SU1430888A1 - Method of stereospecific analysis of triacylglycerols - Google Patents

Description

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The invention relates to the oil and fat industry and relates to the stereoscopic method (an mythical analysis of triacylglycerols.

The aim of the invention is to improve the accuracy of the analysis by increasing the depth of phosphorylation and increasing the purity of the separation of phospholipolysis products.

The method is carried out as follows.

A 50 ml sample of triacylglycerols is placed in a 50 ml conical flask and 4 ml of a t% -Horo polyvinyl alcohol solution, 1 ml of a 22% solution of Kalitsi chloride, 7 ml of ammonia buffer (+ IN, pH 8) are added. The mixture was kept in a thermostat for 10 minutes at 40 ° C, stirring the contents with a stirrer, and then 0.25 g of pancreatic lipase, previously defatted with acetone, was introduced into the flask. The reaction is carried out for 20-30 minutes at 40 ° C, constantly stirring and adding 1 drop of 4M ammonium hydroxide every 10 minutes. To control the hydrolysis depth, a sample is taken every 10 minutes and the next 5 minutes and analyzed by thin layer chromatography in a solvent system of petroleum sfir (40-60 ° C): 7: 3 diethyl ether (by volume). After raising the solvent front and dividing the plate in air, the spots of hydrolysis products in iodine vapors appear, paying attention to the intensity of the Sn-1,2- (2,3) -diacylglycerol stain, whose mobility is characterized by a factor of 3 With high lipase activity, the hydrolysis time is reduced to 5–10 min in order to prevent the de-acylglycerols from being cleaved to free fatty acids, glycerol.

When the desired hydrolysis depth is reached, the reaction is interrupted and 1-1.5 ml of 4N hydrochloric acid is added to the reaction mixture and the products are extracted three times by extraction (10 mp) with diethyl ether, the ether extracts are combined, washed with distilled water until neutral. dried with anhydrous sodium sulfate; the solvent is removed on a rotary evaporator.

The hydrolysis products are separated by preparative thin layer chromatography on plates (20x20 cm) with silica.

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kagel containing 5% gypsum (8 g of sorbent per plate), in the system of petroleum ether (40-60 C): diethyl ether 60:40 (by volume).

After removal of diethyl ether, 10 MP of chloroform are added to the residue of the hydrolyzate and 1 ml of this solution is applied onto 10 plates. In the solvent system, the hydrolyzate is divided into the following components: uncleaned triacylglycerols (Rf 0.6), free fatty acids, cleaved from Sn-1 and Sn-3 positions (Rf 0.6), the sum of Sn-1, 2 (2,3) -Diacylglycerols (Rf 0.3) and 2-monoacylglycerols (Rf 0.07). They are shown by spraying the edge of the plate with 50% sulfuric acid and heating it or in iodine vapor.

Zones of silica gel with the substance are cleaned from the plates, transferred to a SCHOTT funnel with filter No. 4, and the lipids from the silica gel are washed off with a water-jet pump with several portions of diethyl ether. A part of each fraction is taken, fatty acids are analyzed by GLC.

The equality of the acidic compositions of non-degraded and parent triacylglycerols indicates that lipase hydrolysis is not accompanied by isomerization. The coincidence of the composition selected for Sn-1,2 (2,3) -diacylglycerols with the calculated theoretically proves their representativeness, i.e. reflects the distribution of acids in the starting triacylglycerols.

The theoretical calculation of the composition of Sn- -1,2 (2,3) -diacylglycerols% X in Sn-1,2 (2,3) -diacylglycerols is 3/4% X in TAG + 1/4% X in Sn-2- mono-acylglycerols, where X is a fatty acid. . . ,

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The fatty acid composition of the Sn-2-monoacylglycerols characterizes the set of acids in the Sn-2 position.

