WO2020128125A1 - Method for isolating the unsaponifiable fraction of oils or fats by supported liquid extraction - Google Patents

Method for isolating the unsaponifiable fraction of oils or fats by supported liquid extraction Download PDF

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Publication number
WO2020128125A1
WO2020128125A1 PCT/ES2019/070840 ES2019070840W WO2020128125A1 WO 2020128125 A1 WO2020128125 A1 WO 2020128125A1 ES 2019070840 W ES2019070840 W ES 2019070840W WO 2020128125 A1 WO2020128125 A1 WO 2020128125A1
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oils
isolation
procedure
unsaponifiable fraction
fraction
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PCT/ES2019/070840
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Spanish (es)
French (fr)
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Mª Carmen PÉREZ CAMINO
Manuel LEÓN CAMACHO
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Consejo Superior De Investigaciones Científicas (Csic)
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/14—Diatomaceous earth
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A23D9/04—Working-up
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/16—Alumino-silicates
    • B01J20/165—Natural alumino-silicates, e.g. zeolites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0008—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of solubilities, e.g. by extraction, by separation from a solution by means of anti-solvents

Definitions

  • the present invention is framed within the sector 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.
  • 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-performance liquid chromatography (HPLC). in English) preparation and its analysis by gas chromatography.
  • ESL supported liquid extraction column
  • HPLC high-performance liquid chromatography
  • the unsaponifiable matter is a source of information for its characterization and authentication. More specifically, the determination of sterols has been frequently used to detect contamination and adulterations in this type of material.
  • composition of sterols of an oil or fat is very characteristic and has therefore become a very useful tool for detecting mixtures with other vegetable oils.
  • gas chromatographic analysis of certain fractions of compounds of a more or less complex matrix, such as the unsaponifiable fraction presents certain difficulties, especially when these fractions have a high number of compounds [Se ⁇ oráns, FJ, Jabera, I and Herraiz, M. "Rapid separation of free sterois in edible oils by on-line coupled reversed pbase liquid cbromatograpby-gas chromatography". J. Agr ⁇ e. Food Chem. (1996), 44, 3189-3192]
  • the HPLC can be off-line or on-line, requiring in both cases a liquid chromatograph where to carry out the previous separation.
  • the desired fraction is collected by some procedure, the mobile phase is eliminated and the fraction is transferred to the gas chromatograph using some of the known injection techniques [León Camacho M. and Morales MT, Handbook I heard Olive OH Analysis and Properties; Chapter 7 (2000) Ed Aspen Publishers, inc 159 ⁇ 208].
  • a more or less complicated transfer interface is necessary [Grob, K. (1995) Development of the transfer techniques for on-line high-performance ⁇ chid chromatography-capillary gas chromatography. J.Chromatogr.
  • Supported liquid extraction (ESL) methods have been developed as is the case in patent US2018188142 which describes a cartridge that includes two separate compartments, one of which comprises a solid adsorption phase that is of diatomaceous earth and the other comprises the minus a salt. In this case the method is used for the separation of analytes in biological samples.
  • Phenomenex has developed cartridges for this purpose [Determination of Sterols in Olive Oii using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID] in which it uses a diatomaceous earth cartridge, S ⁇ rata®- DE, adapted to an anhydrous sodium sulfate cartridge for the separation of the unsaponifiable and another of activated silica, Strata Si-1, for their purification.
  • purification by HPLC for the separation of the sterols and triterpenic alcohols does not work, but instead, the separation is achieved from the Si column activated with potash and using a mobile phase of hexane / ether.
  • the object of the present invention is a procedure for the isolation of the unsaponifiable fraction of oils or fats, comprising:
  • the supported liquid extraction is carried out by passing the saponification through a column with a filling material that includes:
  • the phyllosiicate is sepiolite, particularly sepioite of granuiomer ⁇ a between 0.30 and 0.13 mm.
  • the phyllosiicate is attapulgite, particularly calcined attapulgite with a granulometry between 1, 21 and 0.60 mm
  • the desiccant material is anhydrous Na 2 S04.
  • 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.
  • the removal of the free fatty acids from the unsaponifiable fraction isolated in the supported liquid extraction step is carried out on a SiG 2 column impregnated in KOH or filled with a support linked to the amino group (-NH2).
  • Elution in the supported liquid extraction column can be done with solvents such as ethyl ether or a mixture of hexane and ethyl acetate.
  • CaCI 2 is added to the filler in a proportion between 10 and 15%
  • the ratio used is preferably 87:13 v / v.
  • an internal standard is added to the oil or fat sample from which the unsaponifiable fraction is to be isolated.
  • FIG. 1 General diagram of the ELS column comprising the following elements:
  • Figure 2 General diagram of the free fatty acid removal system comprising:
  • the unsaponifiable eluate from the column presented in FIG. 1 is concentrated, redissolved and passed through the column presented in FIG. 2 to remove free fatty acids.
  • Figure 3 HPLC chromatogram of the unsaponifiably obtained in the example of an embodiment of the invention.
  • Figure 4 Gas chromatogram of the fraction of ios derived from silicon from iosterols previously recovered by HPLC in the example of an embodiment of the invention 1: Cholesteroi; IS: Internal Standard; 2: Campesterol; 3: Stigmasterol; 4: Clerosterol; 5: b-Sitosterol; 6: Sitostanoi; 7: A 5 -Avenasterol; 8: A 5 24 -Estigmastadienoi; 9: D 7 - Stigmastenoi; 10: A 7 -Avenasierol; 1 1: Erythrodiol.
