6 Linoleic Acid from Crude Tall Oil

separations

Article Separation and Puriﬁcation of ω-6 Linoleic Acid from Crude Tall Oil

Md Shariful Islam, Lew P. Christopher and Md Nur Alam * Bioreﬁning Research Institute, Lakehead University, 1294 Balmoral Street, Thunder Bay, ON P7B 5Z5, Canada; [email protected] (M.S.I.); [email protected] (L.P.C.) * Correspondence: [email protected]

Received: 28 November 2019; Accepted: 22 January 2020; Published: 2 February 2020

Abstract: Crude tall oil (CTO) is the third largest by-product at kraft pulp and paper mills. Due the large presence of value-added fatty and resin acids, CTO has a huge valorization potential as a biobased, readily available, non-food, and low-cost biorefinery feedstock. The objective of this work was to present a method for the isolation of high-value linoleic acid (LA), an omega (ω)-6 essential fatty acid, from CTO using a combination of pretreatment, fractionation, and purification techniques. Following the distillation of CTO to separate the tall oil fatty acids (TOFAs) from CTO, LA was isolated and purified from TOFAs by urea complexation (UC) and low-temperature crystallization (LTC) in the temperature range between 7 and 15 C. The crystallization yield of LA from CTO in − − ◦ that range was 7.8 w/w at 95.2% purity, with 3.8% w/w of ω-6 γ-linolenic acid (GLA) and 1.0% w/w of ω-3 α-linolenic (ALA) present as contaminants. This is the first report on the isolation of LA from CTO. The approach presented here can be applied to recover other valuable fatty acids. Furthermore, once the targeted fatty acid(s) are isolated, the rest of the TOFAs can be utilized for the production of biodiesel, biobased surfactants, or other valuable bioproducts.

Keywords: crude tall oil; essential fatty acids; linoleic acid; γ-linolenic acid; α-linolenic acid; distillation; urea complexation; low-temperature crystallization

1. Introduction The pulp and paper industry has joined the global movement toward cleaner and greener products in their effort to transition to forest biorefineries. This transition is based on the circular bioeconomy concept, which implies the most efficient recycling of wastes, co-products, and residues as a strategy to preserve our bio-based resources and minimize the use of fossil fuel-derived products of environmental concern. As bio-based fuels and products are carbon-neutral, they do not result in net green-house gas (GHG) emissions due to the balance between CO2 emitted and CO2 trapped back by biomass. After lignin and hemicellulose, CTO is the third largest chemical by-product in a kraft pulp and paper mill, with a yield from highly resinous coniferous species in the range of 30–50 kg per ton pulp [1]. CTO is extracted from the partially concentrated black liquor, derived from kraft pulping of softwoods, by skimming off the kraft oil soap. CTO has a complex composition of saponified fatty acids (30–60%), resin acids (40–60%), and unsaponifiables (5–10%) [2]. However, the exact yield and composition of CTO depend on a number of factors such as pulping conditions, wood species, and their length of storage prior to pulping, geographical location, climate conditions, etc. [3]. If not used internally as a lime kiln hoq fuel, mills most commonly sell CTO at a profit of 1–1.5% of their total revenue to chemical companies for further refining to various products. For example, CTO is applied in producing metal-working fluids for metal-rolling and metal-working operations, in producing drill fluids and separating fluids for the concrete products industry as well as to obtain corrosion inhibitors [4]. The TOFAs are primarily used in the production of soaps, detergents, paints,

