ES2772803A1 - PROCEDURE FOR THE ISOLATION OF THE INSAPONIFICABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION (Machine-translation by Google Translate, not legally binding) - Google Patents
PROCEDURE FOR THE ISOLATION OF THE INSAPONIFICABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION (Machine-translation by Google Translate, not legally binding) Download PDFInfo
- Publication number
- ES2772803A1 ES2772803A1 ES201831256A ES201831256A ES2772803A1 ES 2772803 A1 ES2772803 A1 ES 2772803A1 ES 201831256 A ES201831256 A ES 201831256A ES 201831256 A ES201831256 A ES 201831256A ES 2772803 A1 ES2772803 A1 ES 2772803A1
- Authority
- ES
- Spain
- Prior art keywords
- isolation
- oils
- procedure
- unsaponifiable fraction
- fraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000002955 isolation Methods 0.000 title claims abstract description 29
- 239000003925 fat Substances 0.000 title claims abstract description 28
- 239000007788 liquid Substances 0.000 title claims abstract description 27
- 239000003921 oil Substances 0.000 title claims abstract description 25
- 238000000605 extraction Methods 0.000 title claims abstract description 23
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Substances [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 10
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 9
- 239000005909 Kieselgur Substances 0.000 claims description 8
- 229960000892 attapulgite Drugs 0.000 claims description 8
- 229910052625 palygorskite Inorganic materials 0.000 claims description 8
- 239000004113 Sepiolite Substances 0.000 claims description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 7
- 235000021588 free fatty acids Nutrition 0.000 claims description 7
- 229910052624 sepiolite Inorganic materials 0.000 claims description 7
- 235000019355 sepiolite Nutrition 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 229910052615 phyllosilicate Inorganic materials 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- 239000002274 desiccant Substances 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 3
- 238000003379 elimination reaction Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 229910052681 coesite Inorganic materials 0.000 claims description 2
- 229910052906 cristobalite Inorganic materials 0.000 claims description 2
- 238000001033 granulometry Methods 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 235000012239 silicon dioxide Nutrition 0.000 claims description 2
- 229910052682 stishovite Inorganic materials 0.000 claims description 2
- 229910052905 tridymite Inorganic materials 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 11
- 238000004458 analytical method Methods 0.000 abstract description 8
- 238000004817 gas chromatography Methods 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 5
- 239000002904 solvent Substances 0.000 abstract description 5
- 239000008157 edible vegetable oil Substances 0.000 abstract description 4
- 229930182558 Sterol Natural products 0.000 description 13
- 150000003432 sterols Chemical class 0.000 description 13
- 235000003702 sterols Nutrition 0.000 description 13
- 238000000926 separation method Methods 0.000 description 12
- 239000012071 phase Substances 0.000 description 11
- 235000019198 oils Nutrition 0.000 description 8
- 239000007789 gas Substances 0.000 description 7
- 239000004006 olive oil Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- 235000008390 olive oil Nutrition 0.000 description 4
- XUARCIYIVXVTAE-ZAPOICBTSA-N uvaol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(CO)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C XUARCIYIVXVTAE-ZAPOICBTSA-N 0.000 description 4
- 238000003965 capillary gas chromatography Methods 0.000 description 3
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- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 3
- 235000015320 potassium carbonate Nutrition 0.000 description 3
- PSZDOEIIIJFCFE-UHFFFAOYSA-N Oleanolic alcohol Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(CO)CCC(C)(C)CC5C4=CCC3C21C PSZDOEIIIJFCFE-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- PSZDOEIIIJFCFE-OSQDELBUSA-N erythrodiol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(CO)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C PSZDOEIIIJFCFE-OSQDELBUSA-N 0.000 description 2
- HTZRWCSRPTWJCT-UHFFFAOYSA-N erythrodiol Natural products CC1(C)CCC2(CO)CCC3C(CCC4C3(C)CCC5C(C)(C)C(O)CCC45C)C2C1 HTZRWCSRPTWJCT-UHFFFAOYSA-N 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000014593 oils and fats Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- SYFNOXYZEIYOSE-UHFFFAOYSA-N uvaol Natural products CC1CCC2(O)CCC3(C)C(=CCC4(C)C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C SYFNOXYZEIYOSE-UHFFFAOYSA-N 0.000 description 2
