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    • 专利摘要:
    Procedure for the isolation of the unsaponifiable fraction of oils or fats by supported liquid extraction. The object of the invention is a procedure for the isolation of the unsaponifiable fraction of edible oils or fats by means of a supported liquid extraction column (ESL), and the subsequent purification of the different fractions by means of high performance liquid chromatography and their analysis by gas chromatography. The main advantage is that it allows the use of small amounts of sample and the rapid extraction of the unsaponilicable fraction using a much smaller volume of solvent than the techniques described up to now. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2772803A1
    申请号:ES201831256
    申请日:2018-12-20
    公开日:2020-07-08
    发明作者:CAMINO Mª CARMEN PEREZ；Camacho Manuel Leon
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0002] PROCEDURE FOR THE ISOLATION OF THE INSAPONIFICABLE FRACTION OF
    [0004] SECTOR OF THE TECHNIQUE AND OBJECT OF THE INVENTION
    [0006] The present invention is framed within the field of instrumental analytical chemistry and in particular in the preparation of columns used for the separation of compounds, specifically for the isolation of the unsaponifiable fraction of edible oils or fats by supported liquid extraction.
    [0008] The object of the invention is a procedure for the isolation of the unsaponifiable fraction of oils or fats by means of a supported liquid extraction column (ESL), and the subsequent purification of the different fractions by means of high efficiency liquid chromatography (HPLC, for its acronym in English) preparative and its analysis by gas chromatography. The main advantage is that it allows the use of small amounts of sample and the rapid extraction of the unsaponifiable fraction using a much smaller volume of solvent than the techniques described for this purpose.
    [0010] STATE OF THE ART
    [0012] Food authentication has evolved according to market trends and analytical techniques have evolved to solve adulterations of each moment.
    [0014] In the specific case of edible oils and fats, the unsaponifiable matter is a source of information for their characterization and authentication. More specifically, the determination of sterols has been used frequently to detect contamination and adulterations in this type of materials.
    [0016] The composition of sterols in an oil or fat is very characteristic and has therefore become a very useful tool for the detection of mixtures with other vegetable oils.
    [0017] On many occasions the analysis by gas chromatography of certain fractions of compounds of a more or less complex matrix, as is the case of the unsaponifiable fraction, presents certain difficulties, especially when these fractions have a high number of compounds [Señoráns, FJ, Tabera, I and Herraiz, M. "Rapid separation of free sterols in edible oils by on-line coupled reversed phase liquid chromatography-gas chromatography". J. Agrie. Food Chem. (1996), 44, 3189-3192].
    [0019] Furthermore, there is an added problem if it is impossible to carry out chemical reactions and derivatizations on the matrix to separate the desired fraction, due to alteration, contamination or loss of this. In this case, the prior isolation of the fraction by means of liquid-liquid extraction, despite being the standardized or official procedure [European Union Commission Regulation EEC 183/93, Off. J. Eur. Commun L22 (1993) 58], it is an unsuitable, very tedious and time-consuming procedure to carry out; It is necessary to use a large amount of sample (between 5 and 20 g) and solvents for the extraction (of the order of 300 ml), the time required is also excessive, especially if emulsions are formed as in fact it usually happens. Finally, all the solvent used in the extraction must be removed; then a purification has to be carried out so, before carrying out the analysis by gas chromatography, preliminary separations must be carried out, usually using thin layer chromatography (TLC), column (CC) [Brewington CR, Caress, EA and Schwartz DP (1979). "Isolation and identification of new milk fat." J. of Lipid Research 11, 355-361], solid phase extraction (SPE) [Amelio M., Rizzo, R. and Varazini F. (1992). "Determination of sterols , erythrodiol, uvaol and alkanols in olive oils using combined solid-phase extraction, high-performance liquid chromatographic and high-resolution gas chromatographic techniques. " J. Chromatography, 606, 179-185] or high performance liquid chromatography (HPLC) techniques [EEC, 1993; Cert, A., Moreda, W and García-Morena, J. (1997). Determination of sterols and triterpenic dialcohols in olive oil by separation of the fraction by high performance liquid chromatography and analysis by gas chromatography. Standardization of the analytical method. Fats and oils. 48, 207-218]. In summary, to obtain the unsaponifiable fraction of an oil or fat and purify it by a conventional procedure, it is required to use a large amount of sample, organic solvents and time. All this leads to frequent losses or contamination.
