Methanolic saponification extraction

Heemken and eo-workers in Germany reported on percent recovery results for the isolation and reeovery of PAHs and aliphatic hydrocarbons from marine samples (47). Results were eompared using accelerated solvent extraetion, ASE, SFE, ultrasonieation (U-LSE), methanolic saponification extraction (MSE), and classical Soxhlet (S-LSE). Both ASE and SFE compared favorably to the more eonventional methods in terms of a relative percent reeovery against the eonventional methods, so that extracted analytes from ASE and SFE were eompared to the same extracted analytes from S-LSE and U-LSE. Relative pereent recoveries ranged from 96% to 105% for the 23 two, through seven (coronene) fused ringed PAH and methyl-substituted PAHs. To evaluate the percent recoveries, the authors defined a bias, D eb according to... 

The mode of extraction for PAHs is highly dependent on the matrix. For solid-based matrices such as food samples, sediments, soil, marine organisms, etc. extraction methods such as Soxhlet extraction with nonpolar solvent [35 6], hollow fiber membrane solvent microextraction (HFMSME) [10], pressimzed hquid extraction (PLE) [37,38], sonication extraction [3], microwave-assisted extraction (MAE) [3], supercritical fluid extraction, (SEE) [39], accelerated solvent extraction (ASE) [40], cold extraction [41], soxtec extraction [42], microwave-assisted alkaline saponification (MAAS) [43], dynamic microwave-assisted extraction (DMAE) [44], add-induced cloud point extraction (ACPE) [45], methanolic saponification extraction (MSE) [7], etc. are employed. Of all these, Soxhlet extraction is the most common for solid samples and has achieved excellent extraction with high-level recovery but its setback is the high consmnption of solvent and time associated with it. 

Carrots Extract sample with hexane/acetone/ab-solute EtOH /toluene (10 7 6 7) + 40% methanolic KOH. Leave for 16 h in the dark for saponification. Extract unsaponifiables with hexane. Vydac 201 TP54 C,8 5 /zm 250 X 4.6 mm... 

Solvent Recovery. A mixture of methanol and methyl acetate is obtained after saponification. The methyl acetate can be sold as a solvent or converted back into acetic acid and methanol using a cationic-exchange resin such as a cross-linked styrene—sulfonic acid gel (273—276). The methyl acetate and methanol mixture is separated by extractive distillation using water or ethylene glycol (277—281). Water is preferred if the methyl acetate is to be hydroly2ed to acetic acid. The resulting acetic acid solution is concentrated by extraction or a2eotropic distillation. 

The ester (70 g, 0.1 mol) was saponified in a boiling mixture of 250 ml methanol and 250 ml water to which 100 ml N sodium hydroxide solution was added in small batches with stirring. The methanol wasdistilled from the saponification mixture, the residue was mixed with water and extracted with ethyl acetate. The aqueous phase was acidified with hydrochloric acid in the presence of sodium bisulfite. 

A general procedure that our laboratory generally employs is the addition of an equal amount of methanolic 10% potassium hydroxyde (KOH) to an ethereal carotenoid extract. This solution is bubbled with N2 and allowed to stand overnight at room temperature. Other conditions that shorten time at room temperature have also been used, such as saponification of the dichloromethane (CH2CI2) extract with the same amount of 10% KOH in MeOH for 1 hr (peppers and fruits ) and ethereal extract treated with 30% methanolic KOH under N2 for 3 hr (green leaves, vegetables and fruits ). 

More severe conditions, 35 ml of 35% methanolic KOH added to 10 mL extract in EtOAc and shaken for 20 min at 50°C, are necessary for the total conversion of bixin, an ester of a carotenoid acid, to norbixin in snacks. Since saponification yields the norbixin salt (K or Na, depending on the alkali) that is soluble in the aqueous phase, the pH should be decreased to 3.5 or even lower to allow extraction of the protonated norbixin by EtOAc and diethyl ether. ... 

Saponification (see Section 7.4) is carried out to extract more recalcitrant lipids, and the yields are higher than for conventional solvent extraction (Stern et al. 2000). 3 ml of 0.5 M methanolic NaOH is added to 0.1 g of the shard powder and heated at 70°C for 3 hours in a sealed glass vial. After cooling, the supernatant is acidified with HC1 and extracted with three aliquots of 3 ml //-hexane. The hexane will not mix with the methanolic solution (unlike the DCM MeOH used above), but will absorb the lipids and can be transferred into a new clean vial. The removal of excess hexane is carried out as above. Saponification will hydrolyze and methylate any ester functionalities, which removes the requirement to derivatize the samples (Section 7.4) unless other molecules are suspected of being present. However, any wax esters or triacylglycerols will also be hydrolyzed to their fatty acid methyl esters and alcohols therefore, if information on their composition is required, then conventional solvent extraction is recommended as a first step. For subsequent characterization of the lipid extract, see Chapter 7. 

Mono-, di- and triacylglycerols may all be measured by determination of the amount of glycerol released by hydrolysis. The lipid is first extracted into chloroform-methanol (2 1) and saponification is performed under conditions that will not affect any phosphate ester bonds, otherwise glycerol originating from phosphoglycerides would also be measured. Heating at 70°C for 30 min with alcoholic potassium hydroxide (0.5 mol l-1) has been shown to be satisfactory. However, the phospholipids may be removed prior to saponification either by extraction or by adsorption on activated silicic acid. 

