Journal of Analytical and Applied Pyrolysis 113 (2015) 288–295

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Journal of Analytical and Applied Pyrolysis

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Determination of glyceride and free fatty acid residuals in biodiesel bythin layer chromatography combined with on-line pyrolyticmethylation gas chromatography�

Zhongping Huanga, Peipei Zhanga, Yang Sunb, Yilei Huanga, Zaifa Pana, Lili Wanga,∗

a College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR Chinab Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PRChina

a r t i c l e i n f o

Article history:Received 4 December 2014Received in revised form 30 January 2015Accepted 30 January 2015Available online 2 February 2015

Keywords:On-line pyrolytic methylation gaschromatographyThin layer chromatographyBiodieselGlycerideFree fatty acid

a b s t r a c t

A novel method on basis of thin layer chromatography (TLC) combined with on-line pyrolyticmethylation gas chromatography (OPM–GC) in the presence of trimethylsulfonium hydroxide(TMSH) was developed and applied to the determination of glyceride and free fatty acidresiduals in biodiesel. Both glycerides and free fatty acids in biodiesel were separated by TLC aluminasheets, and then the cut spots were analyzed directly by pyrolytic methylation gas chromatographymethod in a pyrolyzer at 350 ◦C without complex pretreatment. In this process, glycerides and free fattyacids were converted into their corresponding fatty acid methyl esters, and quantified well by the totalpeak areas of formed esters. Thus obtained calibration curves of monostearin, distearin, tristearin andoleic acid exhibited good linearity with the regression coefficients from 0.9766 to 0.9950 at the concen-trations ranged from 300 to 5000 mg L−1. The limits of detection were in the range of 100–200 mg L−1,and the RSDs (n = 3) ranged from 3.7% to 12.5%, calculated for monostearin, distearin, tristearin and oleicacid at the concentration level of 2000 mg L−1. The potential of the proposed method was assessed bythe determination of glycerides and free fatty acids in real biodiesel samples. The results prove that thisTLC–OPM–GC technique is a simple, accurate and low solvent consuming method for the simultaneousdetermination of glyceride and free fatty acid residuals in biodiesel.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Biodiesel is an alternative fuel for diesel and petroleum cur-rently for its renewable, biodegradable and nontoxic properties[1,2], especially in European countries and the United States ofAmerica. It is mainly produced from vegetable oil and animal fatvia a transesterification reaction with alcohols, usually methanol,to form fatty acid methyl esters (FAME) [3,4]. However, the resid-ual by-products such as monoglycerides (MG), diglygerides (DG),triglycerides (TG) and free fatty acids (FFA) can lead to severe prob-lems for the engine, such as engine deposits, filter clogging, fueldeterioration and engine failures [5]. Therefore, it is necessary todevelop a method to determine glyceride and free fatty acid resid-

Abbreviations: TG, triglycerides; FFA, free fatty acids; DG, diglycerides;MG, monoglycerides.� Selected Paper from Pyrolysis 2014, Birmingham, U.K. 19–23 May 2014.∗ Corresponding author. Tel.: +86 571 88320797; fax: +86 571 88320797.

E-mail address: lili wang@zjut.edu.cn (L. Wang).

uals in biodiesel for the quality control of biodiesel in productionprocess.

Chromatographic methods such as TLC, HPLC, and GC had beenreported for the analysis of glycerides and free fatty acids inbiodiesel samples [6–11]. TLC–FID was a well-established methodfor the determination of these residuals, but showed low accuracyfor quantification [6]. HPLC–ELSD was available for the separationof glycerides and free acids, but the poor separation of the isomersof glycerides and the relatively narrow linear range as well as lowsensitivity restricted the application of the method [7]. High tem-perature gas chromatography with cold on-column injection wasfeasible for the analysis of free fatty acids and glycerides with highboiling points [8]. But high temperature resulted in high consump-tion of instrument as well as baseline drift that might negativelyaffect the GC determination accuracy.

Recently, thermally assisted hydrolysis methylation-gas chro-matography (THM-GC) had been widely applied to the analysis offatty acids and their esters in food, insects, oils and so on [12], usingdifferent pyrolysis devices [13–16] and methyl donors [17–19]. Reiset al. [15] applied the method to the fatty acid profile analysis of

http://dx.doi.org/10.1016/j.jaap.2015.01.0280165-2370/© 2015 Elsevier B.V. All rights reserved.

