Subscriber access provided by MAHIDOL UNIVERSITY (UniNet)

Article
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

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Industrial & Engineering Chemistry Research is published by the American Chemical

Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 23 Industrial & Engineering Chemistry Research

1
2
3 1 Separation and Purification of Triacylglycerols from Nyamplung (Calophyllum
4
5 2 inophyllum) Oil by Batchwise Solvent Extraction
6
7
3
8
9
10 4 Hakun Wirawasista Aparamartaa, Teguh Saputrab, Anggita Claratikab, Yi-Hsu Jua*, Setiyo
11
12 5 Gunawanb*
13
14 6
15
16 7
17
18 a
19 8 Department of Chemical Engineering
20
21 9 National Taiwan University of Science and Technology, Taipei 10607, Taiwan
22
b
23 10 Department of Chemical Engineering
24
25 11 Institut Teknologi Sepuluh Nopember, Surabaya 60111, Indonesia
26
27
12
28
29 *
30 13 Corresponding Author’s E-mail: yhju@mail.ntust.edu.tw
31
32 14 gunawan@chem-eng.its.ac.id
33
34 15
35
36
16
37
38
39 17
40
41 18
42
43 *
19 To whom correspondence should be addressed at the Department of Chemical Engineering,
44
45 20 Faculty of Industrial Technology, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo,
46
47
48 21 Surabaya 60111, Indonesia, Tel: +62-31-5946240, Fax: +62-31-5999282, E-mail:
49
50 22 gunawan@chem-eng.its.ac.id
51
52 23
53
54
55
56
57
58
59
60 1
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 2 of 23

1
2
3 24 Abstract
4
5 25
6
7
26 Nyamplung (Calophyllum inophyllum) oil is one of the the promising raw materials due to its
8
9
10 27 high oil content. Previous researches were limited to its characterization and conversion into
11
12 28 biodiesel. In this work, triacylglycerols (TAG) was separated from the crude oil via
13
14 29 batchwise solvent extraction. By using a mixture of petroleum ether – methanol (methanol
15
16 30 25%) and a solvent to oil mass ratio of 5, high recovery and purity of TAG were obtained. It
17
18
19 31 was found free fatty acids (FFA) content was decreased from 8.51% to 2.16% and 0.35%
20
21 32 after one-stage and eight- stage extraction, respectively. Moreover, TAG content was
22
23 33 increased significantly from 78.30% to 91.46% and 98.53% after one-stage and eight-stage
24
25 34 extraction, respectively. It was shown that the proposed method can replace degumming,
26
27
28
35 neutralization and bleaching steps in chemical refining process. Although the proposed
29
30 36 method requires large amount of organic solvents that are flammable and environmentally
31
32 37 unfriendly, the solvents can be recovered easily.
33
34 38
35
36
39 Keywords: batchwise solvent extraction; C. inophyllum oil; fatty acids, polarity;
37
38
39 40 triacylglycerols.
40
41 41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 2
ACS Paragon Plus Environment
Page 3 of 23 Industrial & Engineering Chemistry Research

1
2
3 42 1. Introduction
4
5 43 With increasing priority of coastal and marine resources in national economic
6
7
44 development, science and environmental aspects are playing greater roles in Indonesia
8
9
10 45 development policy. Mangroves are highly beneficial for Indonesia’s sustainable economic
11
12 46 development. They produce many valuable products and have many important functions that
13
14 47 support dense coastal populations. However, Indonesia’s mangroves are being lost
15
16 48 increasingly due to unsustainable utilization and habitat conversion.
17
18
19 49 C. inophyllum is a mangrove plant species from the family of Calophyllaceae. Among
20
21 50 mangrove seed oils, C. inophyllum oil is becoming an important raw material of biodiesel
22
23 51 production. This is because C. inophyllum has a high oil content (40-73%) and yield (4.68 kg
24
25 52 oil per ha).1 Therefore, an alternative way to achieve development of mangrove areas is an
26
27
28
53 utilization of nyamplung (Calophyllum inophyllum) as low cost material for producing
29
30 54 biodiesel and pharmaceutical products.
31
32 55 Previous works on C. inophyllum were limited to investigation of its bioactive
33
34 56 compounds and conversion of C. inophyllum oil into biodiesel. Lim2 reported that various
35
36
57 parts of C. inophyllum are used in traditional herbal remedies, such as the sap for treating
37
38
39 58 wound, the bark for serving antiseptic and disinfectant, the root for treating wounds and heart
40
3,4
41 59 stroke, the leaves for treating eye inflammation, and the seed oil for treating rheumatism,
42
43 60 producing soap, and healing wound in skin.5 In Indonesia, the half-ripe fruit is pickled but
44
45 61 only the seed is eaten. Caution is needed as toxic compunds may be present in the fruit and
46
47
48 62 seed.6 Moreover, the seed oil can be used as edible oil after refinement and detoxification,7
49
50 63 as a pharmaceutical product and as a feedstock of biodiesel.8,9
51
52 64 The biggest challenge in using crude C. inophyllum oil as biodiesel feedstock is that it
53
54 65 is prone to saponification reaction leading to soap formation during alkaline-catalyzed
55
56
66 transesterification. Saponification results in reduced biodiesel yields, hinders separation of
57
58
59
60 3
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 4 of 23