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- VGSSUFQMXBFFTM-UHFFFAOYSA-N (24R)-24-ethyl-5alpha-cholestane-3beta,5,6beta-triol Natural products C1C(O)C2(O)CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 VGSSUFQMXBFFTM-UHFFFAOYSA-N 0.000 description 1
- VARLNHXKTAMJPE-GAICQMAKSA-N (3S,6R)-6-[(8R,9S,10S,13R,14S,17R)-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-3-propan-2-ylhepta-1,4-dien-3-ol Chemical compound C1CC2CCCC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)C=C[C@@](O)(C=C)C(C)C)[C@@]1(C)CC2 VARLNHXKTAMJPE-GAICQMAKSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- GHGKPLPBPGYSOO-FBZNIEFRSA-N Clerosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@H](CC)C(C)=C)[C@@]1(C)CC2 GHGKPLPBPGYSOO-FBZNIEFRSA-N 0.000 description 1
- DMPCQZBAZOKWKU-UHFFFAOYSA-N Clerosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(=O)C DMPCQZBAZOKWKU-UHFFFAOYSA-N 0.000 description 1
- -1 Erthyrodiol Natural products 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- LGJMUZUPVCAVPU-JFBKYFIKSA-N Sitostanol Natural products O[C@@H]1C[C@H]2[C@@](C)([C@@H]3[C@@H]([C@H]4[C@@](C)([C@@H]([C@@H](CC[C@H](C(C)C)CC)C)CC4)CC3)CC2)CC1 LGJMUZUPVCAVPU-JFBKYFIKSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- YYLFLRIUDMIWTD-UHFFFAOYSA-N ethyldesmosterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)=C(C)C)C1(C)CC2 YYLFLRIUDMIWTD-UHFFFAOYSA-N 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008240 homogeneous mixture Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- LGJMUZUPVCAVPU-HRJGVYIJSA-N stigmastanol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]2(C)CC1 LGJMUZUPVCAVPU-HRJGVYIJSA-N 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 239000006200 vaporizer Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
Landscapes
- 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
Description
DESCRIPCI NDESCRIPTION
PROCEDIMIENTO PARA EL AISLAMIENTO DE LA FRACCIÓN INSAPONIFICABLE DEPROCEDURE FOR THE ISOLATION OF THE INSAPONIFICABLE FRACTION OF
ACEITES O GRASAS MEDIANTE EXTRACCION LIQUIDA SOPORTADAOILS OR FATS BY SUPPORTED LIQUID EXTRACTION
SECTOR DE LA TÉCNICA Y OBJETO DE LA INVENCIÓNSECTOR OF THE TECHNIQUE 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 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.
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 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.
ESTADO DE LA TÉCNICASTATE 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 evolved according to market trends and analytical techniques have evolved to solve adulterations of each moment.
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 esteroles 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 their characterization and authentication. More specifically, the determination of sterols has been used frequently to detect contamination and adulterations in this type of materials.
La composición de esteroles 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. 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.
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., Tabera, I y 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].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].
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 tal 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 ml), 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). "Isolation 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). "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] o las técnicas de cromatografía líquida de alta eficacia (HPLC) [EEC, 1993; Cert, A., Moreda, W y García-Morena, J. (1997). Determinación de esteroles 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 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.
En el caso de utilizar como técnica de separación previa, la HPLC puede ser off-line u online, 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 al cromatógrafo de gases mediante algunas de las técnicas de inyección conocidas [ León Camacho M. y Morales M.T., Handbook of 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). Development of the transfer techniques for on-line high-performance liquid 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 coupled on-line with GC: state of the art. J. AOAC. Int., 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 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, F.J.; 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].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].
