Preparation of Hydroxylauric Acids for Environmental and ...philjournalsci.dost.gov.ph/images/pdf/pjs_pdf/vol...To monitor the eluents from flash chromatography, a mixture of 50:50

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Philippine Journal of Science142 (2): 175-184, December 2013ISSN 0031 - 7683Date Received: ?? Feb 20??

Key Words: biodegradable polymer, biomaterial, hydroxylauric acids, trimethylsilyl ether methyl ester

*Corresponding author: [email protected] [email protected]

Loida O. Casalme1 and Florentino C. Sumera2*

Preparation of Hydroxylauric Acids for Environmental and Biomaterial Use

1Natural Sciences Research InstituteUniversity of the Philippines, Diliman, Quezon City 1101

2Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101

Biodegradable polymer polyhydroxyacid (PHA) is composed of hydroxyacids such as glycolic, lactic, hydroxybutyric, hydroxyvaleric and hydroxycaproic, the properties of this bioplastic or biomaterial can be improved or modified with the addition of another long chain hydroxyacid such as hydroxylauric acids. The availability of long chain hydroxyfatty acids such as hydroxylauric acids is therefore desirable. Thus, a straightforward method of production, purification, and characterization of hydroxylauric acids was carried out as an alternative to the costly and tedious method of preparing hydroxyfatty acids by other methods. Hydroxylauric acids were synthesized from lauric acid using the trifluoroacetic acid-hydrogen peroxide system. The total product yield of the reaction was 96.00±1.18% of which 68.09+2.03% are hydroxylauric acids HOLAS composed of ϖ-1 (11.61+0.75%), ϖ-2 (13.17+0.57%), ϖ-3 (12.93+0.80%), ϖ-4 (12.99+0.58%), ϖ-5 and ϖ-6 (17.39+1.22%) and the rest are lactones and other oxidized products from gas chromatography data. The major products transformed into trimethylsilyl ether methyl ester derivatives were successfully identified by Gas Chromatography - Mass Spectrometry as a mixture of monohydroxylauric acids with hydroxyl groups remote to the carboxylic acid moiety. Product yield of pure hydroxylauric acids after flash chromatography was 28.85±3.12%, composed mainly of ϖ-1 (16.49+0.91%), ϖ-2 (19.20+0.63%), ϖ-3 (19.31+0.41%), ϖ-4 (20.30+0.79%), ϖ-5 and ϖ-6 (24.71+0.97%) hydroxylauric acids. Their presence and structures were confirmed by Proton and Carbon Nuclear Magnetic Resonance Spectroscopy.

INTRODUCTIONInterest in petroleum oil substitute coming from renewable resources has been the driving force behind the search for new derivatives from plants and microbial products (Sharma & Kundu 2008; Raquez et al. 2010; Vilela et al. 2010). These researches can provide novel compounds for use in industrial processes, add value to agricultural crops, and cause the development of new uses and applications from improvement of physicochemical properties of known biomaterials.

In addition to being a possible substitute for fuel resources, plant crops provide a clear advantage because they can be converted to a range of biomaterials that can provide farmers with new opportunities. Besides being low green house gas generating, they are sustainable, renewable, and favorable to agri-business (Luzier 1992; Ojumu et al. 2004; Sharma & Kundu 2008).

The coconut tree is one of these potential plant sources because its products, coconut oil and derivatized cocomethylesters have been used as fuel substitutes. Coconut oil, however, has a major component, which is lauric acid, together with other medium chain saturated fatty acids, that can be used also as an important

biomaterial constituent in biodegradable polymer. It is thus of technical and economic significance for a country like the Philippines and many other nations of the Pacific region to find new technological uses for coconut oil derivatives.

Transformation of its lauric acid into a hydroxyfatty acid can provide a major material in the synthesis of polyhydroxyalkanoates (PHA) about which literature abounds with respect to the study of their uses and biodegradability. Examples of these are polyglycolic, polylactic, polyhydroxybutyric, polyvaleric, and polycaproic acid used as biomaterial, bioplastic, or biodegradable polymer. All of these come from related studies of hydroxy acids (Domb & Nudelman 1995; Slivniak & Domb 2005).

