Improvement of saponification extraction method for fatty acids separation from geological samples

Jiang Chang·Yanqing Xia·Suping Ma·Xuan Fang·Minzhuo Sun



Improvement of saponification extraction method for fatty acids separation from geological samples

Jiang Chang1，2·Yanqing Xia1·Suping Ma1·Xuan Fang1·Minzhuo Sun1

The conventional saponification method could result in lower recoveries and artificial changes of longchain fatty acids.The main reason is the error judgment of the intermediate layer suspended between the aqueous and organic layer during the liquid-liquid extraction process. This study shows that the intermediate layer consists of lots of medium-to long-chain carboxylic salts for their special physical and chemical properties.An improved saponification extraction method is also developed and the results show that the carboxylic salts distributed in the intermediate layer could be obtained completely，which greatly enhances the authenticity and accuracy of fatty acid analysis.Additionally，the possible reasons of formation of the intermediate layer are also discussed.

Fatty acid·Separation·Saponification

extraction·Carboxylic salt·Geological sample

1 Introduction

Fatty acids are key constituents of natural occurring lipids which are used as energy stores，cellular membrane components and external coatings by plants and animals. Meanwhile，as important precursors of hydrocarbon，they also attract many concerns from scientists in the field of geochemistry（Xu et al.2001；Barakat et al.2000；Wakeham 1999；Paul et al.2002）.The investigation of fatty acids in various depositional environments can provide a lot of information about the sources and diagenetic processes of organic matter（Russell et al.1997；Gong and Hollander 1997；Sessions et al.2002；Huang et al.2002；Hu et al.2003；Hughen et al.2004）.Therefore，it is particularly significant to isolate fatty acids from samples efficiently and completely.

Fatty acids can be separated by many methods，such as column chromatography，thin layer chromatography，high performance liquid chromatographic，solid layer extraction and so on.They share similar working principles，as they all utilize the different distribution capacity of materials between the solid phase and the fluent phase.But geological samples contain multiple compounds，and some complex structures will lead to greater steric hindrance to limit the elution efficiency（Zhang et al.2004）.Besides，researchers will spend lots of effort and time on each sample，or each fraction，in order to obtain optimized chromatographic conditions.

In contrast to the above methods，the saponification extraction method shows great advantages in terms of the quantity and purity of fatty acids，which is widely used in many research fields.The conventional steps are listed as follows（Tommaso et al.2014；Sitindra and Mark 2013；Pisani et al.2013；Guan et al.2013；Leefmann et al.2013）：①extraction of total lipids from samples；②saponificationof free fatty acids；③ separation of neutral and acidic fractions；④ acidification of the aqueous layer（acidic fractions）；⑤extraction of free fatty acids.

✉ Yanqing Xia yqxia@lzb.ac.cn Jiang Chang asd116@163.com

1Key Laboratory of Petroleum Resources，Gansu Province/Key Laboratory of Petroleum Resources Research，Institute of Geology and Geophysics，Chinese Academy of Sciences，Lanzhou 730000，People’s Republic of China

2University of Chinese Academy of Sciences，Beijing 100049，People’s Republic of China

Its theoretical basis is that fatty acids can react with alkali and turn into water-soluble carboxylic salts.After extraction with organic solvents（step③），all of the carboxylic salts could be isolated from other organic matter（unsaponifiable matter）.Then the pure fatty acid fraction will be obtained by subsequent treatments（step④and⑤）.

However，serious problems have been found by our many experiments，especially in step③.Actually，the carboxylic salts are amphiphilic molecules and have special physical and chemical properties，which is closer to anionic surfactants.During the liquid-liquid extraction process（step③），complex micro interfacial phenomena will occur and，in most cases，an intermediate layer between the organic and the aqueous layers will appear.On the one hand，the intermediate layer will hinder the exchange of matter between the organic and the aqueous layers，resulting in lower extraction efficiency and operational difficulties（Logsdail and Slater 1993；Liu et al.2002）.On the other hand，lots of medium-to long-chain carboxylic salts can be found in this layer with further study.Unfortunately，the intermediate layer is often regarded as unsaponifiable matter，or even an impurity，and is always discarded，leading to great loss and artificial changes of medium-to long-chain fatty acids，which seriously affects the authenticity and accuracy in the laboratory analysis of fatty acids.