Separations 2020, 7, 9; doi:10.3390/separations7010009 www.mdpi.com/journal/separations Separations 2019, 6, x FOR PEER REVIEW 2 of 11 producing drill fluids and separating fluids for the concrete products industry as well as to obtain corrosion inhibitors [4]. The TOFAs are primarily used in the production of soaps, detergents, paints, coatings,Separations plastic2020, 7, additives, 9 fuel additives, lubricants, and adhesives [5–10]. Alkyd resins account2 of 11 for the largest demand among all intermediates manufactured from TOFAs, whereas soaps, detergents, and coatings hold the largest market share of TOFAs in terms of volume. More recently, TOFAs have beencoatings, investigated plastic additives,for the production fuel additives, of lubricants,biofuels including and adhesives drop-in [5–10 hydrocarbon]. Alkyd resins fuels account [11] for and the largest demand among all intermediates manufactured from TOFAs, whereas soaps, detergents, biodiesel [12,13] The market for TOFAs is expected to reach USD 1 billion in 2022 [14]. In the U.S., and coatings hold the largest market share of TOFAs in terms of volume. More recently, TOFAs CTO has a floor price the equivalent of the value of natural gas as CTO can be used as a substitute to have been investigated for the production of biofuels including drop-in hydrocarbon fuels [11] and fossil fuels as a pulp mill lime kiln process fuel. The current global annual production of CTO is biodiesel [12,13] The market for TOFAs is expected to reach USD 1 billion in 2022 [14]. In the U.S., approximately 1.8–2 million tons (t), however, the 320 chemical pulp mills worldwide have the CTO has a floor price the equivalent of the value of natural gas as CTO can be used as a substitute potentialto fossil to fuels produce as a pulp2.6 million mill lime t per kiln year process [15]. About fuel. 65% The currentof the total global CTO annual production production is concentrated of CTO in isNorth approximately America. In 1.8–2 Canada, million 12 tonskraft (t), mills however, currentl they generate 320 chemical 75,000 pulp t CTO, mills whereas worldwide in the have U.S., the two thirdspotential of the to TOFA produce supplied 2.6 million to the t per chemical year [15]. indust Aboutry 65% are of derived the total from CTO CTO, production with another is concentrated 100,000 t of inCTO North expected America. to become In Canada, available 12 kraft by mills2020 [16]. currently generate 75,000 t CTO, whereas in the U.S., twoThe thirds main of theTOFAs TOFA in supplied CTO are to theLAchemical (C18:2n6) industry (Figure are 1), derived oleic acid from (C18:1), CTO, with GLA another (C18:3n6), 100,000 and t palmiticof CTO acid expected (C16:0) to become[17]. Typically, available the by 2020TOFAs [16]. consist of 75–85% unsaturated fatty acids (UFAs), mainlyThe polyunsaturated main TOFAs in fatty CTO areacids LA (PUFAs), (C18:2n6) (Figurewith LA1), comprising oleic acid (C18:1), about GLA half (C18:3n6), the total TOFAs and palmitic [13,18]. LAacid is an (C16:0) 18 carbon [17]. Typically,polyunsaturated the TOFAs fatty consist acid with of 75–85% the two unsaturated double bonds fatty at acids C9 (UFAs),and C12 mainly in a cis- configurationpolyunsaturated (all-cis-9,12-octadecadienoic fatty acids (PUFAs), with LA acid comprising) (Figure about1). LA half is denoted the total TOFAsas an omega [13,18]. ( LAω)-6 is anfatty acid18 carbonand together polyunsaturated with ALA fatty ω-3 acid acid with (18:3n3) the two (Figure double bonds1) form at C9theand two C12 essential in a cis-configuration fatty acids that mammals(all-cis-9,12-octadecadienoic (humans and animals) acid) cannot (Figure synthesize1). LA is denoted and must as anbe omegaacquired ( ω through)-6 fatty acidtheir and diet. together Another ω-6with fatty ALA acidω -3of acid health (18:3n3) importance (Figure 1is) formGLA the (18:3n6), two essential which fattyis classified acids that as mammals a conditionally (humans essential and fattyanimals) acid that cannot can synthesize become essential and must under be acquired developme throughntal their or disease diet. Another conditionsω-6 fatty(Figure acid 1). of LA health as an essentialimportance fatty is acid GLA plays (18:3n6), an whichimportant is classified role in as metabolic a conditionally processes essential related fatty acidto human that can health become and nutrition,essential and under may developmental be a factor in or a disease number conditions of degenerative (Figure1 illnesses). LA as an such essential as cancer, fatty acidatherosclerosis, plays an osteoporosis,important role and in cardiovascular metabolic processes disease related [19–21]. to human Nowadays, health the and public nutrition, awareness and may of bethe a nutritional factor in a number of degenerative illnesses such as cancer, atherosclerosis, osteoporosis, and cardiovascular and health benefits of LA is growing, and the market demand is expected to rise in the future. Aside disease [19–21]. Nowadays, the public awareness of the nutritional and health benefits of LA is from its health benefits, LA is used in making oil paints and varnishes, in cosmetics, and as a growing, and the market demand is expected to rise in the future. Aside from its health benefits, LA is surfactant [22,23]. Sources of LA include fish and shellfish, vegetable oils, nuts, and seeds. However, used in making oil paints and varnishes, in cosmetics, and as a surfactant [22,23]. Sources of LA include most of these sources are food-based, seasonal, of limited availability, and fairly expensive. In fish and shellfish, vegetable oils, nuts, and seeds. However, most of these sources are food-based, addition, fish may be contaminated with methyl mercury and lead, which are heavy metals with high seasonal, of limited availability, and fairly expensive. In addition, fish may be contaminated with environmentalmethyl mercury and and human lead, whichtoxicity are [24]. heavy metals with high environmental and human toxicity [24].