1
2
3 67 ester from glycerin and reduces the formation rate of biodiesel.10,11 This is because crude C.
4
5 68 inophyllum oil usually contains large amount of free fatty acids (FFA). FFA level in the oil
6
7
69 should be below a desired level, less than 3% for alkaline-catalyzed transesterification,12,13
8
9
10 70 and less than 0.3% for edible oils.14 Therefore, degumming, neutralization, bleaching, and
11
12 71 deodorization steps are applied in the isolating of triacylglycerols (TAG) that is used as a
13
14 72 feedstock of biodiesel production (refined vegetable oil). However, the process involves
15
16 73 chemicals that can be dangerous in handling and detrimental to environment.
17
18
19 74 Extraction is relatively new method in the chemical engineering. This method is used
20
21 75 to separate the desired substance when mixed with other substances. The mixture is brought
22
23 76 into contact with solvent in which the desired substance will dissolve, but other substances
24
25 77 present in the solution will be insoluble. Gunawan et al15 reported that binary solvent
26
27
28
78 extraction provides better separation of fatty acid steryl esters with higher purity dan recovery
29
30 79 when applied to lipid fraction with high initial fatty acid steryl esters content. This method
31
32 80 has some advantages like enviromental friendly because of the solvents can be recovered
33
34 81 easily and less sophisticated equipment.
35
36
82 The objective of this work was to develop an effective separation method to isolate
37
38
39 83 TAG from crude C. inophyllum seed oil. In this work, the crude oil was separated into two
40
41 84 fractions based on differences in polarity of the constituent components by single-stage and
42
43 85 multi-stage solvent extraction (batch-wise solvent extraction). It was based on the principle of
44
45 86 distribution of a substance between a polar solvent (methanol) and a non polar solvent
46
47
48 87 (hexane or petroleum ether). The effects of various factors on the separation, such as of
49
50 88 solvent to oil mass ratio, solvent mixture and methanol content in solvent mixture were
51
52 89 systematically investigated.
53
54 90
55
56
91
57
58
59
60 4
ACS Paragon Plus Environment
Page 5 of 23 Industrial & Engineering Chemistry Research

1
2
3 92 2. Experimental
4
5 93 2.1. Materials
6
7
94 Crude C. inophyllum oil was obtained from Koperasi Jarak Tani Lestari (Central Java,
8
9
10 95 Indonesia). Thin-layer chromatography (TLC) aluminium plates (20 cm x 20 cm x 250 µm)
11
12 96 were purchased from Merck (Darmstadt, Germany). Silica gel was purchased from Merck
13
14 97 (New York, USA). Standard sitosterols, squalene, fatty acids, triolein, and tripalmitin were
15
16 98 obtained from sigma Chemical Company (St. Louis, MO). All solvents and reagents were
17
18
19 99 either of HPLC grade or analystical grade and were obtained from commercial sources.
20
21 100
22
23 101 2.2. Wax and Gum Content
24
25 102 Wax and gum content in C. inophyllum seed oil was measured according to previous
26
27
28
103 work.16 Crude C. inophyllum seed oil (1.2 g) was added in 40 ml of acetone in a 100 ml glass
29
30 104 stoppered flask, and the mixture was stirred at 40 °C for 30 min. The solution was cooled to
31
32 105 room temperature and was decreased at 10 °C for 24 h. Afterwards solid and liquid phases
33
34
106 were separated immediately by vacuum filtration. The solid phase was washed with acetone
35
36
37 107 (3x10 mL) and was filtered. The solid phase on the filter paper was dried under vacuum and
38
39 108 was designated as the wax and gum.
40
41 109
42
43
110 2.3. High Temperature GC (HT-GC) Analysis
44
45
46 111 Components in C. inophyllum seed oil, such as FFA, squalene, TAG, diacylglycerols
47
48 112 (DAG), and monoacylglycerols (MAG) were identified using pure standard solution as
49
50 113 described by Gunawan et al.17 Quantitative analyses of sample were performed on a
51
52 114 Shimadzu GC-2010 gas chromatograph (Kyoto, Japan), equipped with a flame ionization
53
54
55
115 detector. Separations were carried out on a ZB-5HT (5%-phenyl)-methylpolysiloxane
56
57 116 nonpolar column (15 m × 0.32 mm i.d.; with a film thickness of 0.1µm, Phenomenex, USA).
58
59
60 5
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 6 of 23

