoids included 95% fucoxanthin (Fluka, Switzerland), >95% zeaxanthin (Fluka, Switzerland), 95% trans␤-apo-8 -catotenal (Fluka, Switzerland), and 95% mix isomers of ␣-Carotene: ␤-Carotene = 2:1 (Sigma–Aldrich, USA). 2.2. Ultrasonic extraction A stirred ultrasonic extractor similar to the one used in the work of Chen et al. [23] was employed without modification. 5 g of freezedried microalgae were extracted in 50 mL of the deionized water and were obtained by using an apparatus equipped with a frequency of 40 kHz and a power of 300 W. The equipment was set to 303 K and 323 K with an extraction time of 30 min. After extraction, the solution was filtered through a 0.45 ␮m syringe filter. Then, solvent was removed under a vacuum and the residue was weighed. The extracts were stored in a −80 ◦ C refrigerator before column chromatography fractionation was performed. All extractive and quantitative procedures were carried out under dimmed lights. 2.3. Column chromatography fractionation The freeze-dried microalgae (3.00 ± 0.01 g) were exhaustively extracted in 303 mL of acetone. The algal extracts were concentrated under a vacuum to yield a loading sample (0.210 g) for two open-type small-scale column chromatographs. The loading sample was dissolved in the pharmaceutical grade ethanol. The solution was subjected to a 3 cm (ID) × 30 cm (L) glass column which was packed with polystyrene based resin as the stationary phase. Isocratic elution was carried out using the mobile phase of the EtOH and acetone (98:2). Finally, a total of 3 fractions were collected, and the solvent of each fraction was removed under vacuum and weighed individually. The results of these two chromatographs were used for the precursor of the large-scale chromatography. To increase the productivity, a 10 cm (ID) × 30 cm (L) glass column was used. Approximately 3.01 g of the extracts were dissolved in ethanol in preparation for column fractionation. Isocratic elution was carried out using EtOH and acetone (98:2) solutions at the flow rate of 67 mL/min and a column pressure of 2 kg/cm2 . Finally, a total of 7 volume fractions were collected, and the solvent of each fraction was removed under vacuum and then weighed. The purified samples were stored in a −80 ◦ C refrigerator before HPLC analysis and SC-CO2 precipitation.

2.1. Materials 2.4. The SAS precipitation process Nannochloropsis oculata was cultivated in seawater in four 5 ton polypropylene tanks. After harvest and sedimentation, 500 g of algae in dry basins were obtained from a marine research center at the Institute of Marine Biology of National Sun Yat-sen University (Kaohsiung, Taiwan) and from Genereach-biotech Company (Central science park, Taichung, Taiwan). Microalgae were ground for 5 s into powder using a blade-type grinder and then collected through a 100 mesh stainless steel sieve under dimmed lights. They were then freeze-dried and stored in a freezer at −80 ◦ C before extraction. Analytical grade solvents used for the extractions, the column chromatographs and the SAS processes, included 99.8% ethanol (Mallinckrodt, USA), 99.5% acetone (Mallinckrodt, USA), and 99.5% dichloromethane (DCM) (Mallinckrodt, USA). HPLC grade solvents used for the mobile phase in HPLC included 99.5% methanol

A SC-CO2 precipitation setup similar to the work of Wu et al. [10] was employed with minor modifications by using the counter-current flow nozzle. Liquid CO2 was compressed using a high-pressure pump (Spe-ed SFE, Applied Separations, USA) (4) into the first surge tank (75 mL, L/D = 30) (8). It entered a second surge tank (750 mL, L/D = 10) (11) at a constant flow rate after it was preheated using a heat exchanger (7). A counter-current coaxial flow nozzle with an inner diameter of 0.0007-in. was inserted into the entrance of the precipitator to act as the inlet for the solution feed. Then, CO2 flowed (54 g/min) through a metering valve (SS-31RS4A, Swagelok, USA) (6-3) into the visible precipitator (TST, Taiwan) (12). Feed solutions with concentrations that ranged from 0.4 to 0.8 mg/mL were delivered into the precipitator at flow rates that ranged from 1 to 2 mL/min through a counter-current flow type