In fact，early researchers had already proven that saponification extraction would result in loss of fatty acids（Van der Veen et al.1968）.But they only chose several standard materials of fatty acids and did not give the theoretical analysis that accounted for such loss.So the problem did not attract enough attention and many researchers still used this conventional method for fatty acid separation（Shinya et al.2013；Tommaso et al.2014；Robert et al.2014；Jorge et al.2014；Shailesh et al.2014）.

In this study，an improved method of saponification extraction is proposed.Some standard substances of fatty acids are chosen to prove the repeatability of the improved method，and the applicability is proven by three kinds of geological samples.By comparing the obtained fatty acids with the two methods，the advantages of the improved method is highlighted.The reasons for the formation of the intermediate layer are also explained theoretically.

2 Materials and methods

2.1 Sample and reagents

The geological samples：Huangxian lignite was collected from the Beizao mine of the Huangxian coalfield in the Shandong Province；marsh sediment was obtained from a 4 m core located in the west of the Ruoergai marsh；peat sample was from a 4.5 m core collected at a location situated 2 km southeast of the city of Hongyuan.

Standard substances：palmitic acid，eicosanoic acid，tetracosanoic acid and octacosanoic acid were purchased from the Sinopharm Group Corp.（Shanghai，China）；sodium palmitate，sodium arachidate and sodium nervonate were obtained from NU-CHEK-PREP，INC（USA）.

The reagents used in the experiments：chloroform，dichloromethane，methanol，hydrochloric acid and sodium hydroxide were all analytical pure.They were obtained from the Sinopharm Group Corp.（Shanghai，China），and underwent second distillation before use.The boron trifluoride/methanol solution and erucic acid were bought from Fisher Corp.（Pittsburg，Pennsylvania，USA）.The sodium palmitate，sodium arachidate and sodium nervonate were chromatographically pure（＞99%）and purchased from UN-CHEK-PREP，INC（Elysian，U.S.A）.The vacuum rotary evaporator was bought from Labtechgroup Corp.（Beijing，China）.Highly pure water was acquired from the Milli-Q water system（Millipore Corp.，Billerica，MA，USA）.

2.2 Analytical methods

2.2.1 Conventional saponification extraction method

All conventional method procedures are strictly in accordance with the reported literature（Tommaso et al.2014；Sitindra and Mark 2013；Pisani et al.2013；Guan et al. 2013；Leefmann et al.2013）.

2.2.1.1 Standard substances of fatty acidThe quantitative palmitic acid，eicosanoic acid，tetracosanoic acid and octacosanoic acid were saponified with 6%sodium hydroxide in methanol（w/v）for 2 h，.Then these saponified substanceswere extractedthreetimes withdichloromethane three times.The aqueous layers were acidified to pH 1-2 with aqueous HCl 15%（v/v）and extracted three timeswithdichloromethane.Afterevaporatingand weighing the solvent，we obtained the quantity of obtained fatty acids.

2.2.1.2 Geological samplesThe fresh geological samples were dried at room temperature and ground to 100 mesh. Powdered samples were extracted in a Soxhlet apparatus with chloroform for 72 h.The extracts were saponified overnight with 6%sodium hydroxide in methanol（w/v）. Both the neutral and acidic fractions were successively recovered with dichloromethane three times，respectively. During every extraction process，three layers（aqueous layer，organic layer and intermediate layer）appeared.Onlythe aqueous layer was acidified to pH＜2 with hydrochloric acid（15%，v/v），then dichloromethane was used to extract the fatty acid fraction.The separation procedures were repeated three times and the combined extracts were dried under a gentle stream of N2.A certain amount of erucic acid was added as an internal standard（IS）（no erucic acid component in these samples by our analysis）.Then the fatty acids were esterfied overnight with 14%a boron trifluoride/methanol solution prior to the GC/MS analysis and identification.

Table 1 The recoveries of standard substances of fatty acid by the conventional and improved methods

Table 2 Distribution of carboxylic salts in the aqueous and the intermediate layers

2.2.2 Improved saponification extraction method

The procedures of saponification and extraction of these fatty acids（standard substances and geological samples）were the same as Sects.2.2.1.1 and 2.2.1.2.But during the separation process，the aqueous and the intermediate layers were combined and all were acidified to pH 1-2 with HCl 15%（v/v）and extracted three times with dichloromethane. After evaporating and weighing the solvent，the quantity of obtained fatty acids was obtained.