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 esteroles 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 Gerat im analytischen Labor. Gordian 73, 198-204] publicaron uno de los 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 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.
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 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.
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 (Chem Elut, 20 ml, no tamponado, Agilent Technologies, Santa Clara, 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 (Agilent Mega BE-1, 1 g, 6 ml, Agilent Technologies, Santa Clara, CA) activada con potasa y eluyendo las fracciones con disoluciones de diferentes proporciones de hexano/ éter dietílico. Las fracciones de esteroles y dialcoholes se silanizaron y se analizaron mediante cromatografía de gases [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].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].
La empresa Phenomenex ha desarrollado unos cartuchos para este fin [Determination of Sterols in Olive Oil using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID] en el que utiliza un cartucho de tierra de diatomeas, Strata®-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 falta la purificación mediante HPLC para la separación de los esteroles 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.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.
BREVE EXPLICACIÓN DE LA INVENCIÓNBRIEF 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 process for the isolation of the unsaponifiable fraction of oils or fats that comprises:
- 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 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 insaponificable aislada en la etapa anterior mediante una extracción en fase sólida.- removing 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 saponify through a column with a packing material comprising:
- un filosilicato que se selecciona entre sepiolita y atapulgita en proporción comprendida entre un 20% y un 35%- a phyllosilicate that is selected from sepiolite and attapulgite in a proportion between 20% and 35%
- un material desecante que selecciona entre silica gel y sulfato sódico en proporción comprendida entre un 30% y un 50%- a desiccant material that selects between silica gel and sodium sulfate in a proportion between 30% and 50%
- tierra de diatomeas en proporción comprendida entre un 20% y un 35%. - diatomaceous earth in a proportion between 20% and 35%.
En realizaciones preferentes de la presente invención:In preferred embodiments of the present invention:
- el filosilicato es sepiolita, particularmente sepiolita de granulometría comprendida entre 0,30 y 0,13 mm.- the phyllosilicate is sepiolite, particularly sepiolite with a particle size between 0.30 and 0.13 mm.
- el filosilicato es atapulgita, particularmente atapulgita calcinada de granulometría comprendida entre 1,21 y 0,60 mm.- the phyllosilicate is attapulgite, particularly calcined attapulgite with a granulometry between 1.21 and 0.60 mm.
- el material desecante es Na2SO4 anhidro.- the desiccant material is anhydrous Na2SO4.
- 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 SiO2 impregnada en KOH o rellena de un soporte ligado al grupo amino (-NH2).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).
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 on the supported liquid extraction column can be done with solvents such as ethyl ether or a mixture of hexane and ethyl acetate.
Cuando la elución se hace con éter etílico, se añade CaCl2 al material de relleno en proporción comprendida entre un 10 y un 15 %.When the elution is done with ethyl ether, CaCl2 is added to the packing material 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 elution on the column 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 FIGURASBRIEF 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)- Fritted disc (3)
- Relleno de la 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 elimination system comprising:
- Columna de vidrio (5)- Glass column (5)
- Sílice impregnada en potasa (6) - Silica impregnated in potash (6)
- Colector ("Manifold”) para recoger el eluido de la columna (7)- Collector ("Manifold") to collect the eluate from the column (7)
El insaponificable eluido de la columna presentada en la figura 1 se concentra, se redisuelve 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 Figure 1 is concentrated, redissolved and passed through the column presented in Figure 2 to remove free fatty acids.
Figura 3: Cromatograma de HPLC del insaponificable obtenido en el ejemplo de un modo de realización de la invención.Figure 3: HPLC chromatogram of the unsaponifiable obtained in the example of an embodiment of the invention.