. Figure 1. Chemical structures of essential (LA, ALA) and conditionally essential (GLA) fatty acids. Figure 1. Chemical structures of essential (LA, ALA) and conditionally essential (GLA) fatty acids. Despite the well-documented uses of LA and the growing markets for CTO and TOFAs, there are noDespite reports onthe the well-documented isolation of LA from uses CTO of LA to date.and the LA hasgrowing mainly markets been extracted for CTO from and vegetable TOFAs, there oil areseeds no reports by applying on the extraction isolation techniquesof LA from such CTO as to supercritical date. LA has CO mainly2 [25,26 been], cold extracted pressing from alone, vegetable and in oilconjunction seeds by applying with enzymatic extraction extraction techniques and/or such solvent as supercritical extraction [27 CO–292 ].[25,26], For example, cold pressing Mario et alone, al. [27 and] in extractedconjunction 62% with of LA enzymatic from sunﬂower extraction seeds and/or using aso singlelvent screwextraction extruder, [27–29]. whereas For example, cold pressing Mario of oilet al. seeds followed by extraction with iso-hexane was used to separate LA from other UFAs [29]. Separations 2019, 6, x FOR PEER REVIEW 3 of 11

[27] extracted 62% of LA from sunflower seeds using a single screw extruder, whereas cold pressing ofSeparations oil seeds2020 followed, 7, 9 by extraction with iso-hexane was used to separate LA from other UFAs3 of[29]. 11 The objective of this work was to present a method for the isolation of LA from CTO, an alternative,The objective readily of thisavailable, work was non-food, to present and a method low-cost for the feedstock isolation of by LA fromusing CTO, a ancombination alternative, of fractionation,readily available, separation, non-food, and and purification low-cost feedstock techniques by using. Following a combination fractional of fractionation, distillation of separation, CTO to the majorand puriﬁcationCTO components, techniques. the LA Following in TOFAs fractional was sepa distillationrated by of UC CTO and to LTC. the major The CTOapproach components, presented herethe LAcan inbe TOFAs applied was to separated the isolation by UC of andother LTC. essential The approach fatty acids presented such hereas ω can-3 ALA be applied and ω to-6 theGLA. Furthermoreisolation of other, once essential the targeted fatty acids fatty such acid(s) as ω-3 are ALA isol andated,ω-6 the GLA. rest Furthermore, of the TOFAs once theincluding targeted the remainingfatty acid(s) PUFAs, are isolated, saturated the restfatty of acids the TOFAs (SFAs), including and monounsaturated the remaining PUFAs, fatty saturatedacids (MUFAs) fatty acids can be utilized(SFAs), for and the monounsaturated production of fattybiodiesel, acids (MUFAs)biobased cansurfactants, be utilized or for other the productionvalue-added of biodiesel,bioproducts (Figurebiobased 2). surfactants, or other value-added bioproducts (Figure2).

Figure 2. Separation of ω-6 LA from CTO. Figure 2. Separation of ω-6 LA from CTO. 2. Materials and Methods 2. Materials and Methods 2.1. Materials 2.1. MaterialsCTO was sourced from a pulp and paper mill in Canada and stored in a refrigerator at a temperature belowCTO 4 ◦wasC until sourced use. Urea, from ethanol, a pulp hexaneand paper (ACS mill grade), in Canada LA, sodium and chloride,stored in sodium a refrigerator hydrogen at a temperaturecarbonate, methanol, below 4 °C and until acetone use. (HPLC-grade)Urea, ethanol, were hexane purchased (ACS grade), from Sigma-Aldrich LA, sodium chloride, (Oakville, sodium ON, Canada). Sulfuric acid (99.9%) was obtained from Fisher Scientific (Ottawa, ON, Canada). hydrogen carbonate, methanol, and acetone (HPLC-grade) were purchased from Sigma-Aldrich (Oakville,2.2. CTO PretreatmentON, Canada). Sulfuric acid (99.9%) was obtained from Fisher Scientific (Ottawa, ON, Canada). Prior to distillation, CTO was pretreated with 1% w/v NaCl solution to remove any unwanted 2.2.impurities CTO Pretreatment from the raw CTO (residual pulping chemicals, sulfuric acid, etc.) that may affect the distillation process. A total of 40% v/v of a 1% w/v NaCl solution and 60% v/v of raw CTO were vigorouslyPrior to mixeddistillation, together CTO in was a separating pretreated funnel with for1% 10w/v min. NaCl Two solution distinct to remove layers were any formed:unwanted impuritiesa bottom layerfrom ofthe aqueous raw CTO salt (residual solution containingpulping chem dusticals, matters, sulfuric and acid, a top etc.) layer that ofnon-aqueous may affect the distillationCTO. The aqueousprocess. bottomA total layer of 40% was v/v discarded of a 1% whereas w/v NaCl the upper solution layer and was 60% centrifuged v/v of raw (Sorvall CTO RT1 were vigorouslyCentrifuge, mixed ThermoFisher together Scientific,in a separating Germany) funnel at 10,000 for 10 rpm min. for 10Two min. distinct The yield layers of thewere pretreated formed: a bottomCTO was layer 93% of waqueous/w. salt solution containing dust matters, and a top layer of non-aqueous CTO. The aqueous bottom layer was discarded whereas the upper layer was centrifuged (Sorvall RT1 Separations 2020, 7, 9 4 of 11