  • the object of the present invention is a filling 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 diagram of the ELS column that includes the following elements:
  • the unsaponifiable eluate from the column presented in figure 1 is concentrated, redissolved and passed through the system outlined in figure 2 to eliminate the free fatty acids.
  • 80 m are poured! of an internal standard (which corresponds according to the determination) of concentration 1 mg / ml in a vial of 4 ml. It is brought to dryness with nitrogen and it is weighed with a precision of mg between 0.2-0.4 g of sample. Add 1.5 ml of 1M KOH in ethanol, close the vial and heat at 8 ° C for 45 minutes.
  • the collected solution is brought to dryness on a rotary evaporator and redissolved in 1 ml of 87:13 v hexane / ethyl acetate mixture, passed through a 1 g column of silica gel 60 (0.063-0.200 mm) impregnated with 0 KOH, 2M in ethanoi and dried, a first fraction is discarded which elutes with 10 ml of the 87:13 v / v hexane / ethyl acetate mixture, and finally 6 ml of ethyl ether are passed, which is collected.
  • the unsaponifiable fraction is brought to dryness on the rotary evaporator.
  • the Emponification obtained is redissolved in 500 ml of the mobile phase of the HPLC. 300 ml is injected into the liquid chromatograph. This step is essential, since, as mentioned, there are important interferences between the families of compounds of Examponificabie, especially in the case of olive oils, if it is injected without prior isolation.
  • HPLC and! gas chromatograph mobile phase hexa no: 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 refractive index at 30 ° C, chromatogram time 40 minutes.
  • the resulting chromatogram is shown in Figure 3.
  • the fraction of sterols for example, it is collected between 16 and 31 minutes, it is brought to dryness on the rotary evaporator and 150 ml of siianization reagent are added, waiting 15 minutes and it is injected into the gas chromatograph according to the following conditions: 30-meter DB-5 chromatographic column, 250 pm internal diameter and 0.25 pm phase thickness. Hydrogen carrier gas at constant flow of 1 ml / min. injection in Split mode with a ratio of 10, injector temperature 310C ° Operating conditions of the chromatographic oven:

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Inorganic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Wood Science & Technology (AREA)
  • Geochemistry & Mineralogy (AREA)
  • Fats And Perfumes (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Edible Oils And Fats (AREA)

Abstract

The invention relates to a method for isolating the unsaponifiable fraction of edible oils or fats by means of a supported liquid extraction (SLE) column, and for subsequently purifying the different fractions by means of preparative high-performance liquid chromatography and the analysis thereof by gas chromatography. The main advantage is that the invention 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 thus far for such purpose.

Description

PROCEDIMIENTO PARA EL AISLAMIENTO DE LA FRACCIÓN INSAPONIFICABLE DE ACEITES O GRASAS CEDIANTE EXTRACCION LIQUIDA SOPORTADA PROCEDURE FOR THE ISOLATION OF THE INSAPONIFICABLE FRACTION OF OILS OR FATS CEDIANT SUPPORTED LIQUID EXTRACTION
DESCRIPCIÓN DESCRIPTION
SECTOR DE LA TÉCNICA Y OBJETO DE LA INVENCIÓN TECHNICAL SECTOR AND OBJECT OF THE INVENTION
La presente invención se enmarca dentro del sector de la química analítica instrumental y en particular en la preparación de columnas empleadas para la separación de compuestos, específicamente para el aislamiento de la fracción insaponificable de aceites o grasas comestibles mediante extracción líquida soportada. The present invention is framed within the sector 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.
El objeto de la invención es un procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas mediante una columna de extracción líquida soportada (ESL), y la posterior purificación de las diferentes fracciones mediante cromatografía líquida de alta eficacia (HPLC, por sus siglas en inglés) preparativa y su análisis mediante cromatografía de gases. La principal ventaja es que permite el empleo de pequeñas cantidades de muestra y la rápida extracción de la fracción insaponificable utilizando un volumen de disolvente mucho menor que las técnicas descritas a tal fin. 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-performance liquid chromatography (HPLC). in English) preparation 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.
ESTADO DE LA TÉCNICA STATE OF THE ART
La autentificación de los alimentos ha ido desarrollándose en función de las tendencias del mercado y las técnicas analíticas han ido evolucionando para solucionar las adulteraciones de cada momento. Food authentication has been developed according to market trends and analytical techniques have evolved to solve adulterations at any time.
En el caso concreto de los aceites y grasas comestibles la materia insaponificable es una fuente de información para su caracterización y autentificación. Más concretamente, la determinación de esteróles se ha usado frecuentemente para detectar contaminaciones y adulteraciones en este tipo de materiales. In the specific case of edible oils and fats, the unsaponifiable matter is a source of information for its characterization and authentication. More specifically, the determination of sterols has been frequently used to detect contamination and adulterations in this type of material.