Z. Huang et al. / Journal of Analytical and Applied Pyrolysis 113 (2015) 288–295 289

Fig. 1. Schematic diagram of the novel TLC–OPM–GC system.

Fig. 2. Typical separation profile obtained by TLC for standard solutions. Abbrevia-tions: MES = methyl stearate (FAME); OA = oleic acid (FFA); MS = monostearin (MG);DS = distearin (DG); TS = tristearin (TG).

milk based on thermal desorption integrated with a cold trap inletin the presence of trimethylsulfonium hydroxide (TMSH). Ishidaet al. [17] described the discriminative analysis of zooplankton indi-viduals by THM–GC based on pyrolysis microfurnace using tetram-ethylammonium hydroxide (TMAH). Fabbri et al. [16] profiled thefatty acids in vegetable oils by means of a resistively heated filamentpyrolyser with dimethyl carbonate (DMC) and titanium silicate.

Hudson et al. [20] explored a TLC (chromarod)–THM–GC–MStechnique for marine lipid analysis. A drawback of this tech-nique was the destruction of the chromarod into sections (1–2 cm)in order to isolate individual lipid bands for desorption in thepyrolyzer, while the rod was normally reused up to 100 timesbefore disposal [21]. Estevez and Helleur [21] improved theTLC–THM–GC–MS method by introducing an intact chromarod

(15 cm) into a pyrolyzer without clipping. However, the reuse ofchromarod, after treated by organic bases in THM procedure, wasstill not feasible.

Compared with rod, TLC plate is cheaper and more conve-nient for simultaneous separation of multiple complex samples.In our previous works, Wang et al. [22] developed a method forthe determination of residual glycerides in biodiesel on a basis ofTLC–THM–GC technique. A drawback of this method was that a sol-vent extraction process was necessary to extract glycerides fromthe separated spots which were scrapped from the glass sheetspreviously. The whole solvent extraction process was complex andtime-consuming, involving ultrasonic, centrifugation, evaporationto dryness and redissolution. Furthermore, the free fatty acids inbiodiesel could not be separated and detected in the previousmethod with a TLC developing solvent of toluene/acetone(92/8,V/V), which actually have a negative influence on the quality ofbiodiesel. Therefore, a simple, economic and low solvent consum-ing technique should be taken into account in further study for thesimultaneous analysis of glycerides and free fatty acids in biodiesel.

In this work, for the first time, a novel thin layer chromatographycombined with on-line pyrolytic methylation-gas chromatogra-phy (TLC–OPM–GC) were developed for the determination of bothresidual glycerides and free fatty acids in biodiesel samples. A cheapand tailorable aluminium support silica-gel TLC with a new devel-oping solvent was carefully selected for pre-separation of MG, DG,TG and FFA from FAME. Then, the aluminium sheets, on which thetarget spots were cohered, were cut into individual small pieces,and directly put into a small cup in the presence of organic alkalireagent for pyrolytic methylation reaction, followed by GC analy-sis in a Py–GC system (see in Fig. 1). A validation of the methodwas also carried out. The proposed method was assessed by thedetermination of glycerides and free fatty acids in five real biodieselsamples.

2. Experimental

2.1. Chemical and materials

All reagents used were of analytical-reagent grade. Monos-tearin, distearin, tristearin, methyl stearate and oleic acid standardsas well as a methanol solution of trimethylsulfonium hydroxide(TMSH, 0.2 mol L−1) and tetramethylammonium hydroxide (TMAH,25 wt.%) were purchased from J&K Company (Shanghai, China).Stearic acid standards were purchased from Aladdin Company

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Fig. 3. Typical chromatograms obtained by OPM–GC for a soybean oil sample in the presence of a methanol solution of (a) TMAH (2 wt. %) and (b) TMSH (0.2 mol L−1,equivalent to 1.8 wt.%).

(Shanghai, China). Soybean oil was purchased from supermar-ket (Hangzhou, China). Ethyl acetate, n-hexane, formic acid wereobtained from Sinopharm Company (Shanghai, China). TLC silicagel aluminum sheets 20 cm × 20 cm were purchased from MerckCompany (Germany). Biodiesel samples were supplied by Labora-tory of Biodiesel Preparation, Zhejiang University of Technology.Sample 1 and 4 were both derived from waste oil, sample 3and 5 were derived from palm oil and soybean oil, respectively.Sample 2 was obtained from the market without raw materialinformation.