2.3. CTO Distillation The pretreated CTO was then subjected to fractional distillation to separate the TOFA from the rest of the CTO components. A 5000 mL round-bottom flask containing 200 g of pretreated CTO was placed on a heating mantle and covered with glass wool to prevent heat dissipation. The temperature was controlled at 130–265 ◦C with a temperature sensor that was introduced from the top of the flask. A vacuum pressure of 10 mm Hg was maintained in the flask by air compressor (Vaccuubrand, Model-MZ2CNT, Werthelm, Germany). The fatty acid vapors that were produced under the above temperature and pressure conditions were collected in a condenser. The TOFA composition was analyzed by HPLC as described in Section 2.6.

2.4. UC of PUFAs UC was employed to separate the PUFAs from the SFAs and MUFAs [30,31]. Ethanoic urea was used at a ratio of ethanol:urea of 96:4. The TOFAs were mixed with the ethanoic:urea solution at a 20:80 volume ratio. The mixture was kept at 60 ◦C and stirred until a clear homogeneous solution was formed. Next, this solution was kept at room temperature until urea crystals precipitated. The crystals settled at the bottom, while the PUFAs remained soluble in the ethanol solution. The precipitate was filtered with a Buchner funnel using 70 mm (φ) Whatman filter paper. The filtrate containing the PUFAs was dried at 60 ◦C to remove the ethanol. The solid UC residue, retained on the Buchner funnel, was extracted with hexane to recover the fraction of SFAs and MUFAs, and with water to solubilize the urea. The residual SFAs and MUFAs can be used for biodiesel production, whereas the urea can be recycled in the UC process.

2.5. PUFAs Separation by LTC A 5 g sample of PUFAs was mixed with 45 g acetone. The temperature of crystallization of the resultant acetone-PUFAs mixtures was monitored in the temperature range from 30 C to 4 C − ◦ ◦ (Table1). Before collection, each PUFA-acetone fraction (A–E) was kept for 24 h at the temperature indicated in Table1. Thereafter, fractions were centrifuged at 4100 rpm for 7.5 min. The crystallized portion of each fraction was ﬁltered out from the remaining liquid solution whereas acetone was recovered by evaporation and recycled in the LTC process.

Table 1. PUFAs fractionation by LTC.

LTC Fraction LTC Temperature

A 4 ◦C B 7 C − ◦ C 15 C − ◦ D 20 C − ◦ E 30 C − ◦

2.6. TOFA Identiﬁcation and Quantiﬁcation

2.6.1. High-performance liquid chromatography (HPLC) Analysis Each LTC fraction (A–E) was analyzed using high-performance liquid chromatography (HPLC) with an Agilent Technology 1260 Infinity Series equipped with a refractive index (RI) detector, and a C18 main column (ZORBAX Eclipse XDB-C18, 4.6 150 mm, 5 µm). In a 2 mL vial, 100 µL of each × fraction and 900 µL of HPLC-grade hexane were mixed, and then 10 µL of sample was automatically injected into the HPLC. The mobile phase was 100% ACS-grade hexane, the column temperature was set at 50 ◦C, and the flow rate was fixed at 0.8 mL/min. The total processing time through the column was 20 min. The results from the HPLC-RI detector were sent to a PC-controlled data acquisition system for analysis. Separations 2020, 7, 9 5 of 11

2.6.2. Gas chromatography (GC) Analysis The HPLC results from fraction C (Table1) were veriﬁed using gas chromatography (GC) (Model 6850, Agilent Technologies) equipped with a HP-FFAP column (25 m). In a 2 mL vial, 200 µL of the fraction and 800 µL of isopropyl alcohol were mixed together and then a 20 µL sample was injected manually into the GC. The total time through the column was 24 min. The temperature was increased at 20 ◦C/min up to 240 ◦C, and then held at 240 ◦C for the remainder of the analysis. The outputs were collected by a computer-controlled data acquisition system and the output signal was integrated manually.