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Table 1 Ultrasonic agitation extraction of microalgal N. oculata. Wzea (R) mg/gF (%)

Wfu (R) mg/gF (%)

Wa–c (R) mg/gF (%)

0.7 ± 0.1

1.1 ± 0.0 (69)

0.0 ± 0.0 (0)

0.0 ± 0.00 (0)

1.1 ± 0.0

1.0 ± 0.2

1.4 ± 0.0 (88)

0.0 ± 0.0 (0)

0.1 ± 0.00 (17)

11.0 ± 0.3

0.43 ± 0.1

3.44 ± 0.2

0.2 ± 0.0 (13)

0.0 ± 0.0 (0)

0.1 ± 0.00 (17)

18.7 ± 0.2

1.1 ± 0.1

6.50 ± 0.1

1.6 ± 0.0 (100)

0.1 ± 0.0 (17)

0.6 ± 0.0 (100)

Run

Method

WF (g)

Wext (g)

TY (%)

Czea (mg/g)

Cfu (mg/g)

Ca–c (mg/g)

1

US-Ace1 (0.5 h, 30 ◦ C) US-Ace2 (0.5 h, 50 ◦ C) US-ether (0.5 h, 30 ◦ C) US-THF (0.5 h, 30 ◦ C)

3.02 ± 0.02

0.14 ± 0.01

4.6 ± 0.2

24.3 ± 0.7

0.7 ± 0.1

3.00 ± 0.01

0.21 ± 0.01

6.8 ± 0.2

21.0 ± 0.3

3.03 ± 0.01

0.06 ± 0.00

1.9 ± 0.1

3.06 ± 0.01

0.24 ± 0.01

8.7 ± 0.8

2 3 4

WF : weight of feed; Wext : amount of extract; TY: total yield = (Wext /Wf ) × 100%; Czea : concentration of zeaxanthin in extracts; Cfu : concentration of fucoxanthin in extracts; Ca–c : concentration of ␣-carotene in extracts; Rzea : recovery of zeaxanthin = (Wzea /1.6) × 100%; Rfu : recovery of fucoxanthin = (Wfu /0.6) × 100%; Ra–c : recovery of ␣-carotene = (Wa–c /0.6) × 100%; Wzea : zeaxanthin extracted from algae = (TY × Czea )/100; Wfu : fucoxanthin extracted from algae = (TY × Cfu )/100; Wa–c : ␣-carotene extracted from algae = (TY × Ca–c )/100; US: ultrasonic extraction with stirred (solvent to solid ratio: 80, extraction time: 30 min); triplicate data.

nozzle via a high-pressure liquid pump (L-6200A, Hitachi, Japan) (17). A stainless frit (37 ␮m) and an online filter (0.45 ␮m) were placed at the bottom of the precipitator to prevent the penetration of particles. The operating pressure (150 bar) was regulated using a back-pressure regulator (26-1722, Tescom, USA) (9-3), and the operating temperature (343 K) was controlled using a water bath circulator (5-3). The consumption of CO2 was measured using a wet gas meter (SHINAGAWA, W-NK-A1, Japan) (16).

2.6. Analysis of particle size distributions and morphologies The mean particle size and particle size distribution (PSD) were determined using a light scattering particle size analyzer (Beckman Coulter, Counter F5, USA). The morphologies of the particles were examined using a field emission scanning electron microscope (FESEM) (Zeiss, Ultra-Plus, Germany). Before SEM examination, particles were sputtered with a layer of platinum film and the images were observed under a voltage of 2 kV.

