Separation and Purification of Triacylglycerols from Nyamplung

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Separation and Purification of Triacylglycerols from Nyamplung (Calophyllum inophyllum) Oil by Batchwise Solvent Extraction Hakun Wirawasista Aparamarta, Teguh Saputra, Anggita Claratika, Yi-Hsu Ju, and Setiyo Gunawan Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b04877 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 14, 2016

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Industrial & Engineering Chemistry Research

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Separation and Purification of Triacylglycerols from Nyamplung (Calophyllum

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inophyllum) Oil by Batchwise Solvent Extraction

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Hakun Wirawasista Aparamartaa, Teguh Saputrab, Anggita Claratikab, Yi-Hsu Jua*, Setiyo

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Gunawanb*

6 7 a

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Department of Chemical Engineering

National Taiwan University of Science and Technology, Taipei 10607, Taiwan

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b

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Department of Chemical Engineering

Institut Teknologi Sepuluh Nopember, Surabaya 60111, Indonesia

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*

Corresponding Author’s E-mail: [email protected]

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[email protected]

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*

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Faculty of Industrial Technology, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo,

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Surabaya 60111, Indonesia, Tel: +62-31-5946240, Fax: +62-31-5999282, E-mail:

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[email protected]

To whom correspondence should be addressed at the Department of Chemical Engineering,

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1 ACS Paragon Plus Environment


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Abstract

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Nyamplung (Calophyllum inophyllum) oil is one of the the promising raw materials due to its

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high oil content. Previous researches were limited to its characterization and conversion into

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biodiesel. In this work, triacylglycerols (TAG) was separated from the crude oil via

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batchwise solvent extraction. By using a mixture of petroleum ether – methanol (methanol

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25%) and a solvent to oil mass ratio of 5, high recovery and purity of TAG were obtained. It

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was found free fatty acids (FFA) content was decreased from 8.51% to 2.16% and 0.35%

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after one-stage and eight- stage extraction, respectively. Moreover, TAG content was

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increased significantly from 78.30% to 91.46% and 98.53% after one-stage and eight-stage

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extraction, respectively. It was shown that the proposed method can replace degumming,

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neutralization and bleaching steps in chemical refining process. Although the proposed

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method requires large amount of organic solvents that are flammable and environmentally

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unfriendly, the solvents can be recovered easily.

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Keywords: batchwise solvent extraction; C. inophyllum oil; fatty acids, polarity;

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triacylglycerols.

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1. Introduction

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With increasing priority of coastal and marine resources in national economic

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development, science and environmental aspects are playing greater roles in Indonesia

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development policy. Mangroves are highly beneficial for Indonesia’s sustainable economic

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development. They produce many valuable products and have many important functions that

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support dense coastal populations. However, Indonesia’s mangroves are being lost

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increasingly due to unsustainable utilization and habitat conversion.

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C. inophyllum is a mangrove plant species from the family of Calophyllaceae. Among

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mangrove seed oils, C. inophyllum oil is becoming an important raw material of biodiesel

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production. This is because C. inophyllum has a high oil content (40-73%) and yield (4.68 kg

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oil per ha).1 Therefore, an alternative way to achieve development of mangrove areas is an

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utilization of nyamplung (Calophyllum inophyllum) as low cost material for producing

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biodiesel and pharmaceutical products.

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Previous works on C. inophyllum were limited to investigation of its bioactive

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compounds and conversion of C. inophyllum oil into biodiesel. Lim2 reported that various

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parts of C. inophyllum are used in traditional herbal remedies, such as the sap for treating

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wound, the bark for serving antiseptic and disinfectant, the root for treating wounds and heart

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stroke, the leaves for treating eye inflammation,

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producing soap, and healing wound in skin.5 In Indonesia, the half-ripe fruit is pickled but

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only the seed is eaten. Caution is needed as toxic compunds may be present in the fruit and

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seed.6 Moreover, the seed oil can be used as edible oil after refinement and detoxification,7

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as a pharmaceutical product and as a feedstock of biodiesel.8,9

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and the seed oil for treating rheumatism,

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The biggest challenge in using crude C. inophyllum oil as biodiesel feedstock is that it

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is prone to saponification reaction leading to soap formation during alkaline-catalyzed

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transesterification. Saponification results in reduced biodiesel yields, hinders separation of



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ester from glycerin and reduces the formation rate of biodiesel.10,11 This is because crude C.