1
2
3 117 The temperatures of the injector and the detector were both set at 370 °C. The temperature of
4
5 118 the column was started at 80 °C, increased to 365 °C at a rate of 15 °C/min, and maintained at
6
7
119 365 °C for 8 min. The split ratio was 1:50 using nitrogen as the carrier gas with a linear
8
9
10 120 velocity of 30 cm/s at 80 °C. Twenty milligrams of sample was dissolved in 1 mL of ethyl
11
12 121 acetate and 1 µL sample was taken and injected into the GC.
13
14 122
15
16 123 2.4. TLC Analysis
17
18
19 124 Qualitative analysis of sample was done by TLC using authentic standards as
20
21 125 described by Gunawan et al.18 TLC paper was stained by the sample, then immersed in a
22
23 126 mobile phase of hexane: ethyl acetate: acetic acid at 90: 10: 1 (v/v/v). The immersed part was
24
25 127 not allowed to exceed the height of mobile phase predetermined area on the TLC paper. After
26
27
28
128 soaking with the mobile phase in a sealed bottle, TLC paper was dried at room temperature
29
30 129 and then coloured with a mixture of iodine and silica gel (1/4 tea spoon of iodine and 2 table
31
32 130 spoons of silica gel). The function of the mixture was to give color to the sample. In addition
33
34 131 to reading on paper TLC using iodine, TLC paper was irradiated with UV light at 366 nm.
35
36
132
37
38
39 133 2.5. Extraction of Nonpolar Lipids from Crude C. inophyllum Oil (Single Stage
40
41 134 Solvent Extraction)
42
43 135 Polar solvent (methanol) and nonpolar solvents (petroleum ether and n-hexane) were
44
45 136 used in this study. The percentage of methanol used in solvent mixture were 0, 25, 50, and
46
47
48 137 100. Crude C. inophyllum oil (100 g) was weighed and put into a beaker. A mixture of
49
50 138 solvent (solvent to crude oil mass ratio of 2, 3, and 5) was added to the crude oil. Afterwards,
51
52 139 the mixture was stirred (300 rpm) at room temperature for 15 min, then transferred to a
53
54 140 separatory funnel. Nonpolar lipids, such as hydrocarbons and TAG were extracted by
55
56
141 nonpolar solvents in the upper layer. The extract was separated and concentrated to give the
57
58
59
60 6
ACS Paragon Plus Environment
Page 7 of 23 Industrial & Engineering Chemistry Research

1
2
3 142 nonpolar lipids fraction (NPLF). The methanol extract was designated as the polar lipids
4
5 143 fraction (PLF). The fractions were then analyzed using TLC, HT-GC and GC-MS.
6
7
144
8
9
10 145 2.6. Multistage Solvent Extraction ( batch-wise 8 stages extraction)
11
12 146 Stirred batch-wise method was applied in this study. The NPLF fraction in the previous
13
14 147 step was extracted using methanol at room temperature. The adding of methanol in the NPLF
15
16 148 fraction was done at 8 stages to get the higher content of TG. The methanol extracts (1-8
17
18
19 149 stages fraction) were collected and concentrated to give the polar lipids fraction (PLF). The
20
21 150 hexane extract was concentrated and designated as the nonpolar lipids fraction (NPLF).
22
23 151 Afterwards NPLF and PLF were analyzed using TLC, HT-GC and GC-MS
24
25 152
26
27
28
153 2.7. Statistical Analysis
29
30 154 Reliability of the results was checked by a statistical analysis (Minitab 16, trial
31
32 155 version). The differences in mean values were evaluated by analysis of variance (ANOVA).
33
34 156 Differences associated with p<0.05 were considered significant.19
35
36
157
37
38
39 158 3. Results and Discussion
40
41 159 C. inophyllum fruit is harvested twice a year, in May and November. It is spherical to
42
43 160 ovoid drupe, 25-50 mm in diameter, with a thin, compact and smooth outer layer, and
44
45 161 greyish-green in colour.
46
47
48 162 Crude C. inophyllum seed oil is dark green and liquid at room temperature. The
49
50 163 highest content was TAG was at 78.30%, followed by 8.51% FFA, 5.35% DAG and 2.75%
51
52 164 MAG. TAG content of C. inophyllum seed oil investigated in this study is higher than that
53
54 165 reported in previous works (72.46% 5 and 76.70% 20). The variability of TAG, DAG, MAG
55
56
57
58
59
60 7
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 8 of 23