2.5. Quantification of carotenoids High performance liquid chromatography (HPLC) was performed using a Hitachi 2130 pump and a 2400 UV series system (Hitachi, Ltd., Tokyo, Japan). The analysis was carried out with a reversed-phase YMC C-30 (5 ␮m, 250 mm × 4.6 mm ID) and a Phenomenex Luna security guard cartridge C-18 (5 ␮m, 4 mm × 2.0 mm ID). The microalgal extracts were eluted using mobile phases of water (A) methanol (B) and methyl t-butyl ether (C). The eluent flow rate was maintained at 1 mL/min, the injection volume was 20 ␮L, and the detection wavelength and column temperature were set to 450 nm and 303 K. The elution gradients were as follows: 0 min, 10% A, 90% B, 0% C; 5 min, 4% A, 81% B, 15% C; 25 min, 4% A, 81% B, 15% C; and 50 min, 4% A, 31% B, 65% C. The identities of zeaxanthin and fucoxanthin were confirmed by comparing their HPLC retention times with the analytical standards at a wavelength of 450 nm. The identities of the other analytes were confirmed by comparing their HPLC retention times with the analytical standards. The regression coefficients (R2 ) of the carotenoids were all greater than 0.99.

3. Results and discussion 3.1. Ultrasonic agitation extractions Table 1 lists the experimental data of ultrasonic extractions with three solvents that lasted 0.5 h respectively. Zeaxanthin is a thermal sensitive-labile compound and is particularly easily degraded under prolonged exposure to oxygen and light [13]. Our experimental data showed that ultrasonic extractions yielded high efficiency by recovering zeaxanthin from N. oculata within 0.5 h. (This is when compared with that of Soxhlet extraction [15].) This study also employed tetrahydrofuran (THF) as a solvent in the ultrasonic extraction and it resulted in the highest recoveries from the extractive experiments. It is important to note, however, that the purity of zeaxanthin from the THF extraction was lower than that from the acetone extraction. The US-acetone 1 extract gives a higher concentration of zeaxanthin than the THF extract. The US-acetone 1 extract at 303 K produces a higher content of zeaxanthin than at higher

Table 2 Experimental data from the 3 cm column partition fractionation of algal extract. ˛zea

Run

Entry

WF (g)

CZea,f (mg/g)

Cfu,f (mg/g)

Mobile phase EtOH/ acetone

L (cm)

Fraction

We (mg)

TY (%)

Czea (mg/g)

Cfu (mg/g)

Wzea (Rzea ) mg (%)

Wfu (Rfu ) mg (%)

1

US-ACE1 extract

0.161

23.5

0.7

95/5

20

F-1 F-2 F-3

28.9 11.4 10.3

19.14 7.08 6.82

N.D. 300.3 27.4

2.8 2.8 N.D.

0.0 (0) 3.4 (90) 0.3 (7)

0.1 (71) 0.0 (28) 0.0 (0)

0 13 1

2

US-ACE1 extract

0.159

19.2

0.4

98/2

30

F-1 F-2 F-3

62.3 8.3 14.1

39.18 5.22 8.86

N.D. 303.0 17.9

0.71 N.D. N.D.

0.0 (0) 2.5 (82) 0.3 (9)

0.0 (68) 0.0 (0) 0.0 (0)

0 16 1

3

US-ACE1 extract

0.145

20.3

0.9

92/8

30

F-1 F-2 F-3

57.0 12.2 18.0

35.40 8.41 11.18

N.D. 174.9 40.7

1.1 N.D. N.D.

0.0 (0) 2.1 (72) 0.7 (25)

0.1 (48) 0.0 (0) 0.0 (0)

0 9 2

WF : weight of feed; CZea,f : concentration of zeaxanthin in feed; Cfu,f : concentration of fucoxanthin in feed; L: packing height; We : weight of each fraction; TY: total yield of eluent = (We /WF ) × 100%; Czea : concentrations of zeaxanthin in fraction; Cfu : concentrations of fucoxanthin in fraction; Rzea : recovery of zeaxanthin = (We × Czea )/(WF × Czea,f ) × 100%; Rfu : recovery of fucoxanthin = (We × Cfu )/(WF × Cfu,f ) × 100%; Wzea : amount of zeaxanthin in fraction = (We × Czea )/1000; Wfu : amount of fucoxanthin in fraction = (We × Cfu )/1000; N.D.: not detectable; mobile phase flow rate: 8 mL/min. ˛zea : purification factor of zeaxanthin = (Czea /Czea,f ) = R/TY.