La composición de esteróles de un aceite o grasa es muy característica y se ha convertido, por ello, en una herramienta muy útil para la detección de mezclas con otros aceites vegetales. En numerosas ocasiones el análisis por cromatografía de gases de ciertas fracciones de compuestos de una matriz más o menos compleja, como es el caso de la fracción insaponificable, presenta ciertas dificultades, especialmente cuando estas fracciones poseen un número elevado de compuestos [Señoráns, F.J., Jabera, I y Herraiz, M. “Rapid separation of free sterois in edible oils by on-line coupled reversed pbase liquid cbromatograpby-gas chromatography”. J. Agríe. Food Chem. (1996), 44, 3189-3192] The composition of sterols of an oil or fat is very characteristic and has therefore become a very useful tool for detecting mixtures with other vegetable oils. On numerous occasions, gas chromatographic analysis of certain fractions of compounds of a more or less complex matrix, such as the unsaponifiable fraction, presents certain difficulties, especially when these fractions have a high number of compounds [Señoráns, FJ, Jabera, I and Herraiz, M. "Rapid separation of free sterois in edible oils by on-line coupled reversed pbase liquid cbromatograpby-gas chromatography". J. Agríe. Food Chem. (1996), 44, 3189-3192]
Además, existe un problema añadido si resulta imposible realizar sobre la matriz reacciones químicas y derivatizaciones para separar la fracción deseada, debido a la alteración, contaminación o pérdida de esta. En tai caso el aislamiento previo de la fracción mediante la extracción líquido-líquido, a pesar de ser el procedimiento estandarizado u oficial [European Union Commission Regulation EEC 183/93, Off. J. Eur. Commun L22 (1993) 58], es un procedimiento poco adecuado, muy tedioso y largo de llevar a cabo; es necesario emplear una gran cantidad de muestra (entre 5 y 20 g) y de disolventes para la extracción (del orden de 300 mi), el tiempo requerido también resulta excesivo, sobre todo si se forman emulsiones como de hecho suele ocurrir. Finalmente, es preciso eliminar todo el disolvente empleado en la extracción; a continuación, ha de efectuarse una purificación por lo que, antes de efectuarse el análisis por cromatografía de gases, hay que realizar separaciones previas empleando usualmente cromatografía en capa fina (CCF), en columna (CC) [Brewington C.R., Caress, E.A. y Schwartz D.P. (1979).“isoiation and Identification of new milk fat”. J. of Lipid Research 11 , 355-361], extracción en fase sólida (SPE) [Amelio M., Rizzo, R. y Varazini F. (1992).“Deter ination of sterois, erythrodio!, uvaol and alkanois in olive oils using combined soiid-phase extraction, high-performance liquid chromatographic and high-resolution gas chromatographic techniques.” J. Chromatography, 606, 179-185] o las técnicas de cromatografía líquida de alta eficacia (HPLC) [EEC, 1993; Cert, A., Moreda, W y García-Morena, J. (1997). Determinación de esteróles y dialcoholes triterpénicos en aceite de oliva mediante separación de la fracción por cromatografía líquida de alta eficacia y análisis por cromatografía de gases. Estandarización del método analítico. Grasas y Aceites. 48, 207-218] En resumen, para obtener la fracción del insaponificable de un aceite o grasa y purificarla por un procedimiento convencional se requiere emplear gran cantidad de muestra, disolventes orgánicos y tiempo. Todo esto conlleva que con frecuencia ocurran pérdidas o contaminaciones. 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 its alteration, contamination or loss. In this case, the previous isolation of the fraction by liquid-liquid extraction, despite being the standardized or official procedure [European Union Commission Regulation EEC 183/93, Off. J. Eur. Commun L22 (1993) 58], 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 is actually the case. Finally, it is necessary to eliminate all the solvent used in the extraction; thereafter, a purification has to be carried out so, before carrying out the analysis by gas chromatography, previous separations must be carried out, usually using thin layer chromatography (TLC), column (CC) [Brewington C.R., Caress, E.A. and Schwartz D.P. (1979). "Isoiation 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). “Deter ination of sterois, erythrodio !, uvaol and alkanois in olive oils using combined soiid-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 triterpene 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 necessary to use a large amount of sample, organic solvents and time. All this means that losses or contamination frequently occur.
En el caso de utilizar como técnica de separación previa, la HPLC puede ser off-line u On line, requiriéndose en ambos casos un cromatógrafo líquido donde realizar la separación previa. En la primera modalidad se recolecta la fracción deseada por algún procedimiento, se elimina la fase móvil y se transfiere la fracción ai cromatógrafo de gases mediante algunas de las técnicas de inyección conocidas [ León Camacho M. y Morales M.T., Handbook oí Olive OH Análisis and Properties; Chapter 7 (2000) Ed Aspen Publishers, inc 159~208]. Cuando se emplean técnicas de acoplamiento on-line, es necesario una interfase de transferencia más o menos complicada [Grob, K. (1995) Deveiopment of the transfer techniques for on-line high-performance ¡¡quid chromatography-capillary gas chromatography. J.Chromatogr. A, 703, 265-276]; [ Vreuls, J.J., de Jong. G.J.; Ghijsen, R.T. y Brinkman, U.A.Th. (1994). LC coupied on-line with GC: State of the art. J. AOAC. inl, 77, 306-327] que permita pasar la fracción en estado líquido y alta presión a una fase gaseosa y de baja presión, además, las interfaces son muy diferentes dependiendo de si la cromatografía líquida es de absorción o de reparto [ Señoráns F.J., Herraiz M. y Jabera J. (1995a). On-line reversed-phase liquid chromatography-capillary gas chromatography using a programmed temperature vaporizer as interface.” Journal of Separaíion Science, 18, 433- 438]; [ Señoráns, F.J.; Reglero, G.; Herraiz, M. (1995b)“Use of a Programmed Temperature Injector for On-Line Reversed-Phase Liquid Chromatography-Capiliary Gas Chromatography”. Journal of Chromatographic Science. 