For phosphorylation, Sn-1,2 (2,3) -diacylglycerols, freshly distilled phenyldichlorophosphate is used. A weighed 0.07-0.12 g of Sn-1,2 (2,3) -diacyl-glycerols is dissolved in 0.5-1.0 ml of anhydrous diethyl ether and cooled in ice-salt mixture to (-10) - (- 15) C. A cooled solution of the substance is dripped with a syringe to the pre-cooled to (-10) -

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(-15) With a mixture of 2 ml of water-free dosing, and 0.25–0.50 r-ui, desiccated nanophosphate under nitrogen at a gig: duplei and 0.15 MIfa. After stirring, the reaction mixture is left under these conditions for 8-10 hours at 3-8 ° C,

Then, in T yaktsioin k) a mixture of L and 1) phosphate; lylsnolo 3 is added with cooling 5 ml of pyridine, 3 nl of diethyl gopher and 5-7 drops of distilled water., Transferred to the healing funnel, -shnnuyu 30 ml met anola, 25 MJt distilled) 5th water 30 ml

CHILDREN II 1 ml

sodium sppe 5 ml), clapofor; ny m rast

ver fosfE1T uP g,;: -: OTG0.1N |; - -; О:} -: - У 1 У - from

a solution (, 1x water, chlorine 5; u1 1- N V ar; -; vg1 iOT p rotoro; - evaporator ts-mg heating and pipie 5O С,

For the best; ench side products

Phosphorus LIBBY5 {; - ABOUT CHISSY T.-: -1 1)

- (i) about the Russian Federation g ty depol o y s g and i gjhy hl OD o form;.;: 1b; th 1) apnote 1 g1: osL) g; IgL1, lantals skip 1 ler: a column of aalified;; nJi iKa: i ejieM ohico va column ;; 12 cm

Г; Ка,; 1Г p 1 СГП topol - A small layer (2--3 mm) of dry sodium carbonate is poured onto the top layer of the sorbent, and a chloroform solution of phosphatidylphenols is added to the top layer. The product column is: - from chloroform; 5, the non-prodigious Sn-1 (2 , 3) -diacylglycerol and then with solvent system xopopopi i: methanol 9: 1 (in terms of), wash out the sum of L- and D phosphatidylphensov. The purity of the products is controlled by thin-layer chromatography in chloroform: methanol system; 25% ammonium hydroxide 80:20: 2 (by volume) In this system, the sum of L- and B-phosphatidylphenols has Rf O 56.

They exhibit phosphatidiphenol reactants of VaskovskO; that att simultaneously with a qualitative response to phosphorus.

Phospholipolysis of L-- and 1) -phosphati, tskfe nolov wire, additive to 50-100 mg of phosphate phosphate shphenolone per hour About ml of diethyl ether 0.2 ml of 0.1 M solution of Kapyk chloride and raspor 20 mg and gurzy (yusfopipaza in 1 ml / Tris-buffer with pH 5.0, the complete hydrolysis is controlled by thin-layer chromatography on silica gel in the system chloroform metaiol: 25% ammonium hydroxide (80: 20: 2).

R L Kazanny system products phos-

Phytopolyses have the following chromatographic mobility: unsaturated D-phosphatide gtphenols — Rf 0.6, free acids cleaved from

Sri - 2. Positions of L-phosphatidylphenols - Rf 0.4, lysophospholipids - Rf 0.05.

Phosphalipo. Piz finish after the growth of the growth of the output of free lnarny acids and lysofolol schich,

The reaction mixture is evaporated on a rotated T-opiiOM evaporator at a temperature of nj.iLie -iO С, and added a non-solubilized batch and iso: p dll beats ;; li; azeotro- n; i main water quality. Then

the residue of the phospholipolysis products after dalenmg D 5 ethyl ether and water is dissolved in a mixture consisting of Oj5 K-i chloroform and 0.5 ml of methanol and; az; tslgs: fr preparative thin layer-

IO11 chromatography is carried out sequentially in l, fluff with:; stax. 1) hexane: diethyl sfir (60; - iO by volume), its own (F) tglp: e fatty acids having chromium mobility; in this

ci -vrei-; e (Rf - 0.6). 2) shoroform: ketaiol: 20% ammonium hydroxide in a ratio of 80: 20: 2 (by volume O. The polar fractions are separated in this system: L153OFOSFOLIPIDA (Rf 0.2) and

ijspacFienHBiiniecH D-phosphatidylphenols

(Rf 0.6U,

Sorbent zones containing free fatty acids are stripped and desorbed with several portions of diethyl ether. The eluate is dried, methylated -: by analyzing by gas-liquid chromatography.

Strips of silica gel with D-phosphatidyl-phenol No. 1 and with lysophospholine sciiiiiaioT into separate flasks and add

 3 MP 0.5 M methanol solution of potassium hydroxide. After 15 minutes, 1 ml of 1N hydrochloric acid was added to the mlam and the obtained partially methylated fatty acids were separated by extraction with petroleum ether (AO - 60 s) (3 times 5 in).