Commercially available long chain hydroxyfatty acids are typically produced from saturated fatty acids via biocatalysis (enzymatic transformation carried out by partially purified enzymes or by whole cell catalysts used for the production of a wide variety of chemicals from bulk to fine chemicals [Schulze & Wubbolts 1999]). Hydroxyfatty acids are products of enzymatic reactions catalyzed by oxygenases like cytochrome P450 dependent fatty acid hydroxylases, and are important intermediates in diverse biological processes in plants and animals (Kandel et al. 2006; Tatsuya et al. 2008). In plants, hydroxylases play important roles in reproduction, fatty acid catabolism, cutin synthesis, plant-pathogen interaction, and plant detoxification (Salaun et al. 1982; Petkova-Andonova et al. 2002; Kandel et al. 2006). ϖ - Hydroxylation and in-chain hydroxylation are also exhibited by enzymes (naturally tailored protein catalysts synthesized to perform under physiological conditions [Illanes et al. 2012]) found in amphibians, insects, yeasts, fish, fungi, and bacteria (Lemaire et al. 1992). In humans, a family of Cytochrome P450 eliminates lipid-soluble compounds in the liver (Holland 1991; Reiser 1994).

Biocatalysis using oxygenases is regio-, stereo-, and chemoselective and avoids the use of extremely reactive conditions and multi-step synthetic routes (Reiser 1994; van Beilen et al. 2003). However, practical large-scale application of oxygenases presents more complications compared to biocatalysis using enzymes like hydrolases, isomerases or lyases (van Beilen et al. 2003). Among the practical issues being faced by oxygenases are: they require expensive co-factors (e.g. NADPH); they are very complex mixtures that need elaborate screening procedures; and several of them have the capacity to overoxidize (from alcohol to aldehyde to carboxylic) the substrate (Duetz et al. 2001; van Beilen et al. 2003). Thus, unless the challenge of translating biocatalysis by oxygenases into practical large-scale applications is met, there is a constant need for an economical process of preparing hydroxyfatty acids in

order to meet market demand.

High cost and limited availability of hydroxyfatty acids hinder its potential applications as raw material for the development of novel products. Hydroxyfatty acid, aside from its important role as a metabolite in physiological processes, is a potential component of environmental polymers and biomaterials. Biomaterials with fatty acid moieties are predictably biodegradable and non-toxic as they have natural metabolites as building blocks that may be degraded and later eliminated by the body. As biodegradable polymers, they can easily be biodegraded through microbial enzyme activities (Luzier 1992; Domb & Nudelman 1995; Ojumu et al. 2004). Hydroxyfatty acids find applications also in the production of resins, coatings, waxes, plastics, nylons, corrosion inhibitors, and cosmetics (Hou et al. 1998). Presently, castor oil and its derivatives are the only source of these industrial hydroxyfatty acids (McKeon et al. 2006) .This is besides some studies done on unsaturated fatty acids where the double bond is biocatalytically hydroxylated (Hou et al. 1998; Tatsuya et al. 2008)

This study aims, therefore, to develop an economical way of preparing a hydroxyfatty acid, hydroxylauric acids (HOLAs) from lauric acid for commercial use, in particular as a target raw material for biomaterials or even biodegradable plastics. While there is an available process to obtain the HOLAs using the reaction described by Deno et al. (1977) the process was not optimized for the production of HOLAs. Thus, straightforward two-step synthesis that employs fluoroperoxy acid, a highly reproducible purification procedure, and a simple characterization step using GC-MS (Claeys 1989) will be carried out to prepare the purified HOLAs.

MATERIALS AND METHODSAnalytical reagent grade (AR) lauric acid (Fluka) was from Sigma-Aldrich Co. (Buchs, Switzerland). AR grade trifluoroacetic acid, pyridine, and hydrogen peroxide (30% upon assay) were from Merck Inc. (Darmstadt, Germany). AR grade hexane, methanol, anhydrous diethyl ether, and ethyl acetate were all purchased from J.T. Baker Inc. (New Jersey, USA). AR grade trimethylsilyl imidazole (Aldrich) and ϖ hydroxylauric acid (Aldrich) were purchased from Sigma-Aldrich (Buchs, Switzerland).