33, 446-450]; [Homberg E. (1993). Vitamin D-Bestimmung in Lebertram. Fat Sci. Technol. 95, 228-230] In the case of using as a prior separation technique, the HPLC can be off-line or on-line, requiring in both cases a liquid chromatograph where to carry out the previous 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 using some of the known injection techniques [León Camacho M. and Morales MT, Handbook I heard Olive OH Analysis and Properties; Chapter 7 (2000) Ed Aspen Publishers, inc 159 ~ 208]. When using online coupling techniques, a more or less complicated transfer interface is necessary [Grob, K. (1995) Development of the transfer techniques for on-line high-performance ¡chid chromatography-capillary gas chromatography. J.Chromatogr. A, 703, 265-276]; [Vreuls, JJ, de Jong. GJ; Ghijsen, RT and Brinkman, UATh. (1994). LC coupied on-line with GC: State of the art. J. AOAC. inl, 77, 306-327] that allows to pass the fraction in a liquid and high pressure state to a gaseous and low pressure phase. Furthermore, the interfaces are very different depending on whether liquid chromatography is absorption or distribution [Señoráns FJ , Herraiz M. and Jabera 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-Capiliary Gas Chromatography". Journal of Chromatographic Science. 33, 446-450]; [Homberg E. (1993). Vitamin D-Bestimmung in Lebertram. Fat Sci. Technol. 95, 228-230]
El aislamiento de los compuestos de interés del insaponificable, mediante las técnicas mencionadas es un paso crítico, específicamente en el caso de los aceites de oliva donde esteróles y alcoholes triterpénicos coeluyen en las mismas zonas cromatográficas llevando a resultados erróneos. También lo es el paso previo, el aislamiento de la materia insaponificable de la fracción saponificada y, para ello, Hadorn y Zürcher en 1973 [Hadorn H.; Zürcher (1973). Der Scheidetrichter, ein mangelhaftes Gerát im analytischen Labor. Gordian 73, 198-204] publicaron uno de ios primeros intentos de aislamiento utilizando sistemas de columnas con mezcla de adsorbentes, gel en tetrahidrofurano sobre gel S-832. 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 coelute 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 Gerát im analytischen Labor. Gordian 73, 198-204] published one of the first attempts at isolation using column systems with mixed adsorbents, gel in tetrahydrofuran on S-832 gel.
Se han desarrollado métodos de extracción líquida soportada (ESL) como es el caso de la patente US2018188142 que describe un cartucho que incluye dos compartimentos separados, uno de los cuales comprende una fase de adsorción sólida que es de tierra de diatomeas y la otra comprende al menos una sal. En este caso el método se utiliza para la separación de analitos en muestras biológicas. Supported liquid extraction (ESL) methods have been developed as is the case in patent US2018188142 which describes a cartridge that includes two separate compartments, one of which comprises a solid adsorption phase that is of diatomaceous earth and the other comprises the minus a salt. In this case the method is used for the separation of analytes in biological samples.
Se han desarrollado otras técnicas para la separación de la fracción insaponificable mediante una extracción líquida soportada utilizando para ello cartuchos de tierra de diatomeas (Cberrs Elut, 20 mi, no tamponado, Agüen! Technologies, Santa Ciara, CA) seguida de una filtración a través de sulfato sódico anhidro y aislamiento de las fracciones mediante extracción de fase sólida (SPE) utilizando cartuchos de sílice (Agilení Mega BE-1 , 1 g, 6 mi, Agiient Technologies, Santa Clara, CA) activada con potasa y eluyendo las fracciones con disoluciones de diferentes proporciones de hexano/ éter dietílico. Las fracciones de esteróles y dialcoholes se silanizaron y se analizaron mediante cromatografía de gases [Mathison, B. and Holsfege, D. A“Rapid Method to Determine Sterol, Eríhyrodioí, and Uvaol Concentrations in Olive Oii”. J. Agria Food Chem., (2013), 61 , 4506-4513] Other techniques have been developed for the separation of the unsaponifiable fraction by means of a supported liquid extraction using diatomaceous earth cartridges (Cberrs Elut, 20 ml, unbuffered, Agüen! Technologies, Santa Ciara, CA) followed by filtration through anhydrous sodium sulfate and isolation of the fractions by solid phase extraction (SPE) using silica cartridges (Agilení Mega BE-1, 1 g, 6 ml, Agiient Technologies, Santa Clara, CA) activated with potash and eluting the fractions with solutions of different proportions of hexane / diethyl ether. The fractions of sterols and dialcohols were silanized and analyzed by gas chromatography [Mathison, B. and Holsfege, D. A "Rapid Method to Determine Sterol, Eríhyrodioí, and Uvaol Concentrations in Olive Oii". J. Agria Food Chem., (2013), 61, 4506-4513]
La empresa Phenomenex ha desarrollado unos cartuchos para este fin [Determination of Sterols in Olive Oii using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID] en el que utiliza un cartucho de tierra de diatomeas, Sírata®-DE, adaptado a un cartucho de sulfato de sodio anhidro para la separación del insaponificable y otro de sílice activada, Strata Si-1 , para la purificación de estos. En este caso, no hace faifa la purificación mediante HPLC para la separación de los esteróles y alcoholes triterpénicos, sino que a partir de la columna de Si activada con potasa y utilizando una fase móvil de hexano/éter se consigue la separación. Phenomenex has developed cartridges for this purpose [Determination of Sterols in Olive Oii using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID] in which it uses a diatomaceous earth cartridge, Sírata®- DE, adapted to an anhydrous sodium sulfate cartridge for the separation of the unsaponifiable and another of activated silica, Strata Si-1, for their purification. In this case, purification by HPLC for the separation of the sterols and triterpenic alcohols does not work, but instead, the separation is achieved from the Si column activated with potash and using a mobile phase of hexane / ether.