2.2. Sample pretreatment

Working standard solutions were freshly prepared by dilut-ing monostearin, distearin, tristearin and oleic acid with ethylacetate to the concentrations of 300, 1000, 2000, 3000, 4000 and5000 mg L−1, and then stored at 4 ◦C in a refrigerator. Furthermore,all the biodiesel samples were diluted 2 to 6 times with ethyl acetatebefore analysis, in order to make the concentrations of residualcomponents (glycerides and free fatty acids) in those biodiesel sam-ples within the linear range. TMAH methanol solutions (2 wt %)were also prepared by diluting with methanol.

2.3. TLC–OPM–GC (/MS) procedure

As shown in Fig. 1, the procedure of thin layer chromatographycombined with on-line pyrolytic methylation-gas chromatography(/mass spectrometry) (TLC–OPM–GC (/MS)) was as follows:

TLC separation: 1 �L biodiesel samples were spotted onto TLCplates (about 10 cm long) at a distance of 1.8 to 2.5 cm from each

other and 1 to 2 cm from the bottom using a blunt-tipped syringe.The development was carried out in n-hexane/ethyl acetate/formicacid (90:10:2) until the solvent front reached the top of the plate.After treatment with iodine vapors as chromogenic reagent, theplaces containing FAME, MG, DG, TG and FFA appeared as brown-ish spots on TLC plates. Then, the colored spots were cut intothe size of 0.5 cm × 1.0 cm in elliptic type for the on-line pyrolyticmethylation-gas chromatography (OPM–GC) analysis.

OPM–GC (/MS) analysis: A vertical microfurnace pyrolyzer[Frontier Lab (Koriyama, Japan), PY 2020iD] was directly attachedto a gas chromatograph [Varian (Avondale, PA, USA), CP-3800]equipped with a flame ionization detector (FID). The colored spots(cut from the TLC plates) and 3 �L of the organic alkali (TMAHor TMSH) were added to a small cup. The sample cup was firstmounted on the waiting position of the pyrolyzer near room tem-perature, and then dropped into the heated center of the pyrolyzermaintained at 350 ◦C under the flow of nitrogen carrier gas. The30 mL min−1 carrier gas flow rate at the pyrolyzer was reducedto 1 mL min−1 at the capillary column by means of a splitter. Thecolumn temperature was increased from an initial temperatureof 50–230 ◦C at 10 ◦C min−1, maintained at this temperature for10 min. The injector and detector temperatures were kept at 250 ◦C.A DB-23 column (30 m × 0.25 mm i.d. × 0.25 �m film thickness)coated with 50% cyanopropyl – 50% dimethylpolysiloxane was usedfor the separation.

Identification of the peaks was carried out by the use of GC–MS[Thermo Finnigan (Austin, TX, USA), trace DSQ]. For the MS mea-surement, ionization was executed by electron ionization (EI) at70 eV. Samples were analyzed by full scan MS from 40 to 500 amuat 3.6 scan s−1. Identification of methyl derivatives of fatty acids

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Fig. 4. Typical chromatograms obtained by OPM–GC in the presence of TMSH with (a) blank aluminum support silica-gel plate, (b) tristearin (2000 mg L−1) in aluminumsupport silica-gel TLC plates.

Table 1Calibration parameters (n = 3) for glycerides and free fatty acids in standard solutions.

No. Compound Linear range(mg L−1)

Correlation coefficient (r2) Linear equation LOD(mg L−1)

1 Monostearin 300 – 5000 0.9766 y = 3.55x–1915.2 2002 Distearin 300 – 5000 0.9882 y = 6.04x + 2249.3 1003 Tristearin 300 – 5000 0.9901 y = 4.55x + 3019.4 1254 Oleic acid 300 – 5000 0.9950 y = 5.08x–1437.4 175

was carried out through comparison with spectra from the NISTmass spectral library (NIST02) and their retention times.

2.4. Quantitative method

The quantitative calculation was carried out by external stan-dard and area normalization method. With the external standardmethod, the concentrations of monoglycerides were calculatedby the total peak areas of corresponding peaks, according tothe calibration function obtained for monostearin. Diglycerides,triglycerides and free fatty acids were quantified analogouslyaccording to distearin, tristearin and oleic acid, respectively. Thedistributions of fatty acid compositions in each glycerides and freefatty acids were quantified by area normalization method.