Synthesis of hydroxylauric acids (HOLAs)To 75 mL of concentrated trifluoroacetic acid (TFA), 8.58 mL (30% w/v) H2O2 was added and the solution stirred at room temperature for 5 min. Lauric acid (7.305 g) was added with stirring for another 5 min. The mixture was heated at 85ºC-90ºC for 2 h with stirring, poured in ice-

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Philippine Journal of ScienceVol. 142 No. 2, December 2013

Casalme and Sumera.: Preparation of Hydroxylauric Acids for Environmental and Biomaterial Use

water (equivalent volume as the reaction mixture) and extracted with diethyl ether (total of three extractions). The solvent ether was then evaporated in vacuo.

To completely hydrolyze the ester by-products, the product obtained from the previous reaction was reacted with five molar excess of 5M NaOH. The reaction mixture was refluxed for two hours at 85ºC. Afterwards, the product was acidified with 6 M HCl to pH 4 and extracted with diethyl ether. The organic layer was then dried over sodium sulfate overnight and the solvent removed in vacuo.

Purification of HOLAs using Flash ChromatoraphyTo monitor the eluents from flash chromatography, a mixture of 50:50 by volume ratio of hexane and diethyl ether was used as solvent in Thin Layer Chromatography (TLC) of HOLAs while developed plates were sprayed with the visualizing agent (a solution of 0.1 g bromocresol green in 500 mL ethanol and 5 mL of 0.1 M NaOH).

Isocratic elution was carried out in an Isco RediSep disposable flash column with 230-400 mesh (35 to 60 µm particle size). The sample (10 g) was loaded in a 120 g silica-packed column and eluted with 5 column volumes (1000 mL) of 50:50 hexane-diethyl ether, then followed by 6 column volumes (1200 mL) of diethyl ether. Fractions were characterized by gas chromatography installed with flame ionization detector. Target fractions were pooled together and stored at -20°C to -40°C.

Spectroscopic analysis of HOLAsFourier Transform Infrared (FT-IR) Spectrum of crude HOLAs was recorded in an IR-Prestige Fourier Transform Infrared Spectrophotometer. Samples were prepared using KBr smear technique.

Characterization of trimethylsilyl (TMS) ether derivative of crude and purified HOLAs was carried out using a Shimadzu GC-14B gas chromatograph installed with an Equity-5 (poly(5%diphenyl/95%dimethylsiloxane)) capillary column (Sigma-Aldrich Co.), 30 m x 0.25 mm x 0.5 µm film thickness and flame ionization detector (FID). Samples (5-10 mg) were silylated with trimethylsilyl imidazole (TMSI) in pyridine (Sigma-Aldrich Co.) prior to injection to the column. The reaction vials were placed in a 60ºC water bath for an hour to facilitate the reaction. The derivatives were extracted with hexane, washed with distilled water, and dried over sodium sulfate. Column initial temperature was set to 128°C, held for 1 min, and then ramped to 280°C at a rate of 15°C/min. The total run time was 30 min. Nitrogen set to a flow rate of 0.88 mL/min was the carrier gas. Injection port temperature was held at 250C, while the FID was operated at 280°C. Data gathering and analysis was done using Class GC 10 software.

TMS ether methyl ester derivatives of crude HOLAs were

characterized using a Shimadzu Gas Chromatograph-Mass Spectrometer (GC-MS) Model QP 2010 (Natural Sciences Research Institute, Research and Analytical Services Laboratory) installed with Supelco SPB-5 (poly(5%diphenyl/95%dimethylsiloxane)) fused capillary column (Sigma-Aldrich Co.) of 30 m x 0.32 mm x 0.25 µm dimension. Column initial temperature was 50°C, held for 2 min, ramped at 5°C/min to 100°C, ramped at 10°C/min to 200°C, held for 2 min, ramped at 20°C/min to 300°C, and finally held for 10 min. Injection port temperature was set at 250°C. The mass spectrometer ion source temperature used was 200°C. Scanning range was from 8 to 37 min at 40 to 350 m/z with a scanning interval of 0.5 s. Using the same instrument setting, TMS ether methyl ester derivatives of purified HOLAs were characterized in a Varian 450-GC Gas Chromatograph/Varian 240-MS IT Mass Spectrometer (UP Diliman, Institute of Chemistry, Analytical Services Laboratory).