BREVE EXPLICACIÓN DE LA INVENCIÓN BRIEF EXPLANATION OF THE INVENTION
Constituye el objeto de la presente invención un procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas que comprende: The object of the present invention is a procedure for the isolation of the unsaponifiable fraction of oils or fats, comprising:
saponificar el aceite o grasa mediante la adición de una base y aplicación de temperatura en un rango comprendido entre 75 y 80 °C saponify the oil or fat by adding a base and applying a temperature in a range between 75 and 80 ° C
someter el saponificado procedente de la etapa anterior a una extracción líquida soportada para el aislamiento de la fracción insaponificable subjecting the saponification from the previous stage to a supported liquid extraction for the isolation of the unsaponifiable fraction
eliminar los ácidos grasos libres de la fracción insaponificable aislada en la etapa anterior mediante una extracción en fase sólida. remove the free fatty acids from the unsaponifiable fraction isolated in the previous step by means of a solid phase extraction.
La extracción líquida soportada se realiza haciendo pasar el saponificado a través de una columna con un material de relleno que comprende: The supported liquid extraction is carried out by passing the saponification through a column with a filling material that includes:
- un filosilicafo que se selecciona entre sepioiifa y atapulgifa en proporción comprendida entre un 20% y un 35% - a philosophical that is selected between sepioiifa and atapulgifa in a proportion between 20% and 35%
- un material desecante que selecciona entre silica ge! y sulfato sódico en proporción comprendida entre un 30% y un 50% - a desiccant material that selects from silica ge! and sodium sulfate in a proportion between 30% and 50%
- tierra de diatomeas en proporción comprendida entre un 20% y un 35%. En realizaciones preferentes de la presente invención: - diatomaceous earth in a proportion comprised between 20% and 35%. In preferred embodiments of the present invention:
- el filosiiicato es sepiolita, particularmente sepioiita de granuiomeíría comprendida entre 0,30 y 0,13 mm. - the phyllosiicate is sepiolite, particularly sepioite of granuiomería between 0.30 and 0.13 mm.
- el filosiiicato es atapulgita, particularmente atapulgita calcinada de granulometría comprendida entre 1 ,21 y 0,60 mm - the phyllosiicate is attapulgite, particularly calcined attapulgite with a granulometry between 1, 21 and 0.60 mm
- el material desecante es Na2S04 anhidro. - the desiccant material is anhydrous Na 2 S04.
- la tierra de diatomeas es una tierra de diatomeas calcinada con un tamaño de malla de entre 0,25 mm y 3,35 mm, más preferentemente entre 0,3 mm y 1 mm. - 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.
- la eliminación de los ácidos grasos libres de la fracción insaponificable aislada en la etapa de extracción líquida soportada se realiza en columna de SiG2 impregnada en KOH o rellena de un soporte ligado al grupo amino (-NH2). - the removal of the free fatty acids from the unsaponifiable fraction isolated in the supported liquid extraction step is carried out on a SiG 2 column impregnated in KOH or filled with a support linked to the amino group (-NH2).
La elución en la columna de extracción líquida soportada puede hacerse con disolventes como éter etílico o una mezcla de hexano y acetato de etilo. Elution in the supported liquid extraction column can be done with solvents such as ethyl ether or a mixture of hexane and ethyl acetate.
Cuando ¡a elución se hace con éter etílico, se añade CaCI2 al material de relleno en proporción comprendida entre un 10 y un 15 % When elution is done with ethyl ether, CaCI 2 is added to the filler in a proportion between 10 and 15%
Cuando la elución en la columna se hace con una mezcla de hexano y acetato de etilo, la proporción empleada es preferentemente 87:13 v/v. When the column elution is done with a mixture of hexane and ethyl acetate, the ratio used is preferably 87:13 v / v.
Opcionalmente, se añade un patrón interno a la muestra de aceite o grasa de la que se va a aislar la fracción insaponificable. Optionally, an internal standard is added to the oil or fat sample from which the unsaponifiable fraction is to be isolated.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1 : Esquema general de la columna de ELS que comprende los siguientes elementos: Figure 1: General diagram of the ELS column comprising the following elements:
Reservorio de la columna (1) Column reservoir (1)
Disco filtrante (2) Filter disc (2)
Disco fritado (3) Fried disc (3)
Relleno de ia columna (4) Column filling (4)
Figura 2: Esquema general del sistema de eliminación de ácidos grasos libres que comprende: Figure 2: General diagram of the free fatty acid removal system comprising:
Columna de vidrio (5) Glass column (5)
Sílice impregnada en potasa (6) Colector (“Manifoid”) para recoger ei eluido de la columna (7) Potash-impregnated silica (6) Collector (“Manifoid”) to collect the eluate from the column (7)
El insaponificable eluido de 1a columna presentada en ¡a figura 1 se concentra, se redisueive y se pasa por la columna presentada en la figura 2 para eliminar los ácidos grasos libres. The unsaponifiable eluate from the column presented in FIG. 1 is concentrated, redissolved and passed through the column presented in FIG. 2 to remove free fatty acids.
Figura 3: Cromatograma de HPLC del insaponificable obtenido en ei ejemplo de un modo de realización de ia invención. Figure 3: HPLC chromatogram of the unsaponifiably obtained in the example of an embodiment of the invention.