3. Results and discussion

3.1. Optimization of TLC–OPM–GC conditions

A simultaneous determination of residual glycerides and freefatty acids in biodiesel samples was investigated on a basis

of TLC–OPM–GC technique. Several parameters related to theTLC–OPM–GC process (i.e., TLC developing solvent type, organicalkali type and pyrolytic methylation temperature) were examinedand optimized.

3.1.1. Selection of TLC developing solventIt is well known that the separation of MG, DG, TG, and FFA

from FAME on TLC silica gel aluminum sheets is not easy becauseof their similar characteristics or structures. The key to solve theproblem was to choose a suitable developing solvent. Thus, sev-eral kinds of developing solvent such as petroleum ether/diethylether/acetic acid, n-hexane/diethyl ether, petroleum ether/ethylacetate, toluene/chloroform/acetone, n-hexane/ethyl acetate werecarried out in this study. The results showed that n-hexane/ethylacetate had a better performance for the separation of glyceridesand FFA. Furthermore, the addition of a little amount of acid(formic acid) could contribute to the separation of TG and FAME.Fig. 2 shows the typical separation profile for standard solutionsobtained by TLC with the developing solvent of n-hexane/ethylacetate/formic acid (90:10:2). As shown in Fig. 2, the components ofmethyl stearate, oleic acid, monostearin, distearin, tristearin had a

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Fig. 5. Typical chromatograms obtained by TLC–OPM–GC for tristearin (2000 mg L−1) at the temperatures of (a) 250 ◦C, (b) 350 ◦C, (c) 450 ◦C and (d) 550 ◦C.

complete separation, and their retention factor (Rf) were 0.92, 0.33,0.01, 0.19 and 0.73, respectively. Here, the Rf of glycerides and freefatty acids in real samples may have a small deviation because ofthe presence of unsaturated bonds and isomers.

3.1.2. Selection of organic alkali reagentThe most commonly used organic alkali reagent is tetram-

ethylammonium hydroxide (TMAH, pKb 4.2), which is a relativelystrong organic base [23]. However, the high alkaline nature ofTMAH would bring about the isomerization or degradation ofpolyunsaturated fatty acids (PUFA) [13,24], a large amount of whichcan be found in biodiesel samples.

In order to decrease the isomerization of PUFA in on-linepyrolytic methylation procedure, two kinds of derivatizationreagents, TMSH and TMAH, were examined for a soybean oil sam-ple, which contains large proportions of PUFA components, suchas linoleic acid (C18:2) and linolenic acid (C18:3). Fig. 3 shows thetypical OPM-GC chromatograms of the soybean oil sample with (a)a methanol solution of TMAH (2 wt %) and (b) a methanol solutionof TMSH (0.2 mol L−1, equivalent to 1.8 wt %). During the retentiontime from 15 to 17.5 min, five peaks were identified as methylderivatives of palmitic acid (C16:0), stearic acid (C18:0), oleic acid(C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) formed fromthe methylation of carboxyl groups by means of pyrolytic methy-lation reaction. As anticipated, many additional isomer peaks ofC18:2 and C18:3 derivatives were clearly observed after 17.5 minin Fig. 3a (TMAH) while appeared very weakly in Fig. 3b (TMSH).This result suggested that thermal isomerization or degradation of

unsaturated fatty acids (C18:2 and C18:3) could be significantlydecreased in pyrolytic methylation reaction with TMSH as reagent.Therefore, TMSH at a concentration of 0.2 mol L−1 in methanol solu-tion was selected for further investigation.

3.1.3. Effect of aluminum support silica-gel TLC platesAluminum support silica-gel TLC plate was selected in this study

for its flexible and tailorable properties. An experiment was per-formed to investigate whether the silica-gel or aluminum wouldaffect the observation of the target analytes.

Fig. 4 shows the typical chromatograms obtained by OPM-GCin the presence of TMSH with (a) blank aluminum support silica-gel TLC plates, (b) tristearin (2000 mg L−1) in aluminum supportsilica-gel TLC plates. As shown in Fig. 4, only reagent-related peaks,dimethylsulfide and methanol from TMSH, were observed at theretention time of 2–10 min. The peak of methyl derivative of stearicacid (C18:0) was observed at the retention time of 16.5 min with-out any interference peaks. Therefore, it was clear that the glycerideand free fatty acid residuals in biodiesel could be determined suc-cessfully without obvious disturbance by this novel TLC–OPM–GCmethod based on aluminum support silica-gel TLC plates.