3. Results and Discussion

3.1. CTO Pretreatment and TOFAs Separation CTO is extracted from the spent kraft pulping liquor (black liquor) in three processing steps: (1) black liquor is first partially concentrated to 20–30% solids w/v and allowed to settle; (2) the top layer of the partially concentrated black liquor, known as tall oil soap, is skimmed off; and (3) the tall oil soap is acidified with sulfuric acid to form CTO and remove impurities such as sodium salts, dissolved lignin, unreacted soap or acid, residual pulping chemicals, etc. [4]. However, some impurities (0.2–0.4% w/v) still remain in CTO, which affects its further processing [32,33]. For this reason, a pretreatment step with 1% w/v NaCl was applied to CTO prior to fractional distillation. Due to its salting-out effect, NaCl increases the polarity of the water molecules, which facilitates the separation of the hydrophobic dust 3 particles from CTO [32]. Due to the lower density of CTO of 700–870 kg/m at 25 ◦C[34] than water (997 kg/m3), CTO formed a top layer and was collected for distillation, whereas the aqueous bottom layer containing most of the CTO impurities was removed. The yield of the pretreated CTO from the raw CTO was 93% w/w. Although the TOFA boiling points at 10 mm Hg were all between 210 ◦C and 230 ◦C, a broader temperature range (130–265 ◦C) was used for distillation to ensure a more complete recovery of all TOFAs. As we reported previously [13], the TOFA content in CTO was 53.4% w/w. After distillation, the yield of the collected TOFA fraction was 48% w/w, which represented a 89.9% recovery of all TOFAs contained in CTO. Increasing the temperature above 265 ◦C resulted in the co-distillation of resin acids, which complicated the LA purification process and was therefore undesirable for the purpose of this investigation. Figure3 represents the GC chromatogram of the TOFA composition. Due to the high polarity of the column (HP-FFAP), the most polar ALA containing three double bonds (Figure1) was eluted last. Compared to ALA, LA has two double bonds (Figure1), which makes it less polar, hence, LA was detected at a retention time (RT) of 15.5 min. Palmitic acid has no double bonds (C16:0), and as the least polar TOFA, it eluted first. It is well known that the presence of double bonds increases the polarity of compounds [35]. In addition, alkanes have only 25% of ‘s’ character in their sp hybridization compared to 33% of the ‘s’ character of alkenes, which makes alkanes less polar than alkenes [36]. Separations 2019, 6, x FOR PEER REVIEW 6 of 11

FigureFigure 3. GC 3. GC chromatogram chromatogram of of TOFAs. TOFAs. (1)(1) PalmiticPalmitic acid (C16:0), (C16:0), (2) (2) Palmitoleic Palmitoleic acid acid (C16:1), (C16:1), (3) Oleic (3) Oleic acidacid (C18:1), (C18:1), (4) (4) Stearic Stearic acid acid (C18:0), (C18:0), (5) (5) LA LA (C18:2n6),(C18:2n6), (6) GLA GLA (C18:3n6), (C18:3n6), (7) (7) ALA ALA (C18:3n3). (C18:3n3).

Table 2 presents the composition of TOFAs as extracted from CTO. LA comprised almost half the total TOFAs with 42.6% w/w, followed by stearic and palmitic acid. These results confirm previous findings. For example, TOFAs from Farmchem Oy (Finland) contained 49% w/w LA [18]. CTO from Stora Enzo Oyj Mills (Finland) contained 19.1% w/w LA corresponding to 40% w/w LA in the TOFA fraction of CTO [37], and in another report, 27.6% of CTO [38]. Based on the high content and the high value of this ω-6 essential fatty acid, it was decided to isolate LA from the rest of the TOFAs.

Table 2. Composition of the TOFAs.

LTC Fraction LTC Temperature A 4 °C B −7 °C C −15 °C D −20 °C E −30 °C

3.2. Separation of PUFAs The PUFAs were separated from the rest of the TOFAs by UC [30]. In this method, the SFAs and MUFAs create a physical complex with urea whereas the PUFAs remain unbound. SFAs like palmitic and stearic acid and MUFAs such as palmitoleic and oleic acid form a hexagonal crystal structure with urea at 8–12 °A. Due to the presence of multiple double bonds, however, the PUFAs including LA, ALA, and GLA twist to form compacted tetragonal structures that are bulky in space, which prevents their complexation with urea. As a result, the non-complexed PUFAs remain in solution, whereas the urea-complexed SFAs and MUFAs precipitate. The urea-complexed SFAs and MUFAs were separated from the PUFAs by filtration. The filtrate containing PUFAs was collected and analyzed by HPLC. The RT of the TOFAs before and after UC complexation are shown in Table 2. Figure 4 presents the HPLC chromatograms of the TOFAs before and after UC. It can be seen from Figure 4B and Table 3 that after UC, only PUFAs were detected in the mixture, which is indicative of a successful separation. The small fluctuations in the RT may be because of variations in the operational parameters such as temperature variation of the solvent in the stationary phase, elution solvent composition, or concentration of the samples in the mixture.

