游离脂肪酸测定方法

tive results in the analysis of milk and other products containing significant quantities of water. Various procedures have been
proposed for isolating the FFA from the lipid extract after extraction. These methods include column chromatography with supports such as silicic acid-KOH 16, 81, strong anion exchange resins [3, 9-11], and alumina 112, 131 or converting the FFA with tetramethylammonium hydroxide (TMAH) to TMA-soaps [41. Some of these procedures give satisfactory results but most of them are time consuming. Recent developments in capillary gas chromatography make it possible to separate underivatized FFA with chain lengths from C2:O to C2O:O in one run. Tedious procedures such as the quantitative conversion of lower FFA to fatty acid methyl- or ethyl-esters are therefore unnecessary. This paper describes the results of a study to find a rapid, quantitative method for the determination of the individual FFA in milk and cheese. The sturds:
Capillary gas chromatography Solid-phase clean-up Free fatty acids (C2-C20) Quantification/repeatability Dairy products
Summary
A rapid, reliable and precise capillary gas chromatographic method for routine quantification of short- and long-chain free fatly acids (FFA) in milk and cheese is described. Proceduresof (1) lipid extraction, (2) isolation of the FFAfrom milk and cheese capillary gas chromatographic analysis were extracts, and (3) developedand optimized. FFA can be extracted from cheese for 95-100 % with ether-heptane after grinding with sodium sulfate and addRion of 2.5 M sulfuric acid. From milk, 95-100 YOof the FFA (2 C8:O) are also extracted with ether-heptane after addition of ethanol and 2.5 M sulfuric acid. Internal standards are used to compensate for the losses of lower FFA (C2:O-CS:O) in the aqueous phase. Inview of the excellent recovery (98-100 %) and a considerable saving of time, the use of an aminopropyl column is preferred for the isolation of the FFA from lipidextracts. The underivatized FFA are separated directly by capillary gas chromatography making use of columns which enable accurate and rapid (540 min) determination of FFA C2:0-C20:0. With the method described, all major FFA (C2:0-C18:3) in milk and cheese can be quantified with good repeatability(rsdless then 2 %).The method is also applicable to the analysis of short-chain fatty acids in other products.
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separation of FFA with chain lengths from C2:O to C20:O by capillary gas chromatography (CGC).
1 Introduction
Although free fatty acids (FFA) are usually present only at low levels in dairy products, they play an important role in terms of organoleptic quality. FFA are formed mainly as a result of lipolytic enzyme action on glycerides. Many methods for the determination of the individual FFA in dairy products have been published. Most methods involve lipid extraction, isolation of the FFA and gas-chromatographic (GLC) quantificatlon of the individual FFA. A lot of methods have been used to extract FFA and lipids from dairy products [l-51. Salih et al. [21 and Needs et al. [3]have found satisfactory results by extracting FFA from milk with acidified diethyl ether. To extract FFA from cheese, methods have been reported, where a cheese paste acidified with sulfuric acid is extracted with diethyl ether [4, 51. It should be noted that these procedures are (nearly) quantitative for the higher FFA. However, for the lower FFA (5C8 : 0 partitioning of these acids between ) the organic- and the aqueous phase may influence the quantita-
Determination of Free Fatty Acids in Milk and Cheese
Procedures for Extraction, Clean Up, and Capillary Gas Chromatographic Analysis
Catrienus de Jong and Herman T. Badings* Netherlands Institute for Dairy Research (NIZO), PO. Box 20, 6710 BA Ede,The Netherlands
prepared by saponification of milk fat with 0.5 mol/l KOH in ethanol. After converting the potassium salts to FFA by adding a small quantity H2S04 (2.5 molll), the FFA were dissolved in heptane. For some experiments the FFA reference mixture was enlarged by adding the FFA: C2 : 0, C3 : 0, 2Me C3 : 0 and 3Me C4:O.
8 1990 Dr. Alfred Huethig Publishers
94
Journal of High Resolution Chromatography
Free Fatty Acids in Milk and Cheese
A FFA reference mixture (approx. 15 mg total FFA/ml) was
The method is also applicable to the analysis of short-chain FFA in other products.
2 Materials and Methods
2.1 Reagents and Standards
All solvents were analytical grade and obtained from J.T. Baker (Phillipsburg, USA). Alumina: aluminum oxide neutral (M. Woelm, Eschwege, Germany), deactivated with 10 % water. BondElut aminopropyl columns (500 mg, 2.8 ml), empty BondElut columns and the VacElut processing station were obtained from Analytichem Int. (Harbor City, CA, USA). FFA standards used were obtained from Fluka, (Buchs, Switzerland) (> 99 % pure). An internal standard solution was prepared containing C5 :0, C7:0, C13:O and C17:O (0.5 mg/ml each).
- lipid and FFA extraction from milk and cheese - isolation of the FFA with the use of alumina (12)or a weak anion
exchanger (aminopropyl column) (15).