2.2.3 Extraction experiments of standard substances of carboxylic salts

The quantitative sodium palmitate，sodium arachidate and sodium nervonate were dissolved in 6%sodium hydroxide in methanol（w/v）.Dichloromethane was added in and extracted three times.The aqueous and intermediate layers were isolated each time.After evaporating and weighing the solvent，the quantity of the carboxylic salts distributed in the two layers were obtained.

2.2.4 Gas chromatography/mass spectrometry（GC/MS）All fatty acids were analyzed by gas chromatography-mass spectrometry（GC/MS），using a HP5890 N GC interfaced to a HP5973 N MS.An HP-5 capillary column was used（length 30 m；inner diameter 0.25 mm；film thickness 0.25 μm）.Temperature programming：the temperature increases from 80 to 295°C at a rate of 4 degrees per minute，with the initial and final hold times at 1 and 30 min，respectively.Highly pure helium was used as thecarrier gas and fed at a linear velocity of 37 cm/sec.The injector operated at a constant flow rate of 1.0 mL/min. Operating parameters of the mass-selective detector：ionization energy 70 eV，ion source temperature 230°C，electron multiplier voltage 1800 V，and mass range 35-600 daltons.Identifications of individual compounds were based on the retention times of authentic standards and the comparison of their mass spectra with published MS data（Matsuda and Koyama 1977；Volkman et al.1990）.

All experiments were performed in triplicate，and Microsoft Excel software was used for data treatment.

Fig.1 Total ion current chromatogram of fatty acids obtained by the conventional method.Peak assignments are listed in Table 3

Fig.2 Total ion current chromatogram of fatty acids obtained by the improved method.Peak assignments are listed in Table 3

Fig.3 Total ion current chromatogram of fatty acids obtained by the conventional method.Peak assignments are listed in Table 3

3 Results and discussion

3.1 Standard substances of fatty acid

The recoveries of standard substances of fatty acids by conventional and improved methods are shown in Table 1. The recoveries of different carbon-chain length fatty acids were greatly improved with the new method，from 3.28%-20.41%to 69.39%-86.54%.The longer carbon-chain fatty acid is，the higher recovery gets.

In order to further prove the above point，several standard materials of carboxylic salts（sodium palmitate，sodium arachidate and sodium nervonate）were chosen to be extracted in the method mentioned in Sect.2.2.3 and the distribution results in the aqueous and the intermediate layer are shown in Table 2.It is obvious that most of the short carbon-chain carboxylic salts are distributed in the aqueous layer，and most of the long carbon-chain carboxylic salts are distributed in the intermediate layer. And the longer the carbon-chain carboxylic salt is，the more it distributes in the intermediate layer.

Fig.4 Total ion current chromatogram of fatty acids obtained by the improved method.Peak assignments are listed in Table 3

Fig.5 Total ion current chromatogram of fatty acids obtained by the conventional method.Peak assignments are listed in Table 3

Fig.6 Total ion current chromatogram of fatty acids obtained by the improved method.Peak assignments are listed in Table 3

Table 3 The results of fatty acids obtained from geological samples by the conventional and improved methods

Here are some further explanations on the reasons for the distribution of carboxylic salts in the intermediate layer.Liquid-liquid extraction of carboxylic salts relates to many factors，such as the chemical properties of solvents，extracts，organic layer，aqueous layer and so on（Szymaoowski and Cierpiszewski 1992；Szymanowski and Tondre 1994；Menon and Wasan 1988）.Firstly，as akind of salt molecule，short-chain carboxylic salts are easily ionized and dissolved in the aqueous layer completely.But long-chain carboxylic salts are weak electrolytes in their larger hydrocarbon chains，showing weak polarities and difficult ionization.Secondly，the physical and chemical properties of long-chain carboxylic salts are similar to the anionic surfactants，which are amphiphilic moleculeswithlipophilictails（carbonchain）and hydrophilic heads（carboxylate ion）（Rosen 1978）.During the liquid-liquid extraction process，these medium-to long-chain carboxylic salts will form the intermediate layer with the hydrophilic heads facing the aqueous layer and the hydrophobic tails inserting the organic layer. Thus the intermediate layer is actually composed of longchain carboxylic salts.

The repeatability of the improved method has been proven in Table 1.Then three kinds of geological sample were chosen to prove the applicability of this method.