Separations 2020, 7, 9 6 of 11

Table2 presents the composition of TOFAs as extracted from CTO. LA comprised almost half the total TOFAs with 42.6% w/w, followed by stearic and palmitic acid. These results conﬁrm previous ﬁndings. For example, TOFAs from Farmchem Oy (Finland) contained 49% w/w LA [18]. CTO from Stora Enzo Oyj Mills (Finland) contained 19.1% w/w LA corresponding to 40% w/w LA in the TOFA fraction of CTO [37], and in another report, 27.6% of CTO [38]. Based on the high content and the high value of this ω-6 essential fatty acid, it was decided to isolate LA from the rest of the TOFAs.

Table 2. Composition of the TOFAs.

LTC Fraction LTC Temperature

A 4 ◦C B 7 C − ◦ C 15 C − ◦ D 20 C − ◦ E 30 C − ◦

3.2. Separation of PUFAs The PUFAs were separated from the rest of the TOFAs by UC [30]. In this method, the SFAs and MUFAs create a physical complex with urea whereas the PUFAs remain unbound. SFAs like palmitic and stearic acid and MUFAs such as palmitoleic and oleic acid form a hexagonal crystal structure with urea at 8–12 ◦A. Due to the presence of multiple double bonds, however, the PUFAs including LA, ALA, and GLA twist to form compacted tetragonal structures that are bulky in space, which prevents their complexation with urea. As a result, the non-complexed PUFAs remain in solution, whereas the urea-complexed SFAs and MUFAs precipitate. The urea-complexed SFAs and MUFAs were separated from the PUFAs by filtration. The filtrate containing PUFAs was collected and analyzed by HPLC. The RT of the TOFAs before and after UC complexation are shown in Table2. Figure4 presents the HPLC chromatograms of the TOFAs before and after UC. It can be seen from Figure4B and Table3 that after UC, only PUFAs were detected in the mixture, which is indicative of a successful separation. The small fluctuations in the RT may be because of variations in the operational parameters such as temperature variation of the solvent in the stationary phase, elution solvent composition, or concentration of the samples in the mixture. Separations 2019, 6, x FOR PEER REVIEW 7 of 11

(A)

(B)

Figure 4. HPLCFigure 4. chromatogram HPLC chromatogram of theof the TOFAs. TOFAs. Before Before UC UC (A): ( A(7)): Palmitic (7) Palmitic acid; (6) acid;Palmitoleic (6) Palmitoleic acid; acid; (5) Oleic acid; (4) Stearic acid; (1) LA; (3) ALA; (2) GLA. After UC (B): (1) LA; (2) ALA; (3) GLA. (5) Oleic acid; (4) Stearic acid; (1) LA; (3) ALA; (2) GLA. After UC (B): (1) LA; (2) ALA; (3) GLA. Table 3. HPLC RT of TOFAs before and after UC.

TOFA This Work (% w/w) Literature [2,4,33] (% w/w) Palmitic acid 12.3 4–15 Palmitoleic acid 7.5 2–10 Oleic acid 4.5 1–6 Stearic acid 14.8 6–20 LA 42.6 25–53 GLA 2.95 2–10 ALA 1.75 1–3

3.3. Separation and Identification of LA Finally, the mixture of PUFAs obtained by UC was further separated using LTC. We decided to use LTC rather than fractional distillation. The reason for this decision was based on the differences in the freezing and boiling points of the three main fatty acids [39,40] present in the PUFA fraction after UC: LA, GLA, and ALA (Figure 4B). As can be seen from Table 4, the difference between the freezing points of LA (−6.5 °C) and those of GLA-ALA (−11 °C) was 4.5 °C, whereas the boiling points of the three fatty acids differed only by 1 °C. The numbers shown in Table 4 apply for the acids in their pure form, and a shift in their respective freezing points can be expected for acid mixtures [41]. However, this info provides a useful guideline and evidence for the choice of LTC as the preferred separation method for LA.

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Table 3. HPLC RT of TOFAs before and after UC.