1
2
3 166 and FFA contents was attributed to C. inophyllum cultivar, harvesting age, and diversity of
4
5 167 agronomic factors.
6
7
168 In this study, TLC analysis was also applied to know the relative polarity of each
8
9
10 169 compounds in crude C. inophyllum seed oil. It was found that the most non polar one was
11
12 170 TAG, followed by DAG, FFA, MAG, and gum. The observed TLC spots was simillar to
13
14 171 previous work on soybean oil deodorized distillate. Typical result of the TLC analysis of
15
16 172 soybean oil deodorized distillate showed the presence of squalene, fatty acids steryl esters,
17
18 16
19 173 TAG, tocopherols, DAG, FFA, and free phytosterols . It means that the components of C.
20
21 174 inophyllum seed oil can be separated based on their differences in polarity.
22
23 175 Batchwise solvent extraction method was used to replace the degumming,
24
25 176 neutralization, and bleaching steps in the chemical process of the refined of vegetable oils
26
27
28
177 production as can be seen in Figure 1. Furthermore, purity and recovery of TAG and FFA
29
30 178 were chosen as the indicator for the effective separation of non polar lipid fraction from crude
31
32 179 C. inophyllum seed oil.
33
34 180 The development of experimental design matrix was simulated with Design Expert
35
36
181 9.0.3 trial version as shown in Table 1. A single replicate of the factorial design was applied
37
38
39 182 which is an experimental design with 3 factors at different levels at one stage solvent
40
41 183 extraction, (A) effect of solvent to oil mass ratio at levels of 2, 3 and 5, (B) effect of solvent
42
43 184 system at levels of petroleum ether-methanol and hexane-methanol, and (C) effect of
44
45 185 methanol composition in solvent system at levels of 0, 25, 50, and 100%. Twenty four runs
46
47
48 186 were carried out in random order. The responses were recovery of TAG and FFA, purity of
49
50 187 TAG and FFA, and yield of non polar lipid fraction (NPLF).
51
52 188 Moreover, the goodness of model fit for recovery of TAG and FFA was checked by
53
54 189 using normal probability plot as shown in Figure 2. It can be seen that both plots were
55
56
190 straight lines, which are assosiated to normal distribution. The analysis of variance for
57
58
59
60 8
ACS Paragon Plus Environment
Page 9 of 23 Industrial & Engineering Chemistry Research

1
2
3 191 recovery of TAG and FFA are summarized in Tables 2 and 3. The p-value was used to
4
5 192 determine the significance of the effect of factors. It can be seen that the effect of (B) solvent
6
7
193 system was not significant (p>0.05) so that all interactions involving (B) were negligible
8
9
10 194 among all the responses studied. On the other hand, it was found that the important factors on
11
12 195 TAG recovery are (A) solvent to oil mass ratio, and (C) methanol composition in solvent
13
14 196 system (p<0.05). They also involved insignificant interactions (p>0.05) as shown in Table 2.
15
16 197 However, (C) methanol composition in solvent system was only the significant factor on FFA
17
18
19 198 recovery (p<0.05) as can be see in Table 3.
20
21 199 Effect of solvent to oil mass ratio
22
23 200 A lower solvent to oil mass ratio is more desirable in the extraction because less
24
25 201 solvent is required. TAG and FFA recoveries were found to increase as solvent to oil mass
26
27
28
202 ratio was increased from 2 to 3, then they decreased as this ratio was increased from 3 to 5 as
29
30 203 shown in Figure 3. High TAG recoveries (>90%) were obtained by using a solvent to oil
31
32 204 mass ratio of 3 in all solvent systems and methanol percentage studied. Moreover, the lowest
33
34 205 FFA recovery (17.53%) was obtained by using a solvent to oil mass ratio of 3 in petroleum
35
36
206 ether – methanol system with 50% methanol.
37
38
39 207 Effect of methanol percentage
40
41 208 TAG and FFA recoveries were found to increase with increasing methanol percentage
42
43 209 in solvent system from 0 to 25. Afterward, they decreased as methanol percentage was
44
45 210 increased from 25 to 100 as shown in Figure 4. The lowest FFA recovery (17.53%) was
46
47
48 211 obtained by using 50% methanol in petroleum ether – methanol and a solvent to oil mass
49
50 212 ratio of 5. About 99% TAG recovery can be achieved at solvent to oil mass ratios of 3 and 5
51
52 213 in all solvent systems with 25% and 50% methanol.
53
54 214 In this work, using a single solvent during one stage extraction process gave a
55
56
215 successful separation. Nonpolar compounds such as TAG were weakly dissolved in polar
57
58
59
60 9
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 10 of 23