3.2 Geological samples

From a qualitative point of view，in the Huangxian lignite（Figs.1，2），C14-C28 monocarboxylic acids and C15-C26 dicarboxylic acids were obtained and exhibited bimodal distribution with the main peaks at C16（monocarboxylic acid）and C22（dicarboxylic acid）with the conventional saponification extraction method.Whe using the improved method，C14-C31 monocarboxylic acids and C15-C28 dicarboxylic acids were isolated.Monocarboxylic acids exhibit bimodal distribution with the main peaks at C16 and C26，dicarboxylic acids exhibit unimodal distribution with the main peaks at C22.It is obvious that the carbon number range expanded and the peak distribution changed fundamentally，especially in the monocarboxylic acids. The distribution of monocarboxylic acids varies from unimodal to bimodal peak and the main peak is from C16 to C16 and C26.

With the conventional method，in the peat sediment（Figs.3，4），the C14-C30 monocarboxylic acids and C15-C23 dicarboxylic acids were obtained，and exhibited bimodal distribution，with the main peaks at C24（monocarboxylic acid）.By using the improved method，the C14-C31 monocarboxylic acids and C17-C25 dicarboxylic acids are isolated.Monocarboxylic acids exhibit bimodal distribution with the main peaks at C16 and C24，dicarboxylic acids exhibit unimodal distribution with the main peaks at C21.The carbon number range is expanded and the peak distribution is changed fundamentally.

The distribution of monocarboxylic acids is from unimodal to bimodal peaks and the main peak is from C16 to C16 and C24.

In the marsh sediment（Figs.5，6），the main difference is the peak distribution.The distribution of monocarboxylic acids is from unimodal to bimodal peaks and the main peak is from C16 to C24.

4 Conclusions

The conventional saponification extraction of fatty acids only focuses on the aqueous layer，leading to great loss and artificial changes of the series of fatty acids.The main reason is the error judgment for the intermediate layer during the liquid-liquid extraction process.In fact，the intermediate layer is gathered by plenty of medium-to long-chain carboxylic salts.In this study，an improved saponification extraction method is proposed and the results show that the fatty acids in the intermediate layer could be obtained completely，which greatly reduces the loss of fatty acids，especially in medium-to long-chain fatty acids.The improved method enhances the accuracy and authenticity of fatty acids separation in laboratory analysis，and also provides new ideas or approaches to reduce the formation of the intermediate layer in the liquid-liquid extraction procedure.

AcknowledgmentsThis work supports by the National Natural Science Foundation of China（No.41003021）and the Key Laboratory Project of Gansu Province（Grant No.1309RTSA041）.

Barakat AO，El-Gayar MS，Mostafa AR（2000）Geochemical significance of fatty acids in crude oils and related source rocks from Egypt.Pet Sci Technol 18：635-655

Gong C，Hollander DJ（1997）Differential contribution of bacteria to sedimentary organic matter in oxic and anoxic environments，Santa Monica Basin，California.Org Geochem 26：545-563

Guan HX，Sun YG，Zhu XW，Mao SY，Feng D，Wu NY，Chen DF（2013）Factors controlling the types of microbial consortia in cold-seep environments：a molecular and isotopic investigation of authigenic carbonates from the South China Sea.Chem Geol 354：55-64

Hu FS，Kaufman D，Yoneji S，Nelson D，Shemesh A，Huang Y，Tian J，Bond G，Clegg B，Brown T（2003）Cyclic variation and solarforcing of holocene climate in the Alaskan Subarctic.Science 301：1890-1893

Huang Y，Shuman B，Wang Y，Webb T（2002）Hydrogen isotope ratios of palmitic acid in lacustrine sediments record latequaternary climate variations.Geology 30：1103-1106

Hughen KA，Eglinton TI，Xu L，Makou M（2004）Abrupt tropical egetationresponsetorapidclimatechanges.Science 304：1955-1959

Jorge ES，Montserrat F，Stefanie J（2014）Molecular and isotopic characterization of lipids staining bone and antler tools in the Late Neolithic settlement，Zurich Opera Parking，Switzerland. Org Geochem 69：11-25

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Leefmann T，Heim C，Kryvenda A，Siljestrom S，Sjovall P，Thiel V（2013）Biomarker imaging of single diatom cells in a microbial mat using time-of-flight secondary ion mass spectrometry（ToFSIMS）.Org Geochem 57：23-33

