(Slattery et al., 2000), breast cancer (Kim et al., 2001) and recurrence of breast cancer in women with the early stage disease (Rock et al., 2005). A recent study by Hozawa et al. (2006) also associated diets rich in carotenoids can reduce incidence of diabetes in non- smokers. The photoprotective properties of carotenoids have also been highlighted in several recent studies (Stahl et al., 2000; Stahl and Sies, 2002;
Eichler et al., 2002), and implicating their ability to prevent ultraviolet light-induced erythema in humans.
The conventional refining of palm oil destroys most of the carotenoids during the bleaching process.
Over several decades, various methods have been developed to recover carotenoids from CPO, e.g.
saponification (Eckey, 1945; 1949), selective solvent extraction (Heidlas, 1998; PORIM, 1989) and transesterification followed by molecular distillation and further purification by adsorption using synthetic resins (Lion Corporation, 1986; Tan and Saleh, 1992), silica gel (Ooi et al., 1991; Lion Corporation, 1993) and reverse phase C18 silica (Goh and Toh, 1988). Transesterification, however, is the only process commercially viable and is used by Lion Corporation, Carotech Sdn Bhd and Carotino Sdn Bhd. The process converts the oil irreversibly to methyl esters which are not edible. Further purification is needed to produce crystalline carotenes safe for consumption.
Various studies have shown that carotenoids are more available to humans when ingested in fat/oil
PALM CAROTENE CONCENTRATES FROM CRUDE PALM OIL USING VACUUM LIQUID
CHROMATOGRAPHY ON SILICA GEL
BONNIE TAY YEN PING*
ABSTRACT
Palm carotene was concentrated from crude palm oil by vacuum liquid column chromatography using a commercially available adsorbent silica gel. Two products were produced, palm carotene in oil-based and carotene-depleted oil. The highest carotene concentrates (> 10%) was achieved using a ratio of 1:10 (w/w) crude palm oil:silica gel. For this ratio, activated silica gel improved the carotene recovery to 72%- 92%
compared to 21% –32% by non-activated silica gel. High recovery (99%-99.5%) of the bulk oil recovered from the silica gel was achieved by elution with a polar solvent, e.g. ethanol or 2-isopropanol, followed by a non-polar solvent, e.g. hexane.
INTRODUCTION
Palm oil is one of the richest sources of natural carotenoids and has the highest known concentration of agriculturally-derived carotenoids.
The palm carotenoids concentration varies depending on the species. Crude palm oil (CPO) from tenera, the major oil palm type planted in Malaysia, has a carotene content of 500 – 700 ppm.
The highest carotene content of about 4000 ppm is found in Elaeis oleifera (Yap et al., 1991). The tenera palm carotenoids consist of 91% α- and β-carotenes (including cis and trans) and small amounts of acyclic carotenes (lycopenes, phytofluene, phytoene, neurosporene), and cyclic carotenes (α-and β- zeacarotenes, δ- and γ-carotenes) and lutein (Tay and Ee, 2006). The major carotenes possess high provitamin A activity. The retinol equivalent of palm carotenoids is 15 to 300 times those of carrot, leafy green vegetables and tomato.
Carotenoids also possess other important physiological properties. Consumption of a diet rich in carotenoids was implicated in reducing the risk of lung cancer (Holick et al, 2002), colon cancer
Keywords: adsorption; vacuum liquid column chromatography, palm carotene.
Date received: 29 November 2006; Sent for revision: 23 January 2007; Received in final form: 2 May 2007; Accepted: 20 September 2007.
* Malaysian Palm Oil Board, P. O. Box 10620
50720 Kuala Lumpur, Malaysia.
E-mail: [email protected]
Vacuum pump
(1) Carotene concentrate (elution with non- polar solvent) (2) Residual oil (elution with polar solvent)
Carotene containing oil
adsorbent (Brown et al., 2004; Unlu et al., 2005). Therefore, preserving the palm carotenoids in oil and not extracting them in crystalline forms is preferable.
Thus, a milder refining process using molecular distillation for deodorization to preserve the palm oil carotenoid content was developed. The process produces a carotene-rich edible oil with a concentration of 500 – 600 ppm (Ooi et al., 1996).
Carotenes concentrates extracted directly from CPO without chemical conversion of the oil using synthetic adsorbents has been described in several patents (Hama et al., 1982; Latip et al., 2000).
However, normal phase silica gel is unable to extract carotene from CPO in gravity column (Baharin et al., 1998) or by adsorption (Mamuro et al., 1987).