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Table 3 Experimental data from the 10 cm preparative column partition fractionation of algal extract. Run

3

Entry

Wf (g)

US-ACE 1 Extract

3.01

CZea,f (mg/gext )

Mobile phase (EtOH/acetone)

L (cm)

98/2

23.5

30

Average of fractions of F2-1 to F2-6

Fraction in partition 2 F2-1 F2-2 F2-3 F2-4 F2-5 F2-6 F2-7 F2-1∼F2-6

We (mg)

TY (%)

Czea (mg/g)

Wzea (Rzea ) mg (%)

11.0 13.9 35.5 38.5 52.8 18.4 22.7

0.4 0.5 1.2 1.3 1.8 0.6 0.8

210.1 280.1 354.2 330.4 441.9 250.3 133.2

2.3 (3) 3.9 (6) 12.6 (18) 12.7 (18) 23.3 (33) 4.6 (7) 3.0 (4)

170.1

5.8

349.4

59.3 (85)

WF : weight of feed; CZea,f : concentration of zeaxanthin in feed; Cfu,f : concentration of fucoxanthin in feed; L: packing height; We : weight of each fraction; TY: total yield of eluent = (We /Wf ) × 100%; Czea : concentrations of zeaxanthin in fraction; Cfu : concentrations of fucoxanthin in fraction; Rzea : recovery of zeaxanthin = (We × Czea )/(Wf × Czea,f ) × 100%; Wzea : amount of zeaxanthin in fraction = (We × Czea )/1000; Wfu : amount of fucoxanthin in fraction = (We × Cfu )/1000; P: pressure of column chromatography; Q: mobile phase flow rate = 80 mL/min.

Table 4 Preliminary experimental study of SC-CO2 antisolvent precipitation at 150 bar using two types of nozzles. Entry a

1 2 3 4 5 6 7b 8b 9b

T (K) 323 323 323 323 323 343 343 343 343

t (min) 24 24 24 24 48 12 48 12 12

CF (mg/mL) 0.4 1 1 2 3 3 3 3 3

QF (mL/min) 0.6 0.6 0.4 0.6 0.2 0.2 0.2 0.2 0.1

XF (mol%) −2

1.7 × 10 1.0 × 10−2 8.4 × 10−3 1.0 × 10−2 4.1 × 10−3 4.1 × 10−3 4.1 × 10−3 4.1 × 10−3 2.1 × 10−3

WF (mg)

WP (mg)

TY (%)

CZea (mg/g)

Wzea (RZea ) mg (%)

5.8 14.4 9.6 28.8 28.8 7.2 28.8 7.2 3.6

3.5 5.5 3.7 9.2 9.8 2.6 10.7 2.7 1.3

60.8 38.2 38.5 31.9 34.0 36.1 34.7 37.5 36.1

443.2 523.0 662.7 769.5 785.3 799.6 803.9 833.4 845.4

1.6 (78) 2.9 (58) 2.5 (75) 7.1 (72) 7.7 (80) 2.1 (84) 8.6 (88) 2.3 (91) 1.1 (88)

CF : concentration of feed solution; QF : feed flow rate; XF : molar fraction of feed t: SAS time; WF : total feed weight = (CF × QF × t); T: SAS temperature; WP : amount of solid in precipitator; TY: total yield of precipitates = Wp /(Cf × Qf × t) × 100%; Czea : concentration of zeaxanthin in precipitates; Rzea : recovery of zeaxanthin = (Wp × CZea )/(Cf × Qf × t × 341.0) × 100%; Wzea : amount of zeaxanthin in precipitates = (Ws × Czea )/1000; Cf,zea : concentration of zeaxanthin in feed = 341.0 mg/g; QCO2 : CO2 flow rate; P: SAS pressure; a: feed solution (ethanol), others are THF; b: counter-current feed, others are con-current.