Figura 4: Cromatograma de gases de la fracción de ios derivados de silicio de ios esteróles recuperados previamente mediante HPLC en ei ejemplo de un modo de realización de la invención 1 : Colesteroi; I.S.: Patrón Interno; 2: Campesterol; 3: Estigmasterol; 4: Clerosterol; 5: b-Sitosterol; 6: Sitostanoi; 7: A5-Avenasterol; 8: A5 24-Estigmastadienoi; 9: D7- Estigmastenoi; 10: A7-Avenasierol; 1 1 : Eritrodiol. Figure 4: Gas chromatogram of the fraction of ios derived from silicon from iosterols previously recovered by HPLC in the example of an embodiment of the invention 1: Cholesteroi; IS: Internal Standard; 2: Campesterol; 3: Stigmasterol; 4: Clerosterol; 5: b-Sitosterol; 6: Sitostanoi; 7: A 5 -Avenasterol; 8: A 5 24 -Estigmastadienoi; 9: D 7 - Stigmastenoi; 10: A 7 -Avenasierol; 1 1: Erythrodiol.
DESCRIPCIÓN DETALLADA DE UN EJEMPLO DE REALIZACION DE LA INVENCIÓN DETAILED DESCRIPTION OF AN EXAMPLE OF EMBODIMENT OF THE INVENTION
El objeto de ia presente Invención es un material de relleno para una columna de extracción liquida soportada (ELS) y el uso de dicha columna para el aislamiento de la fracción insaponificable de aceites y grasas libre de ácidos grasos. En ia figura 1 se representa ei esquema general de la columna de ELS que comprende los siguientes elementos: The object of the present invention is a filling 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 diagram of the ELS column that includes the following elements:
Reservorio de la columna (1) Column reservoir (1)
Disco fiitrante (2) Disc fiitrante (2)
Disco fritado (3) Fried disc (3)
Relleno de ia columna (4) Column filling (4)
Ei insaponificable eluido de ia columna presentada en la figura 1 se concentra, se redisuelve y se pasa por ei sistema esquematizado en ia figura 2 para eliminar ios ácidos grasos iibres. The unsaponifiable eluate from the column presented in figure 1 is concentrated, redissolved and passed through the system outlined in figure 2 to eliminate the free fatty acids.
Ejemplo: determinación de esteróles en aceite de oliva Example: determination of sterols in olive oil
Se vierten 80 m! de un patrón interno (el que corresponda según la determinación) de concentración 1 mg/mi en un vial de 4 mi. Se lleva a sequedad con nitrógeno y se pesa con exactitud del mg entre 0,2-0, 4 g de muestra. Se añade 1 ,5 mi de KOH 1 M en etanol, se cierra ei vial y se calienta a 8QC° durante 45 minutos. 80 m are poured! of an internal standard (which corresponds according to the determination) of concentration 1 mg / ml in a vial of 4 ml. It is brought to dryness with nitrogen and it is weighed with a precision of mg between 0.2-0.4 g of sample. Add 1.5 ml of 1M KOH in ethanol, close the vial and heat at 8 ° C for 45 minutes.
Una vez transcurrido e! tiempo se añaden 3 mi de agua para detener ia reacción y se vierte en la columna de ESL. Preparación de ¡a columna de ESL de 50 mi; la columna se rellena con 36 g de una mezcla homogénea compuesta de un 33,3% de Na2S04 anhidro, 11 ,1 % de CaCl227,8% de atapulgita 15/30 y 27,3% de diaiomea calcinada 0,3/1 mm. Once e! Time 3 ml of water is added to stop the reaction and it is poured into the ESL column. Preparation of the 50 ml ESL column; the column is filled with 36 g of a homogeneous mixture composed of 33.3% anhydrous Na 2 S04, 11.1% CaCl 227.8% atapulgite 15/30 and 27.3% calcined diaiomea 0.3 / 1 mm.
Una vez añadida la muestra a la columna se esperan 10 minutos. Transcurrido el tiempo se pasan por la columna 55 mi de éter etílico y se colecta. Once the sample is added to the column, wait 10 minutes. After time, 55 ml of ethyl ether are passed through the column and collected.
La disolución recogida se lleva a sequedad en rotavapor y se redisuelve en 1 mi de mezcla hexano/acetato de etilo 87:13 v, se pasa por una columna de 1 g de sílica gel 60 (0,063- 0,200 mm) impregnada en KOH 0,2M en etanoi y secada, se desecha una primera fracción que eluye con 10 mi de la mezcla hexano/acetato de etilo 87:13 v/v, y finalmente se pasan 6 mi de éter etílico que es recogido. La fracción insaponificabie se lleva a sequedad en el rotavapor. The collected solution is brought to dryness on a rotary evaporator and redissolved in 1 ml of 87:13 v hexane / ethyl acetate mixture, passed through a 1 g column of silica gel 60 (0.063-0.200 mm) impregnated with 0 KOH, 2M in ethanoi and dried, a first fraction is discarded which elutes with 10 ml of the 87:13 v / v hexane / ethyl acetate mixture, and finally 6 ml of ethyl ether are passed, which is collected. The unsaponifiable fraction is brought to dryness on the rotary evaporator.