TOFA This Work (% w/w) Literature [2,4,33] (% w/w) Palmitic acid 12.3 4–15 Palmitoleic acid 7.5 2–10 Oleic acid 4.5 1–6 Stearic acid 14.8 6–20 LA 42.6 25–53 GLA 2.95 2–10 ALA 1.75 1–3

3.3. Separation and Identification of LA Finally, the mixture of PUFAs obtained by UC was further separated using LTC. We decided to use LTC rather than fractional distillation. The reason for this decision was based on the differences in the freezing and boiling points of the three main fatty acids [39,40] present in the PUFA fraction after UC: LA, GLA, and ALA (Figure4B). As can be seen from Table4, the di fference between the freezing points of LA ( 6.5 C) and those of GLA-ALA ( 11 C) was 4.5 C, whereas the boiling points of the − ◦ − ◦ ◦ three fatty acids differed only by 1 ◦C. The numbers shown in Table4 apply for the acids in their pure form, and a shift in their respective freezing points can be expected for acid mixtures [41]. However, this info provides a useful guideline and evidence for the choice of LTC as the preferred separation method for LA.

Table 4. Boiling and freezing points of LA, GLA, and ALA.

PUFA Freezing Point (◦C) [38,39] Boiling Point (◦C) at 10 mm Hg [38] LA 6.5 224 − GLA 11 225 − ALA 11 226 −

The selection of acetone as a solvent for LTC was based on the following: (1) acetone has a low freezing temperature ( 95 ◦C); (2) TOFAs are highly soluble in acetone; and (3) due to its low 3 − density (0.784 g/cm ) and boiling temperature (56 ◦C), acetone is easy to recover and recycle. Table5 presents the LTC yield for each fraction following LTC in the speciﬁed temperature range, while Figure5 displays the HPLC chromatograms for crystallized fraction C (Figure5A) and the LA standard (Figure5B).

Table 5. Separation of PUFAs by LTC.

LTC Fraction LTC Range LTC Yield (% w/w)

A Up to 4 ◦C 10.6 B 4 C to 7 C 12.4 ◦ − ◦ C 7 C to 15 C 26 − ◦ − ◦ D 15 C to 20 C 13.8 − ◦ − ◦ E 20 C to 30 C 37.2 − ◦ − ◦

Figure6 represents the GC chromatograms of fraction C and the HPLC-grade LA standard. The purity of LA was about 95%, with some GLA and ALA present in the crystallized fraction C (Figure6A). In Figure3, the LA in the TOFA fraction (post-distillation of CTO) was identiﬁed at a RT of 15.5 min. In comparison, the GC chromatogram of fraction C (Figure6A), following UC and LTC of the PUFAs, displayed a LA peak area in the same RT region of 15–16 min, however, the LA peak had some tailing and distortion, which may be due to contamination in the GC column. During GC separation, inert, non-active compounds that are relatively non-volatile tend to accumulate in the column over time. As evident from Figure6B, the RT of a standard LA was between 18 and 19 min, Separations 2019, 6, x FOR PEER REVIEW 8 of 11

Table 4. Boiling and freezing points of LA, GLA, and ALA.

PUFA Freezing Point (°C) [38,39] Boiling Point (°C) at 10 mm Hg [38]

LA −6.5 224

GLA −11 225

ALA −11 226

The selection of acetone as a solvent for LTC was based on the following: (1) acetone has a low freezing temperature (−95 °C); (2) TOFAs are highly soluble in acetone; and (3) due to its low density (0.784 g/cm3) and boiling temperature (56 °C), acetone is easy to recover and recycle. Table 5 presents the LTC yield for each fraction following LTC in the specified temperature range, while Figure 5 displays the HPLC chromatograms for crystallized fraction C (Figure 5A) and the LA standard (Figure 5B).

Table 5. Separation of PUFAs by LTC.

LTC Fraction LTC Range LTC Yield (% w/w) Separations 2020, 7, 9 8 of 11 A Up to 4 °C 10.6 B 4 °C to −7 °C 12.4 which representsC a shift of about 2 min,−7 compared °C to −15 to°C Figure6A. The deviation 26 in the RT could be due to the presence ofD impurities such as ALA−15 °C and to GLA −20 °C (Figure 6A), which co-crystallized 13.8 with LA during LTC at 15 C. InE addition, the RT may−20 be °C aﬀ toected −30 by°C diﬀerences in the LA concentration 37.2 between the − ◦ HPLC-grade LA and the fraction C samples.