    [0021] In the case of using as a pre-separation technique, HPLC can be off-line or online, requiring in both cases a liquid chromatograph to carry out the pre-separation. In the first modality, the desired fraction is collected by some procedure, the mobile phase is eliminated and the fraction is transferred to the gas chromatograph by means of some of the known injection techniques [León Camacho M. and Morales MT, Handbook of Olive OH Analysis and Properties; Chapter 7 (2000) Ed Aspen Publishers, Inc. 159-208]. When using on-line coupling techniques, a more or less complicated transfer interface is necessary [Grob, K. (1995). Development of the transfer techniques for on-line high-performance liquid chromatography-capillary gas chromatography. J.Chromatogr. A, 703, 265-276]; [Vreuls, JJ, de Jong. GJ; Ghijsen, RT and Brinkman, UATh. (1994). LC coupled on-line with GC: state of the art. J. AOAC. Int., 77, 306-327] that allows the fraction in liquid state and high pressure to pass to a gas phase and low pressure, in addition, the interfaces are very different depending on whether the liquid chromatography is absorption or distribution [Señoráns FJ, Herraiz M. and Tabera J. (1995a). "On-line reversed-phase liquid chromatography-capillary gas chromatography using a programmed temperature vaporizer as interface." Journal of Separation Science, 18, 433 438]; [Señoráns, FJ; Reglero, G .; Herraiz, M. (1995b) “Use of a Programmed Temperature Injector for On-Line Reversed-Phase Liquid Chromatography-Capillary Gas Chromatography”. Journal of Chromatographic Science. 33, 446-450]; [Homberg E. (1993). Vitamin D-Bestimmung in Lebertram. Fat Sci. Technol. 95, 228-230].
    [0023] The isolation of the compounds of interest from the unsaponifiable, by means of the aforementioned techniques is a critical step, specifically in the case of olive oils where sterols and triterpenic alcohols co-elute in the same chromatographic zones, leading to erroneous results. So is the previous step, the isolation of the unsaponifiable matter from the saponified fraction and, for this, Hadorn and Zürcher in 1973 [Hadorn H .; Zürcher (1973). Der Scheidetrichter, ein mangelhaftes Gerat im analytischen Labor. Gordian 73, 198-204] published one of the first attempts at isolation using column systems with mixed adsorbents, tetrahydrofuran gel on S-832 gel.
    [0025] Supported liquid extraction (ESL) methods have been developed as is the case of patent US2018188142 that describes a cartridge that includes two separate compartments, one of which comprises a solid adsorption phase that is diatomaceous earth and the other comprises the minus one salt. In this case the method is used for the separation of analytes in biological samples.
    [0027] Other techniques have been developed for the separation of the unsaponifiable fraction by a supported liquid extraction using diatomaceous earth cartridges (Chem Elut, 20 ml, unbuffered, Agilent Technologies, Santa Clara, CA) followed by filtration through anhydrous sodium sulfate and isolation of the fractions by solid phase extraction (SPE) using silica cartridges (Agilent Mega BE-1, 1 g, 6 ml, Agilent Technologies, Santa Clara, CA) activated with potash and eluting the fractions with solutions of different proportions of hexane / diethyl ether. The sterol and dialcohol fractions were silanized and analyzed by gas chromatography [Mathison, B. and Holstege, D. A "Rapid Method to Determine Sterol, Erthyrodiol, and Uvaol Concentrations in Olive Oil." J. Agrie. Food Chem. , (2013), 61, 4506-4513].
    [0029] The company Phenomenex has developed cartridges for this purpose [Determination of Sterols in Olive Oil using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID] in which it uses a cartridge of diatomaceous earth, Strata®- DE, adapted to a cartridge of anhydrous sodium sulfate for the separation of the unsaponifiable and another of activated silica, Strata Si-1, for their purification. In this case, purification by HPLC is not necessary for the separation of the sterols and triterpenic alcohols, but the separation is achieved from the Si column activated with potash and using a mobile phase of hexane / ether.
    [0031] BRIEF EXPLANATION OF THE INVENTION
    [0033] The object of the present invention is a process for the isolation of the unsaponifiable fraction of oils or fats that comprises:
    [0034] - saponify the oil or fat by adding a base and applying a temperature in a range between 75 and 80 ° C
    [0035] - subjecting the saponified from the previous stage to a supported liquid extraction for the isolation of the unsaponifiable fraction
    [0036] - removing the free fatty acids from the unsaponifiable fraction isolated in the previous step by means of a solid phase extraction.
    [0038] The supported liquid extraction is carried out by passing the saponify through a column with a packing material comprising:
    [0039] - a phyllosilicate that is selected from sepiolite and attapulgite in a proportion between 20% and 35%
    [0040] - a desiccant material that selects between silica gel and sodium sulfate in a proportion between 30% and 50%
    [0041] - diatomaceous earth in a proportion between 20% and 35%.
    [0042] In preferred embodiments of the present invention:
    [0043] - the phyllosilicate is sepiolite, particularly sepiolite with a particle size between 0.30 and 0.13 mm.
    [0044] - the phyllosilicate is attapulgite, particularly calcined attapulgite with a granulometry between 1.21 and 0.60 mm.