temperatures such as 323 K most likely because the oxidation of zeaxanthin may occur at 323 K. 3.2. Column chromatography fractionation Because ultrasonic acetone fast produced a higher purity of zeaxanthin than did the other solvents, a fixed amount of the ultrasonic acetone extract was dissolved in ethanol and subjected to a small scale packed column for elution chromatography at a fixed flow rate of 7 mL/min over an 3 h period. Table 2 presents the concentrations of zeaxanthin in each partition (with a total of 3 partitions) that changed with the volume ratio of ethanol to acetone

in the mobile phase. A suitable elution was the ethanol/acetone solvent of 98/2 (v/v). The zeaxanthin-rich partition was obtained at the second partition with a concentration of 303.0 mg/g and a ratio of ethanol to acetone of 98:2. Only a small amount of zeaxanthin was lost during column fractionation as was verified by duplicate experiments. The separation factor of zeaxanthin versus other compounds reached 16 in run 2 of the experiment. Subsequently, a pilot-plant preparative column (10 cm inner diameter and 30 cm length) was adopted herein to purify zeaxanthin in the elution chromatography. It took 8 h including packing, elution and drying processes. Table 3 lists the experimental data of these 10 cm preparative column partition fractionations of the algal

Fig. 1. HPLC chromatograms of the samples. (A) 20 ppm of 845.5 mg/g SAS-precipitate; (B) 800 ppm of partition 3; (C) 40 ppm of 349.4 mg/g partition 2; (D) 800 ppm of partition 1; (E) 860 ppm of acetone extract.

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89

extract. The acetone extract was subjected to column chromatography and yielded the zeaxanthin-rich partition (i.e. the second partition, F2) containing 7 fractions. The average concentration of zeaxanthin from the first 6 fractions reached 349.4 mg/g, with a recovery of 85%, shown in Table 3. Elution chromatography spectra of three preparative column partitions (F1 to F3) were analyzed by the HPLC-UV quantification at 450 nm. The HPLC spectra revealed that the amounts of all other carotenoids except for zeaxanthin decreased in the second partition (F2). Finally, the amounts of zeaxanthin in the second partitions were higher than those of the other carotenoids. The preparative elution chromatography partition process showed that the three partitions were completely separated. This was a one-step column fractionation process in refining zeaxanthin efficiently from an algal solution. Finally, preparative column fractionation products, i.e. from the second partition, were dried and collected three times as a feed for the SAS anti-solvent precipitation study. Fig. 2. A descriptive diagram of nozzle configurations used in the supercritical antisolvent process: (A) concurrent flow; (B) counter-current flow.

3.3. Preliminary experimentation of SC-CO2 anti-solvent precipitation Process variables such as type of nozzle, feed concentration, feed flow rate, injection time, temperature and pressure may have significant influences on purity of zeaxanthin recovered from SAS. Table 4 presents preliminary experimental data from the SC-CO2 antisolvent precipitation by fixing temperature and pressure but changing other process variables. SAS experiments at 150 bar were employed herein because a high purity of zeaxanthin was achieved at pressure near 150 bar and 323 K, as shown in the study of Chen et al. [21]. Firstly, two feed solutions were examined for the SC-CO2 antisolvent precipitations; purity and recovery quantities

from the ethanol solution (entry 1) were compared with that of the THF solution (entry 2). THF was a good solvent that easily dissolved zeaxanthin and was therefore suitable for the purification of zeaxanthin in the SAS precipitation thereafter (i.e. the premilinary and RSM designed SAS). Experiments 2–6 were subjected to high feed concentrations (CF ) and low feed flow rates (QF ) and resulted in a noticeable increase in both concentration and recovery of zeaxanthin in the participates. These were compared at a constant injection time. These results might indicate that high feed concentrations and a low molar fraction of the solvent (in this case low flow rate of THF) in the top-stream-low-density

Fig. 3. FESEM images of the SAS precipitates by two flow type nozzles: (A) counter-current, 12 min; (B) counter-current, 48 min; (C) concurrent, 12 min; (D) concurrent, 48 min.