Vacuum liquid chromatography has been in existence since the 1980s (Poole and Schuette, 1984) as a modification of the gravity-fed column, where the sample is eluted by vacuum suction with solvents of increasing polarity. The vacuum enables faster elution of the compounds with comparable resolution to those of gravity columns. Vacuum liquid chromatography uses adsorbents e.g. alumina or silica gel with mesh particle size of 40-63 µm. This paper describes the use of a commercially available adsorbent, silica gel 60 and 100 combined with vacuum liquid column chromatography operated under specific conditions for extraction of high concentrates of palm carotenoids from CPO using different ratios of CPO to silica gel. The use of larger mesh particle size of 63-200 µm, silica gel 60 and 100 with vacuum chromatography reduce pressure
build up in the column while eluting an oily material, e.g CPO. The products obtained are a palm carotenoid concentrates in oil based, ready for encapsulation and consumption plus the carotene- depleted oil.
EXPERIMENTAL Materials
CPO was obtained from MPOB Palm Oil Mill Technology Centre Labu. Silica gel 60 and 100 with pore size of 60 and 100 Å, respectively with mesh particle size of 0.063–0.2 mm mesh grade were purchased from Merck. Hexane and ethanol of analytical grade were purchased from Merck.
Methods
Pre-treatment of adsorbent. The silica gel was activated in an oven at 120oC for 12 hr and cooled in a silica gel dessicator prior to its use in the column.
Pre-treatment of CPO prior carotene extraction.
CPO was first homogenized by immersing the container in warm water (60°-70°C) in a water bath, until it became completely liquid before transfering it into the column.
Separation of carotenes from CPO using vacuum column chromatography on silica gel. Figure 1 shows
Figure 1. Vacuum column chromatography system for palm carotenoids extraction.
the set-up of the vacuum column chromatography system for the palm carotenoids extraction. The column is a polypropylene filtration column with 70 ml capacity fitted with 20 pm porosity polyethylene frits mounted on a vacmaster sample processing station connected to a vacuum pump with a gauge meter (240 V/50 Hz).
The column was packed with a certain weight of silica gel depending on the ratio of CPO: silica gel and compacted by manual pressing. The weights of CPO: silica gel (w/w) used for ratios of 1:5, 1:8, 1:10, 1:15 and 1:20 were 3 g : 15 g ; 1.9 g : 15.2 g ; 2 g : 20 g ; 1 g : 15 g and 1 g : 20 g, respectively. The dry packed column was pre-treated with 400 ml hexane under light suction before the extraction process until the eluant was just above the adsorbent surface. CPO was first homogenized in a water bath at 60oC and then dry fed into the column manually and allowed to sink to the adsorbent bed. The vacuum pressure meter was adjusted to 10-20 mmHg and the pump switched on just after the CPO was loaded into the column. Then 200 ml hexane fractions were collected until no trace of orange colour was detected indicating that all the carotenes had been eluted. The hexane was removed from the fractions in vacuo to give the desired carotene concentrates in small quantities of oil. The remaining oil was then completely recovered from the adsorbent by first eluting with 200 ml polar solvent, e.g. ethanol or 2- propanol, followed by 200 ml non-polar solvent, e.g.
hexane or petroleum ether. All the solvents from the fractions collected and removed in vacuo were reused for the next cycle of carotene extraction.
HPLC-PDA of carotene profiles of CPO and carotene concentrates. The HPLC system consisted of an HP-1100 series on-line degasser connected to a HP 1100 series diode array detector (DAD) equipped with a HP Chemstation (Hewlett Packard, Wilmington, DE) for data processing. The spectral range covered was between wavelengths 190 – 800 nm. The spectral for palm carotenoids were obtained at 200-500 nm. The monitoring wavelengths selected were 444 nm, 347 nm (phytofluene) and 286 nm (phytoene). The isocratic separation consisted of mobile phase with 89:11 (v/v) methanol: tert methyl butyl ether (TBME) at a flow rate of 1 ml min-1 through a Develosil ODS-UG 5 µm polymeric C30 column protected by precolumns with the same stationary phase. The carotene concentrate was dissolved in the mobile phase prior to injection into the HPLC system. Identification was based on comparison of the electronic absorption spectra with published data (Emenhiser et al., 1995; Davies, 1976).
Carotene content and specific extinction at 233 nm and 269 nm in ultra violet light. The extracted carotene concentrate and carotene-depleted oil were further characterized for the presence of primary
(mainly at 233 nm) and secondary (mainly at 269 nm) oxidation products using a Hitachi U 2000 spectrophotometer and according to MPOB p2.14 test methods (MPOB, 2004).
Fatty acid compositions (FAC). The FAC of CPO and the carotene-depleted oil were determined according to MPOB p3.5: methods of test for palm oil and palm oil products: determination of FAC as their methyl esters by capillary column gas-chromatography (MPOB, 2004b).