1
2
3 216 solvent (methanol) and were easily dissolved in non polar solvent (hexane or petroleum ether)
4
5 217 during a single stage solvent extraction. On the other hand, more polar compounds such as
6
7
218 FFA were more easily dissolved in methanol. Our results suggest that high TAG recovery
8
9
10 219 (91.58%) with high purity (91.46%) can be obtained in the NPLF under the following
11
12 220 operation conditions: a solvent to oil mass ratio of 5, solvent system of petroleum ether –
13
14 221 methanol with 25% methanol. Moreover, FFA content in the NPLF was 2.16% with a
15
16 222 corresponding recovery of 18.16%. Most of FFA was partitioned into the PLF. It was also
17
18
19 223 found that the NPLF represents about 74.1% of crude C. inophyllum oil and can be used as a
20
21 224 feedstock of biodiesel production.
22
23 225 Furthermore, in order to increase TAG content (>98%) and decrease FFA content
24
25 226 (<0.5%), a batchwise 8-stage extraction was employed. In the multistage process, NPLF from
26
27
28
227 one extraction stage was used as feed to the next stage with methanol added. The NPLF
29
30 228 obtained was a yellowish liquid at room temperature while the PLF was dark green and semi
31
32 229 solid at room temperature. HTGC and TLC analyses of crude oil, PLF, and NPLF are shown
33
34 230 in Figure 5. It was found that TAG was successfully separated in the NPLF as confirmed by
35
36
231 TLC analysis. Moreover, the composition of NPLF at each stage is shown in Table 5. It was
37
38
39 232 found that FFA content was decreased significantly from 2.16% (1-stage) to 0.35% (8-stage)
40
41 233 while the content of TAG was increased significantly from 91.46% (1-stage) to 98.53% (8-
42
43 234 stage). However, the recovery of TAG was decreased from 91.58% (single stage) to 76.79%
44
45 235 (8-stage) and the yield of the NPLF was decreased from 74.1% (1-stage) to 59.57% (8-stage).
46
47
48 236 These results are comparable with those obtained from corn oil refining (degumming,
49
50 237 neutralization and bleaching). Starting with crude corn oil that contains 95.6% TAG, 2.5%
51
52 238 FFA, and 1.9% others, it was possible to obtain 98.8% TAG, 0.05% FFA, and 1.15% others.
53
54 21
239
55
56
240
57
58
59
60 10
ACS Paragon Plus Environment
Page 11 of 23 Industrial & Engineering Chemistry Research

1
2
3 241 4. Conclusion
4
5 242 C. inophyllum oil was succesfully separated into two fractions based on polarity of their
6
7
243 compounds via batchwise solvent extraction. It was found that the most important parameters
8
9
10 244 affecting the separation are solvent to oil ratio (A) and methanol percentage (C) in solvent
11
12 245 mixture. TAG was obtained in high purity (99%) into nonpolar fraction and almost all FFA,
13
14 246 MAG, and DAG were partitioned into polar fraction after 8 stages of extraction.
15
16 247
17
18
19 248 Acknowledgements
20
21 249 This work was supported by National Taiwan University of Science and Technology
22
23 250 (NTUST) Taiwan and a grant (003246.147/IT2.11/PN.08/2015) provided by the Institute of
24
25 251 Research and Public Services (LPPM), Institut Teknologi Sepuluh Nopember (ITS),
26
27
28
252 Indonesia. The authors thank Mrs. Dewi Puspitasari and Mr. Mukti Utomo for their valuable
29
30 253 technical supports.
31
32 254
33
34 255 References
35
36
256 (1) Singh, S.P.; Singh, D. Biodiesel production through the use of different sources and
37
38
39 257 characterization of oils and their esters as the substitute of diesel : a review. Renew Sust
40
41 258 Energ Rev. 2010, 14(1), 200–16.
42
43
44 259 (2) Lim, T.K. Fruits in Edible medicinal and non-medicinal Plants., Springer. 2012, 2, 7.
45
46
47 260 (3) Bruneton, J. Plantes Medicinales, Technique and Documents Lavoisier, 2nd edition ;
48
49 261 Pharmacognosie–Phytochime: Paris, 1993, 300.
50
51
52 262 (4) Lemmens, R.H.M..J. Calophyllum L. In: Lemmens RHMJ, Bunyapraphatsara N (eds)
53
54
263 Plant resources of South-East Asia no 12(3). Medicinal and poisonous plants 3; Prosea
55
56
57 264 Foundation: Bogor, 2003, 102.
58
59
60 11
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 12 of 23