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inophyllum oil usually contains large amount of free fatty acids (FFA). FFA level in the oil

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should be below a desired level, less than 3% for alkaline-catalyzed transesterification,12,13

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and less than 0.3% for edible oils.14 Therefore, degumming, neutralization, bleaching, and

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deodorization steps are applied in the isolating of triacylglycerols (TAG) that is used as a

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feedstock of biodiesel production (refined vegetable oil). However, the process involves

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chemicals that can be dangerous in handling and detrimental to environment.

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Extraction is relatively new method in the chemical engineering. This method is used

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to separate the desired substance when mixed with other substances. The mixture is brought

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into contact with solvent in which the desired substance will dissolve, but other substances

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present in the solution will be insoluble. Gunawan et al15 reported that binary solvent

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extraction provides better separation of fatty acid steryl esters with higher purity dan recovery

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when applied to lipid fraction with high initial fatty acid steryl esters content. This method

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has some advantages like enviromental friendly because of the solvents can be recovered

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easily and less sophisticated equipment.

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The objective of this work was to develop an effective separation method to isolate

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TAG from crude C. inophyllum seed oil. In this work, the crude oil was separated into two

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fractions based on differences in polarity of the constituent components by single-stage and

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multi-stage solvent extraction (batch-wise solvent extraction). It was based on the principle of

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distribution of a substance between a polar solvent (methanol) and a non polar solvent

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(hexane or petroleum ether). The effects of various factors on the separation, such as of

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solvent to oil mass ratio, solvent mixture and methanol content in solvent mixture were

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systematically investigated.

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2. Experimental

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2.1.

Materials

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Crude C. inophyllum oil was obtained from Koperasi Jarak Tani Lestari (Central Java,

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Indonesia). Thin-layer chromatography (TLC) aluminium plates (20 cm x 20 cm x 250 µm)

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were purchased from Merck (Darmstadt, Germany). Silica gel was purchased from Merck

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(New York, USA). Standard sitosterols, squalene, fatty acids, triolein, and tripalmitin were

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obtained from sigma Chemical Company (St. Louis, MO). All solvents and reagents were

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either of HPLC grade or analystical grade and were obtained from commercial sources.

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2.2.

Wax and Gum Content

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Wax and gum content in C. inophyllum seed oil was measured according to previous

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work.16 Crude C. inophyllum seed oil (1.2 g) was added in 40 ml of acetone in a 100 ml glass

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stoppered flask, and the mixture was stirred at 40 °C for 30 min. The solution was cooled to

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room temperature and was decreased at 10 °C for 24 h. Afterwards solid and liquid phases

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were separated immediately by vacuum filtration. The solid phase was washed with acetone

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(3x10 mL) and was filtered. The solid phase on the filter paper was dried under vacuum and

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was designated as the wax and gum.

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2.3.

High Temperature GC (HT-GC) Analysis

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Components in C. inophyllum seed oil, such as FFA, squalene, TAG, diacylglycerols

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(DAG), and monoacylglycerols (MAG) were identified using pure standard solution as

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described by Gunawan et al.17 Quantitative analyses of sample were performed on a

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Shimadzu GC-2010 gas chromatograph (Kyoto, Japan), equipped with a flame ionization

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detector. Separations were carried out on a ZB-5HT (5%-phenyl)-methylpolysiloxane

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nonpolar column (15 m × 0.32 mm i.d.; with a film thickness of 0.1µm, Phenomenex, USA).



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The temperatures of the injector and the detector were both set at 370 °C. The temperature of

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the column was started at 80 °C, increased to 365 °C at a rate of 15 °C/min, and maintained at

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365 °C for 8 min. The split ratio was 1:50 using nitrogen as the carrier gas with a linear

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velocity of 30 cm/s at 80 °C. Twenty milligrams of sample was dissolved in 1 mL of ethyl

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acetate and 1 µL sample was taken and injected into the GC.

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2.4.