RESULTS AND DISCUSSION
Table 1 shows the influence of different ratios of CPO:
silica gel used on the carotene content in the carotene concentrates, percentage carotene recovery in the concentrates and recovery depleted oil carotene from the silica gel. In all experiments, about 200 ml hexane were collected for each fraction (F1 to F3) until the orange colour of carotene was no longer visible in the column.
Three extractions were carried out for the ratio of 1:5 (CPO:silica gel). In the first extraction 1:5(a), a 200 ml hexane fraction was collected containing 0.51% carotene. For 1:5 (b), two hexane fractions F1 and F2 (200 ml for each) were collected with percentage carotene content 0.56% and 0.54%, respectively. Two hexane fractions (each 200 ml) were also collected for 1:5 (c), and the carotene contents were 0.97% and 0.35%, respectively. As the CPO samples were poured manually into the column, this may have caused uneven distribution of the CPO on the adsorbent bed, thus affecting the orientation of the carotene band in the bed and then the carotene contents of the concentrates collected. The recovery of carotene in the hexane fractions for 1:5 was 82%- 93%. The residual oil was recovered by eluting the hexane free column with 200 ml ethanol and 49%- 52% were recovered.
Two extractions were carried out for the ratio of 1:8. When the ratio of CPO to silica gel was increased to 1:8, the hexane fractions collected had higher carotene concentrations of 1.1%, 1.7%, 2.0% and 3.1%.
Carotene recovery for 1:8 (a) and 1:8 (b) was 87%
and 66%, respectively. The oil recovered from silica gel was 97% and 98% for 1:8(a) and 1:8 (b), respectively. Almost complete recovery of oil was achieved by first removing traces of hexane after the hexane fractions were collected, then followed by elution with 200 ml of a polar solvent, e.g. ethanol or isopropanol, and subsequently eluting with another 200 ml of a non-polar solvent, e.g. hexane.
For the ratio 1:10, two treatments, activated [1:10 (a) to (d)] and non-activated [1:10 (d) to (f)] silica gel, were evaluated. With activated silica gel, the highest carotene concentration found in the hexane fractions was over 10%. Depending on the carotene
distribution in the column, the number of fractions collected varied with the experiments. When three fractions were collected, the carotene content ranged from 0.6% to 14.8% with percentage carotene recovery of 73%-90%. The recovery of carotene- depleted oil was 99% using 200 ml polar solvent, e.g.
ethanol or 2-isopropanol, followed by 200 ml hexane.
For the non-activated silica gel, the highest carotene contents obtained in the fractions was over 10%, although with a much lower recovery of 21%
to 32%.
The influence of the silica gel pore size was explored. Using activated silica gel with a pore size of 100 Å [1:10 (g)], carotene content of 0.04%, 6%
and 15.7% were found in the Fractions 1, 2 and 3, respectively. The recovery of carotene was 54%.
With the ratio of 1:15, the highest carotene extracted in the hexane fraction was 9.8%, with concentration in the first and third fractions of 1.7%
and 6.4%. Although the silica gel was activated, the percentage recovery of carotene was low at 57%.
Further increase in the ratio of CPO: silica gel to 1:20, reduced the carotene eluted in the hexane to 1.2%. The reduction could be because of build-up pressure from the high amount of adsorbent and eluting solvent which slowed down the oil movement, thus affected the carotene concentration.
It was observed that the carotene band was eluted only after 2.4 litre hexane had been passed through.
Fatty Acid Composition (FAC) of the Carotene- depleted Oil
The FAC of the carotene-depleted oil recovered from the column were found to be the same as that for CPO (Table 2). This showed that the extraction was by physical absorption and the oil remained unchanging chemically.