To prepare TMS ether methyl ester derivatives of HOLAs, methylation of HOLAs was first done, followed by silylation. To prepare the methyl ester derivatives, HOLAs were refluxed with 1% sulfuric acid in methanol (2 mL) for 20 min. This was followed by addition of 5% NaCl solution (5 mL) to the mixture. The product was extracted with hexane (2 x 5 mL), later washed with 2% NaHCO3 (4 mL), and dried by standing over anhydrous sodium sulfate. Sodium sulfate was filtered out and hexane was evaporated with a slow stream of nitrogen. Finally, methyl ester products were silylated using the same procedure specified previously in the preparation of samples for GC-FID analysis.

Proton (1H-NMR) and Carbon (13C-NMR) Nuclear Magnetic Resonance spectra of pure HOLAs were recorded using a 400 MHz JEOL Lambda FT-NMR spectrometer (Ateneo de Manila University, National Chemistry Instrumentation Center). The sample was dissolved in deuterated chloroform (CDCl3) and was scanned at 8 kHz and 27.1 kHz sweep widths in the 1H-NMR and 13C-NMR experiments respectively. Trimethylsilane was used as reference standard.

RESULTS AND DISCUSSIONA more detailed synthesis, purification, and characterization of a new potential biomaterial, hydroxylauric acid (HOLA) is here in described. This is made possible by optimizing a reaction to improve the yield of HOLAs as principal product.

Synthesis of HOLAs

According to Deno et al. (1977) the hydroxylation reaction involves an electrophilic attack of the active species, the protonated trifluoroperoxyacid, on the fatty

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acid alkyl chain. In this paper, the principal by-product, the trifluoroacetates were converted to HOLAs directly by base hydrolysis, which was not performed in the original paper by Deno et al. (1977). Through base hydrolysis followed by neutralization with dilute acid, the trifluoroacetate products were converted to HOLAs. The steps of the reaction are illustrated in Figure 1.

Solvent evaporation left a yellow liquid product purified by preparative flash column chromatography and characterized by FTIR, GC-FID, GC-MS and NMR.

Spectroscopic analysis of crude HOLAsThe FTIR profile of the product was characterized by a hydroxy, as well as a carboxylic acid functional group absorption. A broad O—H stretch is present from 3640 to 2500 cm-1 with a C=O stretch at 1713 cm-1 describing the presence of carboxylic acid group. Alcoholic O-H stretch is also apparent at 3435 cm-1 with C-O stretch for 2° alcohol observed at 1113 cm-1.

GC-FID was used to determine the yield and purity of HOLAs. The product was first derivatized with TMSI prior to injection to the instrument. TMSI was used as derivatizing agent because of its capacity to silylate the –COOH group, as well as the less reactive –OH group.

Pyridine was added as solvent-catalyst. Incubation of the mixture at 60ºC for an hour was sufficient to completely derivatize the sample.

The gas chromatogram of the TMS ether derivative of the crude product is shown in Figure 2. A number of peaks along with lauric acid standard, which has a retention time of 13.0 min are present. Deno et al. (1977) identified the product as a mixture of HOLAs, lactones, and vicinal diols. By monitoring the disappearance of the peak corresponding to lauric acid and using area normalization, percent yields of 96.00±1.18% were calculated from the GC-FID spectra with ϖ-1 (11.61+0.75%), ϖ-2 (13.17+0.57%), ϖ-3 (12.93+0.80%), ϖ-4 (12.99+0.58%), ϖ-5 and ϖ-6 (17.39+1.22%) HOLAs.

Using GC-MS this time however was not helpful since the disilylated products easily fragment into smaller mass ions and provide no marker fragments for easy verification of molecules. To easily identify the components of crude HOLAs by GC-MS, products were first derivatized by esterification (not silylation) of the –COOH group, but followed by silylation of the hydroxyl group later. This technique provided stable marker fragment ions that facilitated the interpretation of the mass spectra (Claeys 1989).

Carboxylic group of HOLAs was first methylated by

Figure 1. Synthesis of HOLAs by oxidation of lauric acid using TFA-H2O2 system.

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using a procedure developed by Christie (1989). After methylation of the carboxylic group, the hydroxyl group was silylated with TMSI in the presence of pyridine.