    [0045] - the desiccant material is anhydrous Na2SO4.
    [0046] - the diatomaceous earth is a calcined diatomaceous earth with a mesh size of between 0.25 mm and 3.35 mm, more preferably between 0.3 mm and 1 mm.
    [0047] The elimination of free fatty acids from the isolated unsaponifiable fraction in the supported liquid extraction step is carried out in a SiO2 column impregnated in KOH or filled with a support linked to the amino group (-NH2).
    [0049] Elution on the supported liquid extraction column can be done with solvents such as ethyl ether or a mixture of hexane and ethyl acetate.
    [0051] When the elution is done with ethyl ether, CaCl2 is added to the packing material in a proportion between 10 and 15%.
    [0053] When the elution on the column is done with a mixture of hexane and ethyl acetate, the ratio used is preferably 87:13 v / v.
    [0055] Optionally, an internal standard is added to the oil or fat sample from which the unsaponifiable fraction is to be isolated.
    [0057] BRIEF DESCRIPTION OF THE FIGURES
    [0059] Figure 1: General diagram of the ELS column comprising the following elements:
    [0060] - Column reservoir (1)
    [0061] - Filter disc (2)
    [0062] - Fritted disc (3)
    [0063] - Column filling (4)
    [0065] Figure 2: General diagram of the free fatty acid elimination system comprising:
    [0066] - Glass column (5)
    [0067] - Silica impregnated in potash (6)
    [0068] - Collector ("Manifold") to collect the eluate from the column (7)
    [0069] The unsaponifiable eluate from the column presented in Figure 1 is concentrated, redissolved and passed through the column presented in Figure 2 to remove free fatty acids.
    [0071] Figure 3: HPLC chromatogram of the unsaponifiable obtained in the example of an embodiment of the invention.
    [0073] Figure 4: Gas chromatogram of the fraction of the silicon derivatives of the sterols previously recovered by HPLC in the example of an embodiment of the invention. 1: Cholesterol; I.S .: Internal Pattern; 2: Campesterol; 3: Stigmasterol; 4: clerosterol; 5: p-Sitosterol; 6: Sitostanol; 7: A5-Avenasterol; 8: A5,24-Stigmastadienol; 9: A7-Stigmastenol; 10: A7-Avenasterol; 11: Erythrodiol.
    [0075] DETAILED DESCRIPTION OF AN EXAMPLE OF EMBODIMENT OF THE INVENTION
    [0077] The object of the present invention is a packing material for a supported liquid extraction column (ELS) and the use of said column for the isolation of the unsaponifiable fraction of oils and fats free of fatty acids. Figure 1 represents the general scheme of the ELS column that includes the following elements:
    [0078] - Column reservoir (1)
    [0079] - Filter disc (2)
    [0080] - Fritted disc (3)
    [0081] - Column filling (4)
    [0082] The unsaponifiable eluate from the column presented in Figure 1 is concentrated, redissolved and passed through the system outlined in Figure 2 to remove free fatty acids.
    [0084] Example: determination of sterols in olive oil
    [0086] 80 µl of an internal standard (which corresponds according to the determination) with a concentration of 1 mg / ml are poured into a 4 ml vial. It is brought to dryness with nitrogen and weighed to the nearest mg between 0.2-0.4 g of sample. Add 1.5 ml of 1M KOH in ethanol, close the vial and heat at 80 ° C for 45 minutes.
    [0088] After the time has elapsed, 3 ml of water are added to stop the reaction and it is poured into the ESL column.
    [0089] Preparation of the 50 ml ESL column; the column is packed with 36 g of a homogeneous mixture composed of 33.3% anhydrous Na2SO4, 11.1% CaCl22, 7.8% attapulgite 15/30 and 27.8% calcined diatom 0.3 / 1 mm .
    [0091] After adding the sample to the column, wait 10 minutes. After time, 55 ml of ethyl ether were passed through the column and collected.
    [0093] The collected solution is brought to dryness on a rotary evaporator and redissolved in 1 ml of hexane / ethyl acetate 87:13 v, it is passed through a column of 1 g of silica gel 60 (0.063 0.200 mm) impregnated in 0.2M KOH in ethanol and dried, a first fraction is discarded that elutes with 10 ml of the 87:13 v / v hexane / ethyl acetate mixture, and finally 6 ml of ethyl ether is passed through and collected. The unsaponifiable fraction is brought to dryness on the rotary evaporator.
    [0095] To monitor the profile of the fraction selected and recovered by HPLC, it is operated under the following conditions:
    [0096] - The unsaponifiable obtained is redissolved in 500 µl of the mobile phase of HPLC. 300 µl are injected into the liquid chromatograph. This step is essential, since, as mentioned, there are important interferences between the families of compounds of the unsaponifiable, especially in the case of olive oils, if it is injected without prior isolation.