Figura 4: Cromatograma de gases de la fracción de los derivados de silicio de los esteroles recuperados previamente mediante HPLC en el ejemplo de un modo de realización de la invención. 1: Colesterol; I.S.: Patrón Interno; 2: Campesterol; 3: Estigmasterol; 4: Clerosterol; 5: p-Sitosterol; 6: Sitostanol; 7: A5-Avenasterol; 8: A5,24-Estigmastadienol; 9: A7-Estigmastenol; 10: A7-Avenasterol; 11: Eritrodiol.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.
DESCRIPCIÓN DETALLADA DE UN EJEMPLO DE REALIZACION DE LA INVENCIÓNDETAILED DESCRIPTION OF AN EXAMPLE OF EMBODIMENT OF THE INVENTION
El objeto de la presente invención es un material de relleno para una columna de extracción líquida soportada (ELS) y el uso de dicha columna para el aislamiento de la fracción insaponificable de aceites y grasas libre de ácidos grasos. En la figura 1 se representa el esquema general de la columna de ELS que comprende los siguientes elementos: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:
- Reservorio de la columna (1)- Column reservoir (1)
- Disco filtrante (2)- Filter disc (2)
- Disco fritado (3)- Fritted disc (3)
- Relleno de la columna (4)- Column filling (4)
El insaponificable eluido de la columna presentada en la figura 1 se concentra, se redisuelve y se pasa por el sistema esquematizado en la figura 2 para eliminar los ácidos grasos libres.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.
Ejemplo: determinación de esteroles en aceite de olivaExample: determination of sterols in olive oil
Se vierten 80 pl de un patrón interno (el que corresponda según la determinación) de concentración 1 mg/ml en un vial de 4 ml. 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 ml de KOH 1M en etanol, se cierra el vial y se calienta a 80C° durante 45 minutos.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.
Una vez transcurrido el tiempo se añaden 3 ml de agua para detener la reacción y se vierte en la columna de ESL. After the time has elapsed, 3 ml of water are added to stop the reaction and it is poured into the ESL column.
Preparación de la columna de ESL de 50 mi; la columna se rellena con 36 g de una mezcla homogénea compuesta de un 33,3% de Na2SO4 anhidro, 11,1% de CaCl227,8% de atapulgita 15/30 y 27,8% de diatomea calcinada 0,3/1 mm.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 .
Una vez añadida la muestra a la columna se esperan 10 minutos. Transcurrido el tiempo se pasan por la columna 55 ml de éter etílico y se colecta.After adding the sample to the column, wait 10 minutes. After time, 55 ml of ethyl ether were passed through the column and collected.
La disolución recogida se lleva a sequedad en rotavapor y se redisuelve en 1 ml 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 etanol y secada, se desecha una primera fracción que eluye con 10 ml de la mezcla hexano/acetato de etilo 87:13 v/v, y finalmente se pasan 6 ml de éter etílico que es recogido. La fracción insaponificable se lleva a sequedad en el rotavapor.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.
Para monitorizar el perfil de la fracción seleccionada y recuperada mediante HPLC, se opera en las siguientes condiciones:To monitor the profile of the fraction selected and recovered by HPLC, it is operated under the following conditions:
- El insaponificable obtenido se redisuelve en 500 pl de la fase móvil de1 HPLC. Se inyectan 300 pl en el cromatógrafo líquido. Este paso es fundamental, dado que, como se ha comentado, hay importantes interferencias entre las familias de compuestos del insaponificable, especialmente en el caso de aceites de oliva, si este se inyecta sin un aislamiento previo.- 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.
Condiciones del HPLC y del cromatógrafo de gases: Fase móvil hexano:acetato de etilo 87/13 v/v; fase estacionaria: 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 isocrático, detector de índice de refracción a 30C°, tiempo del cromatograma 40 minutos. El cromatograma resultante se muestra en la Figura 3. Para recoger la fracción de esteroles, por ejemplo, se recolecta entre los 16 y 31 minutos, se lleva a sequedad en el rotavapor y se añaden 150 pl de reactivo de silanización, 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: 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:
Detector de ionización de llama a 320°C y gas auxiliar nitrógeno a 25 ml/min. El cromatograma resultante se muestra en la figura 4. Flame ionization detector at 320 ° C and nitrogen auxiliary gas at 25 ml / min. The resulting chromatogram is shown in Figure 4.