TABLE 2. FATTY ACID COMPOSITION OF CRUDE PALM OIL AND CAROTENE-DEPLETED OIL RECOVERED
FROM SILICA GEL
Oil sample Fatty acid (%)
C14 C16 C18 C18:1 C18:2
CPO 1.1 43.5 4.0 42.2 9.2
Carotene- 1.1 43.6 4.3 41.8 8.6 depleted
oil (F4)
TABLE 1. EFFECT OF CPO/SILICA GEL RATIO ON PERCENTAGE OF CAROTENES RECOVERED IN HEXANE FRACTIONS
Ratio CPO/ Carotene Carotene recovery Oil recovery (%)
silica gel Content (mg) % carotene in hexane
F1 F2 F3 F1 F2 F3
fractions (%)
1.5(a) 1.9 - - 0.51 - - 93 52
1.5(b) 1.3 0.39 - 0.56 0.54 - 82 49
1:5 (c) 1.41 0.29 - 0.97 0.35 - 85 50
1:8 (a) 0.52 0.49 0.04 1.7 2.0 0.01 87 97
1:8 (b) 0.34 0.47 - 1.1 3.1 - 66 98
1:10(a) 0.16 0.41 0.07 2.1 4.4 14.8 73 99
1:10 (b) 1.12 - - 11.2 - - 92 99
1:10 (c) 1.18 0.007 - 12.9 0.6 - 90 99
1:10 (d) 0.10 0.14 0.17 8.3 4.7 11.3 32 -
1:10 (e) 0.15 0.15 - 14.8 5.5 - 21 99
1:10(f) 0.16 0.21 - 14.7 7.1 - 26.6 99.5
1:10(g) 0.06 0.22 0.19 0.04 6.0 15.7 54 99
1:15 0.05 0.14 0.13 1.7 9.8 6.4 57 99
1:20 0.07 0.02 - 1.2 0.03 - 13 -
Notes:
Carotene content of crude palm oil is about 650 ppm.
Letters in parentheses represent the number of extractions for the ratio used.
Ratio 1:10 (g) represent extractions using silica gel with pore size of 100 Å.
All silica gel used was activated except for ratio 1:10 (d) to (f).
UV Absorption at Wavelengths 233 nm and 269 nm Table 3 shows the UV specific extinctions at 233 nm and 269 nm for detection of primary and secondary oxidation products, in CPO and the carotene-depleted oil recovered from the silica gel.
After the extraction, there was no indication of auto- oxidation of the carotene-depleted oil.
HPLC Profiles of the Concentrated Carotenoids Extracted
Figures 2 and 3 show the HPLC-photodiode array profiles for the carotene concentrates from the hexane fractions and CPO, respectively, monitored at 444 nm. Identification of the peaks was based on electronic absorption spectra extracted from a photodiode array detector (Table 4). The geometrical isomers of the major carotenes, α and β, in the concentrates were similar to those in CPO, comprising 13- and 13’-cis α-carotene, 13-cis-β carotene, trans-α-carotene, 9-cis α-carotene and trans β carotene.
CONCLUSION
A one-step process to extract carotene directly from CPO was developed using vacuum liquid column
chromatography with silica gel 60 and 100 as the adsorbents. The process yielded two products, oil- based carotene concentrates and carotene-depleted oil. Oil-based palm carotenoids have higher bioavailability and can be used in pharmaceuticals or incorporated in food and animal feed without further purification. The recovered carotene depleted-oil is easier to refine as less bleaching earth
TABLE 3. UV SPECIFIC EXTINCTION AT 233 nm AND 269 nm FOR CRUDE PALM OIL AND CAROTENE-DEPLETED
OIL RECOVERED FROM SILICA GEL
Oil sample UV specific extinction (nm)
E233 E269
Crude palm oil 1.2 0.41
Carotene- 1.3 0.5
depleted oil (F4)
Figure 2. HPLC profile of carotene concentrates obtained on a C30 column attached to a HPLC-photodiode array detector and monitored at 444 nm. The peaks area 1 (13-cis-α-carotene); 2 (13’-cis-α-carotene); 3 (13-cis-β-carotene);
4 (trans-α-carotene); 5 (9-cis-α-carotene) and 6 (trans−β-carotene).
TABLE 4. ELECTRONIC ABSORPTION MAXIMA OF THE GEOMETRICAL ISOMERS OF ααααα- AND βββββ-CAROTENES OF
CAROTENE CONCENTRATES OBTAINED FROM VACUUM LIQUID CHROMATOGRAPHY SYSTEM Peak Isomer Absorption maxima (nm)
This Previous
study studiesb 1 13- and 330 410 (439) 463 331 nd (438) 466 2 13'- cis-
α-carotene
3 13- cis- 336 419 (444) 470 (443) β-carotene
4 trans- 425 (444) 472 nd (445) 474
α-carotene
5 9- cis- 330 414(440) 464 329 420 (442) 470 α-carotene
6 trans- 421 (450) 475 (450)
β-carotene
Note: Values in parentheses represent the main absorption maxima.
and bleaching time will be needed due to the low content of residual carotene. The carotenes were concentrated by physical adsorption on the silica gel, a milder process than chemisorption and had similar isomers composition as in CPO. The recovered oil was not oxidized as shown by the FAC and oxidative parameters. This extraction process is environment- friendly as all the solvents are reused. However, the process is dependent on the cost of silica gel and the frequency of the silica gel that can be reused. A patent claim by Chandrasekaran et al. (1982) mentioned that silica gel can be repeatedly regenerated.
ACKNOWLEDGEMENT
The author would like to thank the Director-General of MPOB for permission to publish this paper.
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