3.1.4. Effect of pyrolytic methylation temperatureThe pyrolyzer at high temperature served two purposes in

this study: (i) to desorb the analytes (acids and glycerides) fromsilica-gel TLC plates; (ii) to assist the methyl derivatization ofthe analytes with TMSH. Thus, various pyrolytic methylationtemperatures between 250 ◦C and 550 ◦C were examined for

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determining the optimum pyrolytic methylation temperatureempirically. Fig. 5 shows the typical chromatograms for tristearin(2000 mg L−1) obtained by TLC–OPM–GC at temperatures of (a)250 ◦C, (b) 350 ◦C, (c) 450 ◦C and (d) 550 ◦C. As shown in this fig-ure, the peak areas of methyl derivative of stearic acid increasedaccordingly with the increasing of pyrolytic methylation tempera-ture, indicating that high temperature was beneficial to desorb andassist the methyl derivatization of the analytes. However, a series ofsilica-support related peaks could be observed at the retention timefrom 10 to 20 min in Fig. 5c and d when the temperature reachedto 450 ◦C or higher. These small peaks would seriously disturb theidentification of the analytes in biodiesel samples. Therefore, thefollowing determination was carried out at 350 ◦C, though the peakarea of methyl derivative of stearic acid obtained at 350 ◦C wasabout 72% compared to that obtained at 450 ◦C.

3.2. Validation of the method

To evaluate the practical applicability of this proposed method,the linear range, the relative standard deviations (RSDs), andthe limits of detection (LODs) of the TLC–OPM–GC method wereinvestigated using glyceride and free fatty acid standards. Theperformance of this TLC–OPM–GC method under the obtained opti-mum conditions is shown in Table 1. The linearity of the methodwas satisfactory over a concentration range of 300–5000 mg L−1

with correlation coefficients from 0.9766 to 0.9950 for monos-tearin, distearin, tristearin and oleic acid. The RSDs of the peakareas varied from 3.7% to 12.5%, which were calculated from threesuccessive analyses of standard solutions at 2000 mg L−1 concen-tration level for all standards. The detection limits based on thesignal-noise ratio of 3 (S/N = 3) were 200, 100, 125 and 175 mg L−1

for monostearin, distearin, tristearin and oleic acid, which weresignificantly below the limitation of European Union Standards(1740–7010 mg L−1). The results indicated that the TLC–OPM–GCmethod was suitable for the determination of free fatty acid andglyceride residuals in biodiesel.

3.3. Analysis of real biodiesel samples

Five biodiesel samples derived from different feedstock (i.e.,palm oil, waste oil and soybean oil) were analyzed by theTLC–OPM–GC method under the optimized experimental condi-tions (see Chapter 2.3). MG, DG, FFA, TG and FAME in real sampleswere separated well by TLC (see an example in Fig. 6), where thediglycerides appeared as two spots, regarded as structural isomersconsisting of 1, 2-DG and 1, 3-DG [25,26]. As shown in Fig. 6, allresidual component spots including MG, DG, FFA, and TG wereobserved in sample 1. MG, DG and FFA spots were observed inother samples, but only FFA spot in sample 2. Fig. 7 shows the typi-cal chromatograms obtained by OPM-GC for the glyceride and freefatty acid spots in samples 1 (see No. 1 in Fig. 6) derived from wasteoil 1. As shown in Fig. 7, four kinds of fatty acids including C16:0,C18:0, C18:1, C18:2 were all detected in the glycerides and freefatty acids spots of this sample.

With the external standard and area normalization method,the concentrations of glyceride and free fatty acid componentsas well as their distributions of fatty acid compositions in fivebiodiesel samples were determined, and the results are given inTable 2. The contents of glycerides or free fatty acids detected werealmost above the maximum permitted concentrations in EuropeanUnion (EU) (EN 14214:2008(E)) [27], except monoglyceride in sam-ple 3 and those residuals in sample 5. In addition, FFA were alldetected with relatively high contents in given biodiesel samples,while TG were only observed in sample 1 which was made inlaboratory without any purification. Furthermore, the biodiesel

Fig. 6. Typical TLC profile for five biodiesel samples with proper dilution. Sample1: derived from waste oil 1, diluted by 6-fold; sample 2: material-unknown, dilutedby 3-fold; sample 3: derived from palm oil, diluted by 3-fold; sample 4: derivedfrom waste oil 2, diluted by 3-fold; sample 5: derived from soybean oil, diluted by2-fold.

sample derived from waste oil 1 shows high contents of unsatu-rated fatty acids, while the samples derived from palm oil showshigh contents of C16:0. The distributions of fatty acid composi-tions of residual glycerides and free fatty acids vary from biodieselsamples, probably affected by the preparation processes and rawmaterials together.