1
2
3 265 (5) Dweck, A.C.; Meadows, T. 2002. Tamanu (Calophyllum inophyllum) : the African,
4
5 266 Asian, Polynesian and Pacific PanaceaTamanu. Int. J. Cosmet. Sci. 2002, 24, 1.
6
7
8 267 (6) Burkill, I.H.; Birtwistle.W.; Foxworthy F.W.; Scrivenor J.B.; Watson, J.G. A dictionary
9
10
11
268 of the economic products of the Malay peninsula; Ministry of Agriculture and
12
13 269 Cooperatives: Kuala Lumpur, 1966, 1241..
14
15
16 270 (7) Lee, Y.F; Gibot, A . Indigenous edible plants of Sabah; FRC publication, Sabah
17
18 271 Forestry Department: Sandakan, 1986, 25, 9.
19
20
21 272 (8) Mohod,.A.G.; Bhor, P.P.; Kadam; Sengar, S.H.; Khandetod Y.P. Study of properties of
22
23 273 Calophyllum inophyllum L. Oil and its biodiesel. International Journal of Agricucultural
24
25
274 Engineering. 2010, 3,125.
26
27
28
29
275 (9) Hathurusingha,S.; Ashwath,N.; Midmore, D. Periodic variation in kernel oil content and
30
31 276 fatty acid profiles of Callophyllum inophyllum L. : A potential biodiesel feedstock in
32
33 277 Australia. Biomass. Bioenerg. 2011, 35, 3448.
34
35
36 278 (10) Saraf, S.; Thomas, B. Influence of feedstock and process chemistry on biodiesel quality.
37
38 279 J Process Safety and Environmental Protection, Trans. IChemE. Part B. 2007, 85(B5),
39
40
280 360.
41
42
43
281 (11) Thiruvengadaravi, K.V.; Nandagopal, J.; Baskaralingam, P.; Sathya Selva Bala , V.;
44
45
46 282 Sivanesan, S. Acid-catalyzed esterification of karanja (Pongamia pinnata) oil with high
47
48 283 free fatty acids for biodiesel production. Fuel. 2012, 98, 1.
49
50
51 284 (12) Ribeiro, A.; Castro, F. ; and Carvalho, J. Influence of free fatty acid content in biodisel
52
53 285 production on non edible oils. WASTES: Solutions, Treatments and Opportunities, 1St
54
55
286 International Conference, 2011.
56
57
58
59
60 12
ACS Paragon Plus Environment
Page 13 of 23 Industrial & Engineering Chemistry Research

1
2
3 287 (13) Sharma, Y. C. ; Singh, B. Development of biodiesel: current scenario. Renew. Sust.
4
5 288 Energ. Rev. 2009, 13,1646.
6
7
8 289 (14) SNI 01-3741. Indonesian standart of cooking oil, 2002.
9
10
11 290 (15) Gunawan, S.; Fabian, C.; Ju, Y.H. Isolation and purification of fatty acid steryl esters
12
13
14
291 from soybean oil deodorizer distillate. Ind. Eng. Chem. Res. 2008, 47, 7013.
15
16
17 292 (16) Gunawan, S.; Vali, S.R.; Ju, Y.H. Purification and identification of rice bran oil fatty
18
19 293 acid steryl and was esters. J. Am. Oil. Chem. Soc. 2006, 83, 449
20
21
22 294 (17) Gunawan, S.; Wasista, H.W.; Kuswandi, K.; Widjaja, A.; Ju, Y.H. The utilization of
23
24 295 Xylocarpus moluccensis seed oil as biodiesel feedstock in Indonesia . Ind. Crops. Prod..
25
26 296 2014, 52, 286.
27
28
29 297 (18) Gunawan, S.; Kasim, N.S.; Ju, Y.H. Separation and purification of squalene from
30
31
32 298 soybean oil deodorizer distillate. Sep. Purif. Tecnol. 2008, 60, 128.
33
34
35 299 (19) Montgomery DC. Design and analysis of experiments, 6th Ed.; Jhon Wiley and Sons:
36
37 300 New York, 2005; p 96.
38
39
40 301 (20) Crane, S.; Aurore, G.; Joseph, H.; Mouloungui, Z.; Bourgeois, P. Composition of fatty
41
42 302 acids triacylglycerols and unsaponifiable matter in Calophyllum calaba L. oil from
43
44 303 Guadeloupe. Phytochemistry. 2005, 66, 1825.
45
46
47 304 (21) Corn refiners association. Corn oil, 5th edition; Corn Member Companies: Washington
48
49
50 305 D.C, 2006,5805.
51
52
53 306
54
55
56
57
58
59
60 13
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 14 of 23