Claims (13)
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| ES201831256A ES2772803B2 (en) | 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 (en) | 2018-12-20 | 2019-12-12 | Method for isolating the unsaponifiable fraction of oils or fats by supported liquid extraction |
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| ES201831256A ES2772803B2 (en) | 2018-12-20 | 2018-12-20 | PROCEDURE FOR THE ISOLATION OF THE INSAPPONABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION |
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|---|---|
| ES2772803A1 true ES2772803A1 (en) | 2020-07-08 |
| ES2772803B2 ES2772803B2 (en) | 2020-12-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| ES201831256A Expired - Fee Related ES2772803B2 (en) | 2018-12-20 | 2018-12-20 | PROCEDURE FOR THE ISOLATION OF THE INSAPPONABLE FRACTION OF OILS OR FATS THROUGH SUPPORTED LIQUID EXTRACTION |
Country Status (2)
| Country | Link |
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| ES (1) | ES2772803B2 (en) |
| WO (1) | WO2020128125A1 (en) |
Citations (3)
| 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 |
-
2018
- 2018-12-20 ES ES201831256A patent/ES2772803B2/en not_active Expired - Fee Related
-
2019
- 2019-12-12 WO PCT/ES2019/070840 patent/WO2020128125A1/en active Application Filing
Patent Citations (3)
| 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)
| Title |
|---|
| AQEEL, Z. . Determination of Sterols in Olive Oil using Supported Liquid Extraction (SLE), Solid Phase Extraction (SPE) and GC-FID. SelectScience. Applications Notes. , 07.08.2018, Vol. 2017, Páginas 1-8 [en línea][recuperado el 17.10.2019]. Recuperado de 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> ), Ver página 1, Introducción y Materiales y Métodos; página 2. * |
| MATHISON, B. & HOLSTEGE, D. . A Rapid Method to Determine Sterol, Erythrodiol, and Uvaol Concentrations in Olive Oil. Journal of Agricultural and Food Chemistry , 15.04.2013, Vol. 61, Páginas 4506-4513 0021-8561, (DOI: 10.1021/jf400254k) Ver página 4506, resumen; página 4507, columna 2, párrafo 8-página 4508, columna 1, párrafo 3. * |
| OELLIG, C. & RADOVANOVIC, J. . Screening for 16-O-methylcafestol in roasted coffee byhigh-performance thin-layer chromatography-fluorescence detection ¿ Determination of Coffea canephora admixtures to Coffea arabica. Journal of Chromatography A , 12.10.2017, Vol. 1525, Páginas 173-180 [en línea][recuperado el 15.10.2019]. Recuperado de Internet (URL:<https://doi.org/10.1016/j.chroma.2017.10.031>), 0021-9673; 1873-3778 (en línea), (DOI: 10.1016/j.chroma.2017.10.031) Ver página 4507, columna 2, párrafo 8-página 4508, columna 1, párrafo 3. * |
| 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. 06.03.2018, [en línea][recuperado el 18.10.2019]. Recuperado de Internet (URL:<https://phenomenex.blog/2018/03/06/supported-liquid-extraction/?pdf=8621>), * |
| SAHU, S. ET AL. . Isolation of the unsaponifiable matter (squalene, phytosterols, tocopherols, ¿-oryzanol and fatty alcohols) from a fatty acid distillate or rice bran oil. Grasas y Aceites , 2018 (julio-septiembre), Vol. 69, Páginas e262 [en línea][recuperado el 16.10.2019]. Recuperado de 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>), 0017-3495, (DOI: 10.3989/gya.1112172) Ver página 4507, columna 2, párrafo 8-página 4508, columna 1, párrafo 3. * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020128125A1 (en) | 2020-06-25 |
| ES2772803B2 (en) | 2020-12-09 |
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