The slurry is filtered through filter No. 4 from a Schott funnel, from the receiving flask a layer of petropain ether containing fatty acid and lots is removed by pipette into another flask, the solvent is evaporated, the acids are housetilated with diazomethane and analyzed by P1 (X,

The composition of free fatty acids obtained by the cleavage of lysophosphatidylphenols should not differ from the composition of Sn-2-monoacylglycerol acids obtained by lipolytic hydrolysis. The coincidence of the composition of ovs confirms the absence of isomerization in the process of isolating Sn-1,2 (2,3) -dacylglycerols and, therefore, their representativeness.

Of the two products of phospholipolysis, the composition of lysophosphatidylphenol acids directly reflects the set of fatty acids of the 1 position of triacylglycerols. A set of acids in the Sn-3 position is obtained by calculation in two ways: 1) Sn-3-З (initial triacylglycerols) - (lysophosphatides) - (2-mono-Parallel), the experiment was performed according to the modes indicated in the prototype (Table 1).

From the results given in table. 1, it can be seen that under overpressure in a nitrogen atmosphere with a decrease in temperature in the initial phosphorylation period to -10 ° C and separation of the phospholipolysis products successively in two systems, the phosphorylation depth increases by 25%, the content of degradation products g decreases by 20%, the duration

process is reduced by 8 hours and ensures complete separation of the products of phospholipolysis ..

Example 2. 0.1 g of 1.2 (2,3) - -diacylglycerols, obtained by

acylglycerols), 2) Sn-3-2 (D-phosphati-20 hydrolysis of triacylglycerols pancreatylphenols) - (2-monoacylglycerols). tonic lipase, was phosphorylated by phenyldichlorophosphate in a stream of nitrogen at a pressure of 0.13 MPa, at a temperature of –11 ° C, and the mixture was kept at + 7 ° C during phosphorylation and the formation of 25–9 hours before the completion of the reaction. Products

Under the selected modes, an increase in the phosphorylation depth, a decrease in the substrate destruction is provided.

reaction products, reducing the duration of the analysis, increasing the purity of the released products of phospho-, lipolysis. Neutral overpressure /

Rally gas over the reaction medium, with

on the one hand, it prevents oxidative processes in the long phosphorylation reaction, and on the other hand, it prevents the condensation of moisture into the reaction mixture from the gas composition of the flask, which improves the completeness of phosphorylation. The two-stage temperature phosphorylation regime ensures the absence of reaction by-products in the first stage and the depth of the second stage. When going beyond the parameters of the method, the accuracy of the analysis remains at the level of the prototype ...

Example 1. About 1 g of 1,2 (2,3) - -diacylglycerols, obtained by hydrolysis of triacylglycerols with pancreatic lipase, was phosphorylated by phenyldichlorophosphate in a stream of nitrogen at a pressure of 0.10 MPa, temperature — JC was kept until the reaction was completed at +5 C per for 8 hours. The phospholipids obtained after phospholipolysis were separated by thin layer chromatography successively in two solvent systems: hexane: diethyl ether (60:40), chloroform: methanol: 20% ammonium hydroxide (80: 20: 2).

phospholipolysis were successively separated in solvent systems hexane diethyl sfir (60:40), chloroform: methanol: 20% - on the am hydroxide. In parallel, experiments were carried out using the regimes indicated in the prototype (Table D).

From the data given in table. 2, 35, it is clear that under these conditions the depth of phosphorylation increases by 25%, the content of degradation products decreases by 10%, the duration of the phosphorylation process decreases to 9 hours, absolute separation of the products of phospholipolysis is provided.

EXAMPLE 3: 0.1 g of 1,2 (2,3) -di 45 acylglycerols, obtained by hydrolysis of triacylglycerols with pancreatic lipase, were phosphorylated with nitrochlorophosphate I in a stream of nitrogen at

pressure of 0.15 MPa, temperature - 12 C 50 held for 10 hours before completion of the reaction, the products of phospholipolysis were successively separated in solvent systems: hexane: diethyl ether (60:40), chloroform: methanol 55: 20 % ammonium hydroxide (80: 20: 2).

In parallel, an experiment was conducted on the modes indicated in the prototype (Table 3).