1
2
3 307 Table 1 Experimental design of NPLF separation from crude C. inophyllum seed oil
4
5
6 Run (A) Solvent (B) Solvent (C) Methanol TAG TAG FFA FFA NPLF
7 to oil mass system composition in Rec Purify Rec Purify (%)
8 ratio solvent system (%) (%) (%) (%)
9
(%)
10 1 2 PE MEa 25 85.27 73.63 98.17 9.16 86.85
11
12 2 2 HE MEb 50 80.82 81.86 46.65 4.91 77
13
14 3 2 PE ME 50 74.5 78.5 60.8 6.92 71.18
15 4 3 HE ME 100 ND c
ND ND ND ND
16
17 5 5 PE ME 50 68 86.55 30.43 4.18 58.93
18
6 5 HE ME 25 92.51 77.15 88.82 8 89.92
19
20
7 3 HE ME 50 97.51 81.94 95.44 8.09 97.11
21
22 8 2 PE ME 0 ND ND ND ND ND
23
24 9 2 PE ME 100 ND ND ND ND ND
25 10 3 PE ME 50 99.27 81.12 60.8 5.37 91.78
26
27 11 3 PE ME 100 ND ND ND ND ND
28
29
12 5 HE ME 50 67.79 87.88 22.42 2.93 62.95
30 13 2 HE ME 25 86.61 74.51 95.07 9.78 86.02
31
32 14 2 HE ME 0 ND ND ND ND ND
33 15 5 HE ME 0 ND ND ND ND ND
34
35 16 3 HE ME 25 99.06 76.92 80.7 7.45 95.3
36
37 17 3 HE ME 0 ND ND ND ND ND
38 18 3 PE ME 25 94.67 75.56 95.45 8.23 93.96
39
40 19 2 PE ME 0 ND ND ND ND ND
41
20 3 PE ME 0 ND ND ND ND ND
42
43 21 5 PE ME 100 ND ND ND ND ND
44
45 22 5 PE ME 25 91.58 91.46 18.16 2.16 74.1
46 23 2 HE ME 100 ND ND ND ND ND
47
48 24 5 HE ME 100 ND ND ND ND ND
49 a
50 308 Petroleum ether- Methanol system
b
51 309 Hexane – Methanol system
c
310 Not Detected (there was no separation)
52
53
311
54
55
56
57
58
59
60 14
ACS Paragon Plus Environment
Page 15 of 23 Industrial & Engineering Chemistry Research

1
2
3 312 Table 2 Analysis of variance for TAG recovery
4 Source of Variations Sum of Degree of Mean F-Value P-Value
5 squares freedom square
6 Model 46280.59 17 2722.39 894.56 < 0.0001
7 A-Mass Ratio of Solvent to Oil 385.16 2 192.58 63.28 < 0.0001
8
B-Solvent system 5.35 1 5.35 1.76 0.2332
9
10 C-Methanol Composition 44337.23 3 14779.08 4856.32 < 0.0001
11 AB 3.70 2 1.85 0.61 0.5747
12 AC 707.97 6 117.99 38.77 0.0002
13 BC 6.53 3 2.18 0.71 0.5780
14 Residual 18.26 6 3.04
15
Lack of Fit 18.26 5 3.65
16
17 Pure Error 0.000 1 0.000
18 Cor Total 46298.85 23
19 313
20 314
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 15
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 16 of 23

1
2
3 315 Table 3 Analysis of variance for FFA recovery
4 Source of Variations Sum of Degree of Mean F-Value P-Value
5 squares freedom square
6 Model 34078.25 17 2004.60 5.12 0.0263
7 A-Mass Ratio of Solvent to Oil 2399.15 2 1199.58 3.06 0.1212
8
B-Solvent system 188.06 1 188.06 0.48 0.5143
9
10 C-Methanol Composition 30085.42 3 10028.47 25.60 0.0008
11 AB 501.17 2 250.58 0.64 0.5600
12 AC 2093.39 6 348.90 0.89 0.5541
13 BC 253.99 3 84.66 0.22 0.8818
14 Residual 2350.22 6 391.70
15
16 Lack of Fit 2350.22 5 470.04
17 Pure Error 0.000 1 0.000
18 Cor Total 36428.47 23
19
316
20
21
317
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 16
ACS Paragon Plus Environment
Page 17 of 23 Industrial & Engineering Chemistry Research