    [0098] HPLC and gas chromatograph conditions: Mobile phase hexane: ethyl acetate 87/13 v / v; stationary phase: Si 60 column, 250 mm long by 4.6 mm in diameter and 5 pm in particle size, column temperature 20 ° C, mobile phase flow 0.6 ml / min in isocratic regime, detector of refractive index at 30 ° C, chromatogram time 40 minutes. The resulting chromatogram is shown in Figure 3. To collect the fraction of sterols, for example, it is collected between 16 and 31 minutes, it is brought to dryness on a rotary evaporator and 150 µl of silanization reagent are added, waiting 15 minutes and it is injected into the gas chromatograph according to the following conditions: DB-5 chromatographic column of 30 meters, 250 µm internal diameter and 0.25 µm phase thickness. Hydrogen carrier gas at a constant flow of 1 ml / min. Injection in Split mode with a ratio of 10, injector temperature 310C °. Chromatographic oven operating conditions:
    [0100] Flame ionization detector at 320 ° C and nitrogen auxiliary gas at 25 ml / min. The resulting chromatogram is shown in Figure 4.
    权利要求:
    Claims (13)
    [1]
    1. - Procedure for the isolation of the unsaponifiable fraction of oils or fats that comprises:
    - saponify the oil or fat by adding a base and applying a temperature in a range between 75 and 80 ° C
    - subjecting the saponified from the previous stage to a supported liquid extraction for the isolation of the unsaponifiable fraction
    - remove free fatty acids from the unsaponifiable fraction isolated in the previous step by means of a solid phase extraction
    characterized in that the supported liquid extraction is carried out by passing the saponified through a column with a filling material comprising:
    - a phyllosilicate that is selected from sepiolite and attapulgite in a proportion comprised between 20% and 35% by weight.
    - a desiccant material that selects between silica gel and sodium sulfate in a proportion comprised between 30% and 50% by weight.
    - diatomaceous earth in a proportion comprised between 20% and 35% by weight.
    [2]
    2. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 1, where the phyllosilicate is sepiolite.
    [3]
    3. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 2, where the sepiolite is sepiolite with a particle size between 0.30 and 0.13 mm.
    [4]
    4. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 1, where the phyllosilicate is attapulgite.
    [5]
    5. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 4, where the attapulgite is calcined attapulgite with a granulometry between 1.21 and 0.60 mm.
    [6]
    6. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 5, where the desiccant material is anhydrous Na2SO4.
    [7]
    7. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 6, where diatomaceous earth is a calcined diatomaceous earth with a mesh size of between 0.25 mm and 3, 35 mm.
    [8]
    8. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 7, where the diatomaceous earth has a mesh size between 0.3 mm and 1 mm.
    [9]
    9. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 8, where the elution in the supported liquid extraction column is done with ethyl ether.
    [10]
    10. - Procedure for isolating the unsaponifiable fraction of oils or fats according to claim 9, wherein the filler material additionally comprises CaCl2 in a proportion between 10 and 15% by weight.
    [11]
    11. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 8, where the elution in the supported liquid extraction column is done with a mixture of hexane and ethyl acetate.
    [12]
    12. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 11, where the elimination of free fatty acids from the unsaponifiable fraction isolated in the supported liquid extraction stage is carried out in a column of SiO2 impregnated in KOH or filled with a support linked to the amino group (-NH2).
    [13]
    13. - Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 12, where an internal standard is added to the oil or fat sample from which the unsaponifiable fraction is to be isolated.
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    同族专利:
    公开号 | 公开日
    WO2020128125A1|2020-06-25|
    ES2772803B2|2020-12-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2222338T3|2000-01-12|2005-02-01|Laboratoires Expanscience|PROCEDURE FOR THE EXTRACTION OF INSAPONIFIED VEGETABLE OILS THROUGH CHLORINE-1-BUTANE.|
    US20160122687A1|2013-06-04|2016-05-05|Saeml Valagro Carbone Renouvelable Poitou-Charentes|Methods for the selective extraction of unsaponifiable matters from renewable raw materials by solid-liquid extraction in the presence of a cosolvent|
    WO2016198616A1|2015-06-11|2016-12-15|Biotage Ab|Sample prep method|
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    申请号 | 申请日 | 专利标题
    ES201831256A|ES2772803B2|2018-12-20|2018-12-20|PROCEDURE FOR THE ISOLATION OF THE INSAPPONABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION|ES201831256A| ES2772803B2|2018-12-20|2018-12-20|PROCEDURE FOR THE ISOLATION OF THE INSAPPONABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION|
    PCT/ES2019/070840| WO2020128125A1|2018-12-20|2019-12-12|Method for isolating the unsaponifiable fraction of oils or fats by supported liquid extraction|
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