Liu JS，Lan ZY，Qiu GZ（2002）Mechanism of crud format ion in copper solvent extraction.J Cent South Univ Technol 9：169-172

Logsdail DH，Slater MJ（1993）Solvent extraction in the process industries：Isec 93.Elsevier Applied Science，New York，pp 25-35

Matsuda H，Koyama T（1977）Early diagenesis of fatty acids in lacustrine sediments-I.identifcation and distribution of fatty acids in recent sediment from a freshwater lake.Geochim Cosmochim Acta 41：777-783

Menon VB，Wasan DT（1988）A review of the factors affecting the stabilityofsolids-stabilizedemulsions.SepSciTechnol 23：2131-2142

Paul F，Tony G，Efthymios E（2002）Hopanoic acids in esozoic sedimentary rocks：their origin and relationship with opanes.Org Geochem 33：965-977

Pisani O，Hills KM，Courtier MD，Simpson AJ，Mellor NJ，Paul EA，Morris SJ，Simpson MJ（2013）Molecular level analysis of long term vegetative shifts and relationships to soil organic matter composition.Org Geochem 62：7-16

Robert WH，Christopher DKH，Danielle NS（2014）Soluble organic compounds in the Tagish Lake meteorite.Meteorit Planet Sci 49：526-549

Rosen MJ（1978）Surfactants and interfacial phenomena.Wiley，New York，pp 615-616

Russell M，Grimalt JO，Hartgers WA，Taberner C，Rouchy JM（1997）Bacterial and algal markers in sedimentary organic matter deposited under natural sulfurization conditions（Lorca Basin，Murcia，Spain）.Org Geochem 26：605-625

Sessions AL，Jahnke LL，Schimmelmann A（2002）Hydrogen isotope fractionation in lipids of the methane oxidizing bacterium Methylococcuscapsulatus.GeochimCosmochimActa 66：3955-3969

Shailesh A，Valier G，Prasanta S（2014）C4 plant expansion in the Ganga Plain during the last glacial cycle：insights from isotopic composition of vascular plant biomarkers.Organ Geochem 67：58-71

Shinya Y，Kimitaka K，Osamu S，Tadashi K，Meehye L（2013）Influence of aerosol source regions and transport pathway on δD of terrestrial biomarkers in atmospheric aerosols from the East China Sea.Geochim Cosmochim Acta 106：164-176

Sitindra SD，Mark P（2013）Hydrogen isotope fractionation during lipid biosynthesis by Tetrahymena.Org Geochem 64：105-111

Szymanowski J，Tondre C（1994）Kinetic and interfacial phenomena in classical and micellar extraction systems.Solvent Extr Ion Exch 12：873-905

Szymaoowski J，Cierpiszewski R（1992）Kinetic model for interfacial process of copper extract ion with 2-hydroxy-5-alkylbenzaldehyde oximes.Solvent Extr Ion Exch 10：663-683

Tommaso T，Igor S，Gustaf H，Oleg D，Peter K，Orjan G（2014）Composition and fate of terrigenous organic matter along the Arctic land-ocean continuum in East Siberia：Insights from biomarkers and carbon isotopes.Geochim Cosmochim Acta 133：235-256

Van der Veen J，Medwadowski BF，Olcott HS（1968）Losses of fatty acids during the saponification extraction of small samples. Lipids 3：189-190

Volkman JK，Kearney P，Jerey SW（1990）A new source of 4-methyl sterols and 5a（H）-stanols in sediments：prymnesiophyte microalgae of the genus Pavlova.Org Geochem 15：489-497

Wakeham SG （1999）Monocarboxylic，dicarboxylic and hydroxy acids released by sequential treatments of suspended particles and sediments of the Black Sea.Org Geochem 30：1059-1074

Xu YC，Shen P，Liu WH（2001）Isotopic composition characteristics and identification of immature and low-mature oils.Chin Sci Bull 46：1923-1929

Zhang TM，Liang YZ，Cui H（2004）Universal method for the fraction separation and the gain of coupling information of chromatography and mass-spectrum in systematic analyses of structures and contents of non-hydrocarbon compounds in crude oils by combining with chemometrics.Chin J Anal Chem 32：1450-1454

27 October 2014/Revised：14 December 2015/Accepted：25 January 2016/Published online：23 March 2016ⒸScience Press，Institute of Geochemistry，CAS and Springer-Verlag Berlin Heidelberg 2016