TLC Analysis

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Qualitative analysis of sample was done by TLC using authentic standards as

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described by Gunawan et al.18 TLC paper was stained by the sample, then immersed in a

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mobile phase of hexane: ethyl acetate: acetic acid at 90: 10: 1 (v/v/v). The immersed part was

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not allowed to exceed the height of mobile phase predetermined area on the TLC paper. After

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soaking with the mobile phase in a sealed bottle, TLC paper was dried at room temperature

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and then coloured with a mixture of iodine and silica gel (1/4 tea spoon of iodine and 2 table

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spoons of silica gel). The function of the mixture was to give color to the sample. In addition

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to reading on paper TLC using iodine, TLC paper was irradiated with UV light at 366 nm.

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2.5.

Extraction of Nonpolar Lipids from Crude C. inophyllum Oil (Single Stage

Solvent Extraction)

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Polar solvent (methanol) and nonpolar solvents (petroleum ether and n-hexane) were

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used in this study. The percentage of methanol used in solvent mixture were 0, 25, 50, and

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100. Crude C. inophyllum oil (100 g) was weighed and put into a beaker. A mixture of

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solvent (solvent to crude oil mass ratio of 2, 3, and 5) was added to the crude oil. Afterwards,

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the mixture was stirred (300 rpm) at room temperature for 15 min, then transferred to a

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separatory funnel. Nonpolar lipids, such as hydrocarbons and TAG were extracted by

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nonpolar solvents in the upper layer. The extract was separated and concentrated to give the


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nonpolar lipids fraction (NPLF). The methanol extract was designated as the polar lipids

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fraction (PLF). The fractions were then analyzed using TLC, HT-GC and GC-MS.

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2.6. Multistage Solvent Extraction ( batch-wise 8 stages extraction)

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Stirred batch-wise method was applied in this study. The NPLF fraction in the previous

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step was extracted using methanol at room temperature. The adding of methanol in the NPLF

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fraction was done at 8 stages to get the higher content of TG. The methanol extracts (1-8

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stages fraction) were collected and concentrated to give the polar lipids fraction (PLF). The

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hexane extract was concentrated and designated as the nonpolar lipids fraction (NPLF).

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Afterwards NPLF and PLF were analyzed using TLC, HT-GC and GC-MS

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2.7.

Statistical Analysis

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Reliability of the results was checked by a statistical analysis (Minitab 16, trial

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version). The differences in mean values were evaluated by analysis of variance (ANOVA).

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Differences associated with p0.05) so that all interactions involving (B) were negligible

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among all the responses studied. On the other hand, it was found that the important factors on

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TAG recovery are (A) solvent to oil mass ratio, and (C) methanol composition in solvent

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system (p0.05) as shown in Table 2.

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However, (C) methanol composition in solvent system was only the significant factor on FFA

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recovery (p90%) were obtained by using a solvent to oil

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mass ratio of 3 in all solvent systems and methanol percentage studied. Moreover, the lowest

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FFA recovery (17.53%) was obtained by using a solvent to oil mass ratio of 3 in petroleum

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ether – methanol system with 50% methanol.

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Effect of methanol percentage

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TAG and FFA recoveries were found to increase with increasing methanol percentage

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in solvent system from 0 to 25. Afterward, they decreased as methanol percentage was

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increased from 25 to 100 as shown in Figure 4. The lowest FFA recovery (17.53%) was

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obtained by using 50% methanol in petroleum ether – methanol and a solvent to oil mass

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ratio of 5. About 99% TAG recovery can be achieved at solvent to oil mass ratios of 3 and 5

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in all solvent systems with 25% and 50% methanol.

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In this work, using a single solvent during one stage extraction process gave a

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successful separation. Nonpolar compounds such as TAG were weakly dissolved in polar



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solvent (methanol) and were easily dissolved in non polar solvent (hexane or petroleum ether)

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during a single stage solvent extraction. On the other hand, more polar compounds such as

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FFA were more easily dissolved in methanol. Our results suggest that high TAG recovery

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(91.58%) with high purity (91.46%) can be obtained in the NPLF under the following

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operation conditions: a solvent to oil mass ratio of 5, solvent system of petroleum ether –

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methanol with 25% methanol. Moreover, FFA content in the NPLF was 2.16% with a

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corresponding recovery of 18.16%. Most of FFA was partitioned into the PLF. It was also

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found that the NPLF represents about 74.1% of crude C. inophyllum oil and can be used as a

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feedstock of biodiesel production.

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Furthermore, in order to increase TAG content (>98%) and decrease FFA content

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(

Separation and Purification of Triacylglycerols from Nyamplung

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