Figure 3 illustrates the total ion chromatogram of crude HOLAs as TMS ether methyl ester, which is analogous to the order of elution and peak intensities of the GC-FID

chromatogram of the TMS ether derivative (Figure 2). HOLAs with hydroxyl groups close to the ϖ terminus elute last in capillary columns with nonpolar stationary phase poly(5%diphenyl/95%dimethylsiloxane) like Equity 5 in GC-FID and Supelco SPB-5 used in GC-MS. GC chromatograms of TMS ether derivative and

Figure 2. Gas chromatogram of TMS ether derivative of crude HOLAs.

Figure 3. Total ion chromatogram of TMS ether methyl ester derivative of crude HOLAs: (a) lauric acid (18.88 min); (b) δ-lactone (17.85 min), γ-lactone (20.42 min), ϖ-8 HOLA (19.77 min), and ϖ-9 HOLA (20.95 min); (c) ϖ-6 HOLA (21.58 min), ϖ-5 HOLA (21.64 min), ϖ-4 HOLA (21.70 min), ϖ-3 HOLA (21.87 min), ϖ-2 HOLA (22.13 min), ϖ-1 HOLA (22.30 min) (d) ϖ HOLA (22.95 min).

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TMS ether methyl ester derivative of HOLAs have the elution order: Lauric acid, ϖ-6, ϖ-5, ϖ-4, ϖ-3, ϖ-2, and ϖ-1 HOLAs in non-polar columns.

To identify the peaks in the total ion chromatogram, base peaks which resulted from cleavage at the carbon bearing the TMS ether were monitored. This unique fragmentation pattern provides information on the position of the hydroxyl group along the alkyl chain, as illustrated in Figure 4 (Claeys 1989). Figure 4 is a representative mass spectrum showing the marker fragment ion m/z = 117 (CH3 -CH=O+ - Si (CH3)3) of -1 HOLA TMS ether methyl ester derivative.

Table 1 tabulates marker fragment ions that are present in the mass spectra of the derivatized crude sample.

Major structural isomers of HOLAs in the crude product were successfully identified by GC-MS as ϖ-1, ϖ-2, ϖ-3, ϖ-4, ϖ-5 and ϖ-6 HOLAs (Figure 5).

In addition, the crude product had traces of the five-membered and six-membered rings, γ- and δ- lactones respectively, the structures of which are shown in Figure 6. Marker fragment ions of HOLAs with lactonizing carbons, γ- (C4 from terminal carboxylic acid) and δ- lactones (C5 from terminal carboxylic acid) appeared at retention times 20.42 min and 17.85 min respectively (See Figure 3). Molecular ion peak of -lactone was present at m/z 198. Fragment ions with m/z 84 (with migration of 1 proton) and 97 (with migration of 2 protons) that represents the loss of alkyl groups from the five- and six-membered rings via α-cleavage were also located in the mass spectra of γ- and - lactones (Chance et al. 1998). The ϖ HOLA with marker fragment ion of m/z 255 was present in trace amount. The ϖ-8 (retention time: 19.77 min) and ϖ-9 (retention time: 20.95 min) marker fragment ions were also present with m/z 213 (with migration of 2 protons) and 227 (with migration of 2 protons) respectively. Marker fragment ions that correspond to ϖ-10 HOLA was not found in the mass spectra of the crude product.

Figure 4. Mass spectrum of the TMS ether methyl ester derivative of ϖ-1 HOLA.

Table 1. Identified hydroxy group location and associated fragment ions of TMS ether methyl ester derivative of crude HOLAs

Fragment Ion (m/z)Location

of hydroxy group M.+ M-CH3 M-CH3O

Marker Fragment Ion

Retention Time (min)

ϖa 302 287 271 255 22.95ϖ-1 --- 287 271 117 22.30ϖ-2 --- 287 273 131 21.13ϖ-3 --- 287 271 145 21.87ϖ-4 --- 287 271 159 21.70ϖ-5 --- 287 271 173 21.64ϖ-6 --- 287 271 187 21.58

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Figure 7. Total ion chromatogram of TMS ether methyl ester derivative of purified HOLAs: (a) ϖ-6 HOLA (22.379 min); (b) ϖ-5 HOLA (22.446 min); (c) ϖ-4 HOLA (22.532 min); (d) ϖ-3 HOLA (22.726 min); (e) ϖ-2 HOLA (23.076 min); (f) ϖ-1 HOLA (23.283 min).