Linoleic acid Separations 2019, 6, x FOR PEER REVIEW 9 of 11 column over time. As evident from Figure 6B, the RT of a standard LA was between 18 and 19 min, which represents a shift of about 2 min, compared to Figure 6A. The deviation in the RT could be due to the presence of impurities such as ALA and GLA (Figure 6A), which co-crystallized with LA during LTC at −15 °C. In addition, the RT may be affected by differences in the LA concentration between the HPLC-grade LA and the fraction C samples. In summary, the separation of LA from CTO was based on differences in the boiling points of fatty acids and the remaining CTO components(A) (distillation), freezing points (LTC), solvent solubility, and fatty acid degree of unsaturation (UC and LTC). Fractional distillation at 130–265 °C under 10 mm Hg vacuum yielded 48% w/w of TOFAs from CTO. Next, UC separated the TOFAs into 62.5% w/w PUFAs and 37.5% w/w SFAs and MUFAs. The PUFAs were then further fractionated by LTC in the temperature range of 4 °C to −30 °C. The −7 °C to −15 °C fraction yielded 26% w/w LA from the PUFAs or 7.8% w/w from CTO. The purity of LA was 95.2% w/w, with 3.8% w/w GLA and 1.0% ALA present as contaminants. This mixture of essential fatty acids may be applied as is, or LA can be further purified using the above-described method. The approach presented here can be applied to recover other valuable fatty acids. Furthermore(B) , once the targeted fatty acid(s) are isolated, the rest of the TOFAs can be utilized for the production of biodiesel, biobased surfactants, or other valuable bioproducts.Figure 5. HPLC chromatograms of fraction C (A)) andand HPLC-gradeHPLC-grade LALA standardstandard ((BB).).

Figure 6 represents the GC chromatograms of fraction C and the HPLC-grade LA standard. The purity of LA was about 95%, with some GLA and ALA present in the crystallized fraction C (Figure 6A). In Figure 3, the LA in the TOFA fraction (post-distillation of CTO) was identified at a RT of 15.5 min. In comparison, the GC chromatogram of fraction C (Figure 6A), following UC and LTC of the PUFAs, displayed a LA peak area in the same RT region of 15–16 min, however, the LA peak had some tailing and distortion, which may be due to contamination in the GC column. During GC separation, inert, non-active compounds that are relatively non-volatile tend to accumulate in the

(A)

(B)

Figure 6. GC chromatogram of fraction C (A) and HPLC-grade LA standard ((B).).

4. ConclusionsIn summary, the separation of LA from CTO was based on diﬀerences in the boiling points of fatty acids and the remaining CTO components (distillation), freezing points (LTC), solvent solubility, and This study presents an example that demonstrates how the circular bioeconomy concept can be applied to upgrade low-value industry by-products such as CTO. The valorization of CTO at pulp and paper mills provides a route toward product and market diversification that can render the forest industry more competitive and bring about additional revenue. It also offers more flexibility that allows the industry to adequately react to product and market fluctuations by adjusting their product range. The economics of CTO-based technologies would certainly benefit from the increased number of cost-competitive CTO-derived products that become an integral part in the value-chain of the forest products ecosystem.

Author Contributions: The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Separations 2020, 7, 9 9 of 11

fatty acid degree of unsaturation (UC and LTC). Fractional distillation at 130–265 ◦C under 10 mm Hg vacuum yielded 48% w/w of TOFAs from CTO. Next, UC separated the TOFAs into 62.5% w/w PUFAs and 37.5% w/w SFAs and MUFAs. The PUFAs were then further fractionated by LTC in the temperature range of 4 C to 30 C. The 7 C to 15 C fraction yielded 26% w/w LA from the PUFAs or 7.8% ◦ − ◦ − ◦ − ◦ w/w from CTO. The purity of LA was 95.2% w/w, with 3.8% w/w GLA and 1.0% ALA present as contaminants. This mixture of essential fatty acids may be applied as is, or LA can be further puriﬁed using the above-described method. The approach presented here can be applied to recover other valuable fatty acids. Furthermore, once the targeted fatty acid(s) are isolated, the rest of the TOFAs can be utilized for the production of biodiesel, biobased surfactants, or other valuable bioproducts.

4. Conclusions This study presents an example that demonstrates how the circular bioeconomy concept can be applied to upgrade low-value industry by-products such as CTO. The valorization of CTO at pulp and paper mills provides a route toward product and market diversification that can render the forest industry more competitive and bring about additional revenue. It also offers more flexibility that allows the industry to adequately react to product and market fluctuations by adjusting their product range. The economics of CTO-based technologies would certainly benefit from the increased number of cost-competitive CTO-derived products that become an integral part in the value-chain of the forest products ecosystem.

Author Contributions: The manuscript was written through the contributions of all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Natural Science and Engineering Council of Canada (NSERC), grant number EGP 490582-15 (L.P. Christopher) and grant number RGPIN-2017-05355 (Md Nur Alam), and the article processing charge (APC) was funded by RGPIN-2017-05355. Acknowledgments: The authors wish to thank Shrikanta Sutradhar at the Biorefining Research Institute, Lakehead University, Canada, for his assistance with the GC data analysis and the manuscript revision. Conflicts of Interest: The authors declare no conflicts of interest.

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