Para monitorizar el perfil de la fracción seleccionada y recuperada mediante HPLC, se opera en las siguientes condiciones: To monitor the profile of the selected fraction and recovered by HPLC, it is operated under the following conditions:
El insaponificabie obtenido se redisuelve en 500 mI de la fase móvil del HPLC. Se inyectan 300 mI en el cromatógrafo líquido. Este paso es fundamental, dado que, como se ha comentado, hay importantes interferencias entre las familias de compuestos del insaponificabie, especialmente en el caso de aceites de oliva, si este se inyecta sin un aislamiento previo. The insaponification obtained is redissolved in 500 ml of the mobile phase of the HPLC. 300 ml is injected into the liquid chromatograph. This step is essential, since, as mentioned, there are important interferences between the families of compounds of insaponificabie, especially in the case of olive oils, if it is injected without prior isolation.
Condiciones del HPLC y de! cromatógrafo de gases: Fase móvil hexa no: acetato de etilo 87/13 v/v; fase estacionaría: Columna de Si 60 de 250 mm de longitud por 4,6 mm de diámetro y 5 pm de tamaño de partículas, temperatura de la columna 20C°, flujo de fase móvil 0,6 ml/min en régimen ¡socrático, detector de índice de refracción a 30C°, tiempo del cromatograma 40 minutos. El cromatograma resultante se muestra en ia Figura 3. Para recoger ia fracción de esteróles, por ejemplo, se recolecta entre ios 16 y 31 minutos, se lleva a sequedad en el rotavapor y se añaden 150 mI de reactivo de siianización, se esperan 15 minutos y se inyecta en el cromatógrafo de gases de acuerdo con las siguientes condiciones: columna cromatográfica DB-5 de 30 metros, 250 pm de diámetro interno y 0,25 pm de espesor de fase. Gas portador hidrógeno a flujo constante de 1 ml/min. inyección en modo Split con una relación de 10, temperatura del inyector 310C° Condiciones de operación del horno cromatográfico:
Figure imgf000009_0001
HPLC and! gas chromatograph: mobile phase hexa no: 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 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 the rotary evaporator and 150 ml of siianization reagent are added, waiting 15 minutes and it is injected into the gas chromatograph according to the following conditions: 30-meter DB-5 chromatographic column, 250 pm internal diameter and 0.25 pm phase thickness. Hydrogen carrier gas at constant flow of 1 ml / min. injection in Split mode with a ratio of 10, injector temperature 310C ° Operating conditions of the chromatographic oven:
Figure imgf000009_0001
Detector de ionización de ilama a 320°C y gas auxiliar nitrógeno a 25 ml/min. Ei cromaíograma resultante se muestra en ia figura 4. Ilama ionization detector at 320 ° C and nitrogen auxiliary gas at 25 ml / min. The resulting chromatogram is shown in Figure 4.

Claims

REIVINDICACIONES
1.- Procedimiento para ei aislamiento de la fracción insaponificabie de aceites o grasas que comprende: 1.- Procedure for the isolation of the unsaponifiable fraction of oils or fats, comprising:
saponificar el aceite o grasa mediante la adición de una base y aplicación de temperatura en un rango comprendido entre 75 y 80 °C saponify the oil or fat by adding a base and applying a temperature in a range between 75 and 80 ° C
someter ei saponificado procedente de la etapa anterior a una extracción liquida soportada para ei aislamiento de la fracción insaponificabie subjecting the saponified from the previous stage to a supported liquid extraction for the isolation of the unsaponifiable fraction
eliminar los ácidos grasos libres de la fracción insaponificabie aislada en la etapa anterior mediante una extracción en fase sólida remove the free fatty acids from the insaponificabie fraction isolated in the previous step by means of a solid phase extraction
caracterizado porque la extracción líquida soportada se realiza haciendo pasar el saponificado a través de una columna con un material de relleno que comprende: characterized in that the supported liquid extraction is carried out by passing the saponification through a column with a filling material comprising:
- un filosilicato que se selecciona entre sepiolita y atapulgita en proporción comprendida entre un 20% y un 35% en peso. - a phyllosilicate that is selected between sepiolite and attapulgite in a proportion comprised between 20% and 35% by weight.
- un material desecante que selecciona entre silica ge! y sulfato sódico en proporción comprendida entre un 30% y un 50% en peso. - a desiccant material that selects from silica ge! and sodium sulfate in a proportion comprised between 30% and 50% by weight.
- tierra de diatomeas en proporción comprendida entre un 20% y un 35% en peso. - diatomaceous earth in a proportion comprised between 20% and 35% by weight.
2.- Procedimiento para el aislamiento de la fracción insaponificabie de aceites o grasas según la reivindicación 1 , donde el filosilicato es sepiolita. 2.- Procedure for the isolation of the insaponificabie fraction of oils or fats according to claim 1, where the phyllosilicate is sepiolite.
3.- Procedimiento para el aislamiento de la fracción insaponificabie de aceites o grasas según la reivindicación 2, donde la sepiolita es sepiolita de granuiometría comprendida entre 0,30 y 0,13 mm. 3. Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 2, where the sepiolite is sepiolite with a granuiometry between 0.30 and 0.13 mm.
4.- Procedimiento para el aislamiento de ia fracción insaponificabie de aceites o grasas según ia reivindicación 1 , donde el filosilicato es atapulgita. 4.- Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 1, where the phyllosilicate is attapulgite.
5.- Procedimiento para ei aislamiento de ia fracción insaponificabie de aceites o grasas según ia reivindicación 4, donde ia atapulgita es atapulgita calcinada de granuiometría comprendida entre 1 ,21 y 0,60 mm. 5.- Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 4, where the attapulgite is calcined attapulgite of granuiometry comprised between 1, 21 and 0.60 mm.
6.- Procedimiento para el aislamiento de ia fracción insaponificabie de aceites o grasas según una cualquiera de las reivindicaciones 1 a 5, donde ei material desecante es Na S04 anhidro. 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 Na S04.