The effect of saponification on the concentration of carotenoids in fatty foods has also been investigated by RP-HPLC. Sausages containing 5.6 per cent powdered paprika were extracted exhaustively with chloroform-methanol (2 1, v/v). The extracting solvent contained 0.01 per cent butylated hydroxyanisole (BHA). An aliquot of the combined extracts was evaporated to dryness and saponified at 50°C for 5min with 10 per cent KOH in methanol in the presence of 0.01 per cent BHA. Free carotenoid pigments were extracted with diethyl ether, washed with water, dried over anhydrous NajSC and evaporated under... 

Carotenoids A large number of solvents have been used for extraction of carotenoids from vegetables matrices, such as acetone, tetrahydrofuran, n-hexane, pentane, ethanol, methanol, chloroform [427-431], or solvent mixtures such as dichloromethane/methanol, tetrahydrofuran/methanol, -hexane/acetone, or toluene or ethyl acetate [424,432-435], SPE has been used as an additional purification step by some authors [422,426], Supercritical fluid extraction (SEE) has been widely used, as an alternative method, also adding CO2 modifiers (such as methanol, ethanol, -hexane, water, methylene chloride) to increase extraction efficiency [436-438], In addition, saponification can be carried out, but a loss of the total carotenoid content has been observed and, furthermore, direct solvent extraction has been proved to be a valid alternative [439],... 

BASIC PROTOCOL I PREPARATION OF FATTY ACID METHYL ESTERS FROM LIPID SAMPLES CATALYZED WITH BORON TRIFLUORIDE IN METHANOL In this method, lipid samples are first saponified with an excess of NaOH in methanol. Liberated fatty acids are then methylated in the presence of BF3 in methanol. The resulting fatty acid methyl esters (FAMEs) are extracted with an organic solvent (isooctane or hexane), and then sealed in GC sample vials for analysis. Because of the acidic condition and high temperature (100°C) used in the process, isomerization will occur to those fatty acids containing conjugated dienes, such as in dairy and ruminant meat products, that contain conjugated linoleic acids (CLA). If CLA isomers are of interest in the analysis, Basic Protocol 2 or the Alternate Protocol should be used instead. Based on experience, this method underestimates the amount of the naturally occurring cis-9, trans-11 CLA isomer by -10%. The formulas for the chemical reactions involved in this protocol are outlined in Equation D1.2.1 Saponification RCOO-R + NaOH, RCOO-Na + R -OH v 100°C DC Esterification RCOO-Na + CH,OH r 3 v RCOO-CH, + NaOH ioo°c ... 

The removal of triglycerides from the food sample by saponification provides the opportunity to utilize reversed-phase chromatography. The unsaponifiable matter is conventionally extracted into a solvent [e.g., diethyl ether/petroleum ether (50 50) or hexane] that is incompatible with a semiaqueous mobile phase. It then becomes necessary to evaporate the unsaponifiable extract to dryness and to dissolve the residue in a small volume of methanol (if methanol is the organic component of the mobile phase). For the analysis of breakfast cereals, margarine, and butter, Egberg et al. (153) avoided the time-consuming extraction of the unsaponifiable matter and the evaporation step by acidifying the unsaponifiable matter with acetic acid in acetonitrile to precipitate the soaps. An aliquot of the filtered extract could then be injected, after dilution with water, onto an ODS column eluted with a compatible mobile phase (65% acetonitrile in water). 

Saponification is often used to extract xanthophylls as well as remove chlorophylls and lipids from samples prior to analysis, as these compounds can interfere with the chromatographic detection. Although saponification with methanol and potassium hydroxide is routinely used to facilitate carotenoid extraction, numerous studies indicate that saponification can also result in losses of carotenoids. For example, Khachik et al.60 demonstrated that saponification actually caused the loss of total carotenoids in samples. Alternatively, enzymatic saponification using lipase can be used to help prevent the loss and isomerization of some carotenoids. Fang et al.32 suggested that saponification of plasma samples should be avoided to prevent unnecessary lycopene degradation. 

Hullett and Eisenreich [11] used high performance liquid chromatography for the determination of free and bound fatty acids in river water samples. The technique involves sequential liquid-liquid extraction of the water sample by 0.1m hydrochloric acid, benzene-methanol (7 3) and hexane-ether (1 1). The resultant extract was concentrated and the fatty acids were separated as a class on Florasil using an ether-methanol 1 1 and 1 3 elution. Final determination of individual fatty acids was accomplished by forming the chromatophoric phenacyl ester and separating by high performance liquid chromatography. Bound fatty acids were released by base saponification or acid hydrolysis of a water sample from which the fatty acids had been removed by solvent extraction. 

Dimathyl-3-hydroxy-6-heptenoic acid. In a 500-mL flask, the crude ester prepared in Part A (37.00 g, 0.199 mol) is dissolved in a 2 N solution of potassium hydroxide (KOH) in methanol (130 mL, 0.260 mol). The solution is stirred at 25°C and disappearance of the starting material is monitored by GLC (Note 9). After 5 hr saponification is complete and the methanol is evaporated at reduced pressure (Note 11). The residue is taken up with water (500 mL), extracted with diethyl ether (3 x 100 mL), and the organic phase is discarded. The aqueous phase is acidified (pH 2.5 on universal pH indicator paper) with - 6 N hydrochloric acid (about 60 mL) and extracted with diethyl ether (5 x 100 mL). These latter ethereal extracts are washed with water (2 x 30 mL) and then with saturated sodium chloride (2 x 30 mL). The organic phase is dried over sodium sulfate, and filtered. Evaporation at reduced pressure affords crude 3,6-dimethyl-3-hydroxy-6-heptenoic acid as a viscous yellow oil that can be used in... 

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