Recovery studies were carried out for a real biodiesel sam-ple (sample 4) in which the concentrations of monoglycerides,diglycerides, triglycerides and free fatty acids were 7019 mg L−1,2055 mg L−1, 0 mg L−1, 7695 mg L−1, respectively. The diluted sam-ple was fortified monostearin, distearin, tristearin and stearic acidat 2380 mg L−1, 1230 mg L−1, and 2240 mg L−1, 2460 mg L−1. Therecoveries were calculated by subtracting the results for the non-spiked samples from those of the spiked sample, according tothe calibration curve in Chapter 3.2. The results showed that therecoveries for monoglycerides, diglycerides, triglycerides and freefatty acids were 96.8%, 123.7%, 87.5% and 95.1%, respectively,and the RSDs (n = 3) of the recoveries were from 7.9% to 12.2%,illustrating the practical effectiveness of the method. Here, theRSDs were relatively larger than typical GC analysis because themethod was a combination of several techniques (TLC, THM andGC) [21].

294 Z. Huang et al. / Journal of Analytical and Applied Pyrolysis 113 (2015) 288–295

Fig. 7. Typical chromatograms obtained by TLC–OPM–GC for glycerides and free fatty acids spots in sample 1 derived from waste oil 1.

Table 2Total contents of residuals (MG, DG, TG and FFA) and their distributions of fatty acid compositions for five biodiesel samples.

Sample Raw material Residual Fatty acid compositiona(%) Total contentC16:0 C18:0 C18:1 C18:2 (mg L−1)

Sample 1 Waste oil 1 Monoglycerideb 21.4 6.3 33.3 39.0 10161Diglyceridec 28.6 5.3 25.7 40.4 12293Triglycerided 44.5 9.3 34.6 11.6 6774Free Fatty acide 40.0 6.4 33.6 20.0 28409

Sample 2 – Monoglyceride – – – – –Diglyceride – – – – –Triglyceride – – – – –Free Fatty acid 68.1 25.2 6.7 8360

Sample 3 Palm oil Monoglyceride 69.4 12.4 11.5 6.7 4974Diglyceride 74.8 14.0 8.0 3.2 2428Triglyceride – – – – –Free Fatty acid 81.1 13.6 5.3 – 4864

Sample 4 Waste oil 2 Monoglyceride 66.8 13.2 10.9 9.1 7019Diglyceride 59.5 15.8 16.0 8.7 2055Triglyceride – – – – –Free Fatty acid 62.5 13.6 15.8 8.1 7695

Sample 5 Soybean oil Monoglyceride 51.8 13.3 14.4 20.5 1624Diglyceride 40.5 16.6 9.8 33.1 443Triglyceride – – – – –Free Fatty acid 55.7 22.3 15.8 6.2 1969

a Peak areas relative to the total peak areas.b The EU standard establishes the limit of 0.8% (m/m) for monoglycerides, which is converted into concentration of 7010 mg L−1based on the density of biodiesel

(0.86–0.90 g mL−1, 0.87 g mL−1 was used).c The EU standard establishes the limit of 0.2% (m/m) for diglycerides, which is converted into concentration of 1740 mg L−1 like monoglycerides.d The EU standard establishes the limit of 0.2% (m/m) for triglycerides, which is converted into concentration of 1740 mg L−1 like monoglycerides.e The EU standard establishes the limit of 0.5 mg KOH/g for free fatty acids, which is converted into concentration of 2194 mg L−1, calculated by oleic acid.

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4. Conclusions

A novel approach for the determination of glyceride and freefatty acid residuals in biodiesel by thin layer chromatography com-bined with on-line pyrolytic methylation gas chromatogrphy usinga pyrolysis-GC system has been described. The condition optimiza-tion and validation of the method were carried out. The techniquewas proved to be simple, solvent saving and environmentallyfriendly since it avoided off-line derivatization and solvent extrac-tion. It can be applied in the quantitative determination of bothresidual glycerides and free fatty acids in biodiesel, and also thedistributions of fatty acid composition in these compounds. Theproposed method is expected to have potential applications in thequality control of biodiesel.

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