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Table 5 A RSM-designed counter-current flow supercritical antisolvent precipitations at 150 bar, 343 K and 15 L/min of carbon dioxide. RSM#

QF (mg/mL)

XTHF (mol%)

t (min)

WF (mg)

WP (mg)

TY (%)

CZea (mg/g)

Wzea (RZea ) mg (%)

 (nm)

1(F) 2(A) 3(A) 4(F) 5(C) 5(C) 6(A) 7(F) 8(A) 9(F) 10

0.1 0.2 0.2 0.1 0.2 0.2 0.3 0.3 0.1 0.3 0.15

2.1 × 10−3 4.6 × 10−3 4.6 × 10−3 2.1 × 10−3 4.1 × 10−3 4.1 × 10−3 6.2 × 10−3 6.2 × 10−3 2.1 × 10−3 6.2 × 10−3 3.1 × 10−3

12 12 48 48 30 30 30 48 30 12 12

3.6 7.2 28.8 14.4 18.0 18.0 27.0 43.2 9.0 10.8 5.4

1.3 2.7 10.1 5.1 6.6 6.2 9.7 14.9 3.2 3.8 1.9

36.1 37.5 34.7 35.4 36.7 34.4 35.9 35.4 35.6 35.2 35.2

845.5 833.4 803.9 812.7 811.2 827.0 778.1 742.5 834.2 802.4 831.0

1.1 (90) 2.3 (92) 8.1 (83) 4.1 (85) 5.4 (87) 5.1 (84) 7.5 (82) 11.1 (75) 2.7 (87) 3.0 (83) 1.6 (88)

211 323 1093 812 843 777 1335 2567 543 783 243

QF : feed flow rate; t: SAS time; XTHF : molar fraction of THF in feed; WF : total feed weight = (CF × QF × t); WP : amount of solid in precipitator; TY: total yield of precipitates = WP /(CF × QF × t) × 100%; Czea : concentration of zeaxanthin in precipitates; Rzea : recovery of zeaxanthin = (WP × CZea )/(CF × QF × t × 341.0) × 100%; Wzea : amount of zeaxanthin in precipitates = (WP × Czea )/1000; CF,zea : concentration of zeaxanthin in feed = 341.0 mg/g; CF : concentration of feed solution; QCO2 : CO2 flow rate; 10: prediction data from ANOVA analysis.

supercritical phase region yield a high supersaturation of zeaxanthin. However, a long injection time resulting in a large distribution and long retention time of already generated precipitates in the down-stream-high-density CO2 phase might lead to both low purity and recovery of zeaxanthin because of the re-dissolution of the precipitates. Fig. 1 displays HPLC chromatographs of several samples. The SAS-precipitate of 845.5 mg/g presented the same peak area at a 20 mg/L concentration (shown by A) as it did with

the second partition of 349.4 mg/g at 40 mg/L (shown by C) and the acetone extract at 860 mg/L (shown by E). Partitions 1 and 3 did not appear to have peaks at 800 mg/L (shown by B and D). Peaks of impurities were removed by the first chromatography fractionation (shown by D). Fig. 2 indicates con-current (A) and counter-current (B) nozzles of a 0.007 in. inside diameter stainless steel tube installed in the entrance of the precipitator configurated with different flow directions. The latter case avoided the contact

Fig. 4. Three-dimensional representation of the effect of feed flow rates and injection times on (A) recovery, (B) concentration, (C) total yield, (D) mean particle size.