7.- Procedimiento para el aislamiento de ia fracción insaponificable de aceites o grasas según una cualquiera de las reivindicaciones 1 a 6, donde ia tierra de diatomeas es una tierra de diatomeas calcinada con un tamaño de malla de entre 0,25 mm y 3,35 mm. 7. Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 6, wherein the diatomaceous earth is a calcined diatomaceous earth with a mesh size of between 0.25 mm and 3, 35 mm.
8.- Procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas según ia reivindicación 7, donde la tierra de diatomeas presenta un tamaño de malla entre 0,3 mm y 1 mm. 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.- Procedimiento para el aislamiento de ¡a fracción insaponificable de aceites o grasas según una cualquiera de las reivindicaciones 1 a 8, donde ia elución en la columna de extracción líquida soportada se hace con éter etílico. 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.- Procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas según la reivindicación 9, donde el material de relleno comprende adicionalmente GaCb en proporción comprendida entre el 10 y el 15% en peso. 10. Procedure for the isolation of the unsaponifiable fraction of oils or fats according to claim 9, wherein the filler material additionally comprises GaCb in a proportion comprised between 10 and 15% by weight.
11.- Procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas según una cualquiera de las reivindicaciones 1 a 8, donde la elución en la columna de extracción líquida soportada se hace con una mezcla de hexano y acetato de etilo. 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.- Procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas según una cualquiera de las reivindicaciones 1 a 11 , donde la eliminación de los ácidos grasos libres de ia fracción insaponificable aislada en la etapa de extracción líquida soportada se realiza en columna de SÍO2 impregnada en KOH o rellena de un soporte ligado ai grupo amino (-NH2). 12. Procedure for the isolation of the unsaponifiable fraction of oils or fats according to any one of claims 1 to 11, wherein the removal of free fatty acids from the isolated unsaponifiable fraction in the supported liquid extraction step is carried out in a column of SÍO2 impregnated in KOH or filled with a support linked to the amino group (-NH 2 ).
13.- Procedimiento para el aislamiento de la fracción insaponificable de aceites o grasas según una cualquiera de las reivindicaciones 1 a 12, donde se añade un patrón interno a la muestra de aceite o grasa de la que se va a aislar la fracción insaponificable. 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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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2222338T3 (en) * 2000-01-12 2005-02-01 Laboratoires Expanscience PROCEDURE FOR THE EXTRACTION OF INSAPONIFIED VEGETABLE OILS THROUGH CHLORINE-1-BUTANE.
US20160122687A1 (en) * 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 (en) * 2015-06-11 2016-12-15 Biotage Ab Sample prep method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2222338T3 (en) * 2000-01-12 2005-02-01 Laboratoires Expanscience PROCEDURE FOR THE EXTRACTION OF INSAPONIFIED VEGETABLE OILS THROUGH CHLORINE-1-BUTANE.
US20160122687A1 (en) * 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 (en) * 2015-06-11 2016-12-15 Biotage Ab Sample prep method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AQEEL, Z: "Determination of Sterols in Olive Oil using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID", SELECTSCIENCE, 2017, pages 1 - 8, Retrieved from the Internet <URL:https://www.selectscience.net/application-article/determination-of-sterols-in-olive-oil-using-supported-liquid-extraction-(sle)-solid-phase-extraction-(spe)-and-gc-fid/?artid=46975> [retrieved on 20191017] *
MATHISON, B. ET AL.: "A Rapid Method to Determine Sterol, Erythrodiol, and Uvaol Concentrations in Olive Oil", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2013, vol. 61, no. 19, 15 April 2013 (2013-04-15), pages 4506 - 4513, XP055721576, ISSN: 0021-8561 *
OELLIG, C. ET AL.: "Screening for 16-O-methylcafestol in roasted coffee by high-performance thin-layer chromatography- fluorescence detection - Determination of Coffea canephora admixtures to Coffea arabica", JOURNAL OF CHROMATOGRAPHY A 2017, vol. 1525, no. 19, 12 October 2017 (2017-10-12), pages 173 - 180, XP085274454, ISSN: 0021-9673, Retrieved from the Internet <URL:https://doi.org/10.1016/j.chroma> [retrieved on 20191015] *
PHENOMENEX: "Two Unique Options for Supported Liquid Extraction", HAVING TWO DIFFERENT SUPPORTED LIQUID EXTRACTION (SLE) SORBENT OPTIONS CAN MAKE IT EASIER FOR YOU TO GET A PRODUCT THAT WORKS FOR YOUR UNIQUE EXTRACTIONS, 6 March 2018 (2018-03-06), Retrieved from the Internet <URL:https://phenomenex.blog/2018/03/06/supported-liquid-extraction/?pdf=8621> [retrieved on 20191018] *
SAHU, S. ET AL.: "Isolation of the unsaponifiable matter (squalene, phytosterols, tocopherols, y-oryzanol and fatty alcohols) from a fatty acid distillate or rice bran oil", GRASAS Y ACEITES 2018 (JULIO-SEPTIEMBRE, vol. 69, no. 3, July 2018 (2018-07-01), pages e262, ISSN: 0017-3495, Retrieved from the Internet <URL:https://www.researchgate.net/publication/327258278_Isolation_of_the_unsaponifiable_matter_squalene_phytosterols_tocopherols_g-oryzanol_and_fatty_alcohols_from_a_fatty_acid_distillate_of_rice_bran_oil> [retrieved on 20191016] *

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