1
2
3 318 Table 4 Composition of NPLF at each stage of extraction
4
5 Stage Compounds
6
7 TAG DAG MAG FFA Othersc
8
9 1 93.32± 3.05a 2.94 ± 0.97 1.76 ± 1.37 1.66 ± 0.53 0.31 ± 0.22
10 (92.02±0.66)b (39.12±2.53) (43.95±32.84) (14.40±3.88) (3.15±1.80)
11
12 2 91.30 ± 4.54 2.83 ± 0.99 1.01 ± 0.72 1.13 ± 0.18 3.72 ± 2.71
13 (84.04±3.15) (35.09±3.46) (23.68±15.49) (9.22±0.87) (34.86±20.28)
14
15 3 91.71 ± 4.67 2.44 ± 0.75 0.91 ± 0.66 0.79 ± 0.08 4.15 ± 3.27
16 (80.26±3.98) (29.04±2.23) (20.18±13.47) (6.18±0.80) (36.44±24.15)
17
18 4 93.87 ± 3.23 1.91 ± 0.60 0.79 ± 0.57 0.71 ± 0.10 2.72 ± 2.00
19 (78.58±4.05) (21.78±1.67) (16.88±11.11) (5.25±0.41) (23.34±13.12)
20
21 5 96.39 ± 2.09 1.09 ± 0.78 0.52 ± 0.41 0.62 ± 0.17 1.28 ± 0.75
22
(77.72±4.29) (10.81±6.33) (12.76±7.51) (4.38±0.83) (11.16±3.78)
23
24
6 97.70 ± 1.45 0.54 ± 0.34 0.47 ± 0.34 0.50 ± 0.25 0.80 ± 0.52
25
26 (76.84±4.86) (5.41±2.28) (9.34±6.02) (3.39±1.47) (6.79±2.56)
27
28 7 98.20 ± 1.11 0.43 ± 0.28 0.26 ±0.20 0.44 ± 0.21 0.67 ± 0.42
29 (2.98±1.26) (4.27±1.87) (5.08±3.61) (2.98±1.26) (5.72±1.91)
30
31 8 98.53 ± 0.97 0.42 ± 0.30 0.06 ± 0.03 0.35 ± 0.20 0.64 ± 0.44
32 (79.50±5.43) (4.09±2.12) (1.25±0.52) (2.39±1.14) (5.44±2.05)
a
33 319 Content (%)
b
34 320 Recovery (%)
c
35 321 Such as hydrocarbons, calophyllolides, coumarins, phytosterols, gum, and wax
36 322
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 17
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 18 of 23

1
2
3 323 Figure legends
4 324 Figure 1 Full refining processing steps of the vegetable oils.
5
6 325 Figure 2 Normal probability plot of residuals for (A) TAG recovery and (B) FFA recovery
7
8 326 Figure 3 The effect of solvent to oil mass ratio on TAG and FFA recoveries. (A) Petroleum ether-
9 327 methanol system (PEME). (B) Hexane – methanol system (HEME)
10
11 328 Figure 4 The effect of methanol composition in solvent system on TAG and FFA recoveries. (A)
12 329 Petroleum ether-methanol system (PEME). (B) Hexane – methanol system (HEME)
13
14 330 Figure 5 Result of the HTGC (a) and TLC (b) developed in mobile phase (90:10:1)
15
16 331
17
18
19 332
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 18
ACS Paragon Plus Environment
Page 19 of 23 Industrial & Engineering Chemistry Research

1
2
3 333
4 Chemical Physical Proposed method
5 334
6 Degumming Degumming Batch wise liquid
7
335 extraction
8
9
10 336
11
12 337
13 Neutralization
14 338
15
16 339
17
18
19 340
20 Bleaching Degumming
21 341
22
23 342
24
25 343
26
27 Deodorization Deodorization Deodorization
344
28
29
30 345
31
32 346
33 Refined Oils
34 347
35
36
348 Figure 1.
37
38
39 349
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 19
ACS Paragon Plus Environment
 Industrial & Engineering Chemistry Research Page 20 of 23

1
2
3
4
5 (A)
6
7
8
9
Normal % Probability

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24 Standardized Residual
25 350
26
27
28 (B)
29
30
31
32
33
Normal % Probability

34
35
36
37
38
39
40
41
42
43
44
45
46
47
48 Standardized Residual
49 351
50
51 352 Figure 2
52
53 353
54
55
56
57
58
59
60 20
ACS Paragon Plus Environment
354

21
 Industrial & Engineering Chemistry Research Page 22 of 23

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 361
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39 362 ----- = FFA
40 = TG
41 363 = Solvent to Oil Mass Ratio of 2
42 = Solvent to Oil Mass Ratio of 3
43 364 = Solvent to Oil Mass Ratio of 5
44
45 365 Figure 4
46
47 366
48
49
50
51
52
53
54
55
56
57
58
59
60 22
ACS Paragon Plus Environment
Page 23 of 23 Industrial & Engineering Chemistry Research

1
2
3 8.0
uV(x100,000)

Chromatogram TAG
4 7.5 FFA
5 7.0 Crude oil DAG
MAG
6 6.5

6.0
7 TAG
5.5
8
5.0
9
FID Response

4.5 PLF
10 4.0
11 3.5

12 3.0

13 2.5

14 2.0

15 1.5
NPLF
16 1.0

17 0.5

18 0.0

19 -0.5

20 0
1.0 2.0 3.0 4.0 5.0
5
6.0 7.0 8.0 9.0 10.0
10
11.0 12.0 13.0 14.0 15.0
15
16.0 17.0 18.0 19.0 20.0
20
21.0 22.0 23.0 24.0 25.0
25
26.0 27.0 28.0 min
Crude PLF NPLF
21
22 Retention time, min
23 (A) (B)
367
24
25 368 Figure 5
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 23
ACS Paragon Plus Environment