Figure 5. Sites of hydroxylation of lauric acid.

Figure 6. γ- and δ- lactones.

Purification of HOLAs using flash chromatoraphyIsocratic elution of the product was carried out in an Isco RediSep disposable flash column to remove unreacted lauric acid. In addition, minor products were also removed, resulting to purified HOLAs containing only the following major products - ϖ-1, ϖ-2, ϖ-3, ϖ-4, ϖ-5, and ϖ-6 HOLAs.

Initially, Thin Layer Chromatography was carried out to predict the sample’s column behavior. The color reagent, bromocresol green specific for the detection of organic acids, appeared as yellow spots in a blue background. The use of 50:50 by volume ratio of hexane and diethyl ether as eluent resulted to a good separation of lauric acid

from HOLAs, which have Rf’s equivalent to 0.67 and 0.24-0.43 respectively.

Flash chromatography was employed in the bulk purification of the product using the optimized solvent system from TLC experiments (50:50 hexane:diethyl ether). Each fraction (per column volume) was characterized using GC-FID and similar fractions were pooled together. Weight percent yield of purified HOLAs from flash chromatography was 28.85±3.12% of the crude sample loaded. Purified HOLAs were further characterized using GC-MS and NMR.

Spectroscopic analysis of purified HOLAsThe composition of purified HOLAs was determined by employing GC-MS and GC-FID as qualitative and quantitative tools of analysis. Structural isomers of purified HOLAs were successfully identified through the marker fragment ions in their mass spectra. The GC-MS total ion chromatogram of purified HOLAs is shown in Figure 7.

Analysis of the GC-FID spectra of the TMS ether derivative of pooled mixed HOLAs (after flash chromatography) resulted to the following percent composition, reported as means of three trials: ϖ-1 (16.49+0.91%), ϖ-2 (19.20+0.63%), ϖ-3 (19.31+0.41%), ϖ-4 (20.30+0.79%), ϖ-5 and ϖ-6 (24.71+0.97%) HOLAs.

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Figure 8. Methine region in 1H-NMR spectrum showing the presence of HO-C-H proton peaks of ϖ-1 to ϖ-6 HOLAs.

Figure 9. Hydroxy carbon region in 13C-NMR spectrum showing the presence of HO-C-H carbon peaks of ϖ-1 to ϖ-6 HOLAs.

1H-NMR results are in good agreement with results obtained from GC-MS, confirming the presence of a series of structural isomers of HOLA. Figure 8 shows the methine peaks in the 1H-NMR spectrum of mixed HOLAs. Six types of methine hydrogens are present and were assigned as methines of -1, ϖ-2, ϖ-3, ϖ-4, ϖ-5, and ϖ-6 HOLAs.

Figure 9 shows the hydroxy carbon region in the 13C-NMR spectrum of purified HOLAs. Accordingly, six types of hydroxy carbons are present, corresponding to the six HOLA isomers.

In the present study, a mixture of structural isomers of HOLA was successfully synthesized using Deno’s (1977)

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hydroxylation method with yield of 96.00+1.18% of which 68.09+2.03% are HOLAs. Major products, ϖ-1 (11.61+0.75%), ϖ-2 (13.17+0.57%), -3 (12.93+0.80%), ϖ-4 (12.99+0.58%), ϖ-5 and ϖ-6 (17.39+1.22%) HOLAs were isolated and purified using flash chromatography. The identification of their TMS ether methyl ester derivative was made through the marker fragment ions in the mass spectra.

It is hoped that this method would become very useful in the preparation of biodegradable plastics or biopolymers containing long chain fatty acid such as HOLAs that could easily be derived from renewable sources.

ACKNOWLEDGMENTThe authors would like to acknowledge the financial support from the Natural Sciences Research Institute of the University of the Philippines – Diliman.

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Preparation of Hydroxylauric Acids for Environmental and ...philjournalsci.dost.gov.ph/images/pdf/pjs_pdf/vol...To monitor the eluents from flash chromatography, a mixture of 50:50