Y.-C. Cho et al. / J. Chromatogr. A 1250 (2012) 85–91

of precipitates with the down-stream-high-density supercritical phase and reduced the amount of re-dissolution. This produced high purities of zeaxanthin as shown in the last three experiments of Table 4. This counter-current flow type nozzle configuration appeared to have a significant influence on dispersing the particles and on narrowing the particle size as shown in Fig. 3(A) and (B). With the constant flow type nozzle configuration, feed concentration and feed flow rate, experiments by increasing the injection time caused more agglomeration, as shown in Fig. 3(B) and (D). The concurrent coaxial nozzle produced particles with block morphology that became agglomerated at long injection times, as shown in Fig. 3(C) and (D). It is estimated that the counter-current flow type nozzle presented a more effective method for achieving good contact between the feed solution and the supercritical carbon dioxide as well as avoiding re-dissolution of the precipitates. Hence, this counter-current flow nozzle was employed in the response surface methodology (RSM)-designed SAS experiments.

91

This study demonstrated a production of the purest powdered zeaxanthin from N. oculata by using pilot scaled column elution chromatography coupled with a counter-current flow type supercritical anti-solvent (SAS) precipitation process. A polystyrene based resin was necessarily chosen as the absorbent in elution chromatography to yield highly pure zeaxanthin-rich precipitates with a recovery of 85%. Experimental results show that the amount of zeaxanthin in the second partition was near 15-fold higher than that in the extract. High purities of powdered zeaxanthin in the SAS precipitates were obtained by using a counter-current flow type supercritical anti-solvent precipitation at short injection times and low feed flow rates. A RSM-designed SAS revealed that an adequate selection of feed flow rates and injection times could manipulate solute super-saturation and control the purity and recovery of zeaxanthin in the SAS precipitations. It is evident that high purities (>850 mg/g) and high recoveries (>90%) of zeaxanthin with mean particle sizes that ranged from 211 nm to 2567 nm can be generated.

3.4. RSM-designed SAS precipitation Acknowledgements After the preliminary investigations of the SAS precipitations, a response surface methodology (RSM) that adopted a center composite configuration for two design-independent variables (feed flow rate and injection time) with four axial points was employed for the SAS precipitation study. The RSM-designed SAS precipitation was carried out at 341 mg/g of constant feed concentration, 150 bar, 343 K, and at a 15 L/min CO2 flow rate. Table 5 lists the experimental data of the SAS precipitations at injection times that ranged from 12 to 48 min and feed flow rates that ranged from 0.1 to 0.3 mL/min. Four major responses: concentration of zeaxanthin (Czea ), recovery of zeaxanthin (Rzea ), total yield (TY) and mean particle size () were analyzed using a quadratic regression model from the Design-Expert software package (Stat-Ease, Version 6.01). Fig. 4 displays the three-dimensional response surfaces for the concentration of zeaxanthin (Czea ), the total yield (TY) of the precipitates, the recovery of zeaxanthin (Rzea ) and the mean particle size of the precipitates () that changed with injection times and feed flow rates. It is clear that low feed flow rates resulted in a large ratio of CO2 to feed solution and high purities of zeaxanthin as well as a relatively high recovery at a constant injection time. Also a decreasing feed flow rate caused a significantly high super-saturation rate of zeaxanthin and resulted in a noticeably reduced mean particle size. The effect of the injection time is also noticeable in the SAS experiments, and appears to be important in changing both the concentration and recovery of zeaxanthin. When injection time increased, the concentration and recovery of zeaxanthin decreased because the high-density-down-stream supercritical solution redissolved more zeaxanthin. This increase in the injection time also led to a longer growth time of particles and more agglomeration, as shown in Fig. 3. The maximal purity of zeaxanthin obtained from this RSM study was 845.5 mg/g, shown as Run #1 in Table 5. The predicted purity of zeaxanthin was 831 mg/g, as shown in Run #10. 4. Conclusions Since zeaxanthin is a kind of high value and high cost nutraceutical compounds, a suitable hybrid process should be employed to obtain high recovery of zeaxanthin from natural materials.

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