From the results given in table. 3 it can be seen that in this case the depth of phosphorylation is increased by 25%, the content of the decomposition products is reduced by 20%, the duration of the process is reduced by 8 hours, absolute separation of the products of phospholipsis is provided,

Example 4. 0.1 g of 1,2 (2,3) - -diacylglycerols, obtained by hydrolysis of triacylglycerols with pancreatic lipase, were phosphorylated with phenyldichlorophosphate in a stream of nitrogen at a pressure of 0.09 MPa and kept at a temperature C at 10 ° C until the reaction was completed 7 hours, the phospholipolysis products were successively separated in the systems hexane: diethyl ether (50:50), chloroform: methanol (75:25). In parallel, they conducted an experiment on the modes specified in the Stable prototype. four).

From the results given in table. 4, it can be seen that with the implementation of the scientific research institute according to the regimes beyond the optimal values, only one goal is achieved - reduction of the content of degradation products, while the depth of phosphorylation, the purity of the product separation remains at the level of the prototype.

Example 5. 0.1 g of 1,2 (2,3) - -dacylglycerols, obtained by hydrolysis of triacylglycerols by pancreatic lipase, were phosphorylated with phenyldichlorophosphate in a stream of nitrogen at a pressure of 0.20 MPa, temperature, highest, at + 5 C for 14 hours, the products of phospholipolysis were successively separated in the systems hexane-diethyl ether (70:30), chloroform: methanol (85:15).

In parallel, an experiment was conducted on the modes indicated in the prototype (Table 5).

From the results given in tab. 5, it can be seen that in the implementation of the method according to the regimes, the release of pc1m beyond the limits of the optimal regime, the purity of the separation of products is not achieved.

phospholipolysis and depth of phosphorylation.

Claims (1)

Invention Formula The method of stereospecific analysis of tripolyglycerols, including the hydrolysis of a sample of pancreatic lipase, extracting 2-monoacylglycerols from hydrolysis products and 1,2 (2,3) -diacylglycerols hydrolysis products, determination of 2-monoacetic acid-2-fatty acid composition of 2-chyro-chi-chi-chi-h1-2-chromosome-2-monoacylglycerols; chromatography, phosphorylation of a mixture of 1,2 (2,3) -diacyl glycerols phenyldichlorophosphate | atom at. negative temperatures followed by heating to positive temperatures and aging for several hours : methanol, determination of their fatty acid composition in the 1-position by gas-liquid chromatography and determination of the fatty acid composition in the 3-position calculated in order to increase the accuracy of the analysis by increasing the phosphorylation depth and increasing the purity of the separation of phospholipolysis products, phosphorylation is carried out in a nitrogen atmosphere under a pressure of 0.10-0.15 MPa at a temperature of (-10) - (-12) C and heating is carried out to TeMnepaTy ry 5-8 C, when phospholipolysis products are introduced into the solvent system, chloroform: methanol additional 20% ammonium hydroxide is added, and the second solvent system is used hexane and diethipovy ether with a ratio of solvents 80: 20: 2 and 60:40 by volume, respectively. Depth of phosphorylation,% of the amount of dia1D1glycerols The content of degradation products,% The duration of the phosphorylation process, h Pure separation of phospholipolysis products,% Depth of phosphorylation,% of the amount of diacylglycerols  The content of degradation products,% The duration of the phosphorylation process, h Pure separation of phospholipolysis products,% Table 1 65 90 25 ten 70-80 100 table 2 65 90 25 15 70 100 .Way Prototype Offered at Р 0,15 MPa t -} 2 ° С t +8 С I. -. and% catch 65 90 25 .5 phospho 18 10 duktov 70 100 T a b l and c a 4 Way Prototype Offered at Р 0,09 Sha t t n-Y с Depth of phosphorylation, % of the amount of diacylglycerols Product Content Destrukhschki,% The duration of the phosphorylation process, h Pure separation of phospholipolysis products,% 65 18 70 13 Indicators Depth of phosphorylation, % of the amount of diacylglycerols Product content destruction, 7, The duration of the phosphorylation processes, h Pure separation of phospholipolysis products,% Editor M. Tsitkina Compiled by V.Gordeev Tehred l, Serdyukov Proofreader M.Vasilyeva Order 5338/47 Circulation 847 VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 1430888 14 Table 5 Way Offered at Р 0,2 MPa t -З С t 65 15 14 70 Subscription