Fat analysis

Fat ananlysis

1. Fat analysis Dr. Arfaa Sajid Assistant Professor Department of Chemistry,UOL

2. Lipids, proteins, and carbohydrates constitute the principal structural components of foods. Lipids are a group of substances that, in general, are soluble in ether, chloroform, or other organic solvents but are sparingly soluble in water.

3. Other lipids, such as di - and monoacylglycerols , have both hydrophobic and hydrophilic moieties in their molecules and are soluble in relatively polar solvents. (Ice cream, peanut butter, shortening) Some lipids, such as triacylglycerols , are very hydrophobic. (body fat & vegetable fat)

4. Short-chain fatty acids such as C1–C4 are completely miscible in water and insoluble in nonpolar solvents. Formic acid Acetic acid Propanoic acid Butyric acid

5. Lipids comprise a broad group of substances that have some common properties and compositional similarities. Triacylglycerols are fats and oils that represent the most prevalent category of the group of compounds known as lipids. “Lipid” commonly refers to the broad term, total collection of food molecules that are insoluble in water. Fats generally refer to those lipids that are solid at room temperature. Oils generally refer to those lipids that are liquid at room temperature. Oil Fat Liquid at room temperature Solid at room temperature Plant source Animal source Unsaturated Fatty acids Saturated at room temperature

6. Classification of lipids: 1) Simple Lipids: Ester of fatty acids with alcohol: Fats: Esters of fatty acids with glycerol – triacylglycerols Waxes: Esters of fatty acids with long-chain alcohols other than glycerols (e.g., myricyl palmitate , cetyl palmitate , vitamin A esters, and vitamin D esters ) Mericyl palmitate Mericyl alcohol: C 30 H 62 O Cetyl alcohol: C 10 H 34 O

7. Compound Lipids: Compounds containing groups in addition to an ester of a fatty acid with an alcohol: Phospholipids: Glycerol esters of fatty acids, phosphoric acids, and other groups containing nitrogen ( e.g., phosphatidyl choline , phosphatidyl serine, phosphatidyl ethanolamine, and phosphatidyl inositol ) It has glycerol as a backbone. phosphatidyl choline

8. Cerebrosides : Compounds containing fatty acids, a carbohydrate, and a nitrogen moiety ( e.g., galactocerebroside and glucocerebroside ) Sphingolipids : Compounds containing fatty acids, a nitrogen moiety, and phosphoryl group ( e.g., sphingomyelins ). It has ceramide backbone ( sphingosine and a fatty acid).

9. 3) Derived Lipids Derived lipids are substances derived from neutral lipids or compound lipids. They have the general properties of lipids – examples are fatty acids, long chain alcohols, sterols, fat-soluble vitamins, and hydrocarbons. Sterol

10. An accurate and precise quantitative and qualitative analysis of lipids in foods is important for Accurate nutritional labeling, Determination of whether the food meets the standard of identity, To ensure that the product meets manufacturing specifications. Inaccuracies in analysis may prove costly for manufacturers and could result in a product of undesirable quality and functionality. Importance of Analysis:

11. General considerations: Lipids are soluble in organic solvents and insoluble in water. Therefore, water insolubility is the essential analytical property used as the basis for the separation of lipids from proteins, water, and carbohydrates in foods. The wide range of relative hydrophobicity of different lipids makes the selection of a single universal solvent impossible for lipid extraction of foods. For example Glycolipids are soluble in alcohols and have a low solubility in hexane . In contrast, triacylglycerols are soluble in hexane and petroleum ether , which are nonpolar solvents. Some lipids in foods are components of complex lipoproteins and liposaccharides ; therefore, successful extraction requires that bonds between lipids and proteins or carbohydrates be broken so that the lipids can be freed and solubilized in the extracting organic solvents.

12. Solvent extraction methods: Continuous Solvent Extraction Method: Goldfish Method Semicontinuous Solvent Extraction Method: Soxhlet Method Discontinuous Solvent Extraction Methods: Mojonnier Method Non solvent wet extraction methods: Babcock Method Gerber Method Instrumental methods : Infrared Method Specific Gravity (Foss-Let Method) Nuclear Magnetic Resonance Gas Chromatography Methods for fat analysis:

13. Solvent extraction method: Sample preparation: The validity of fat analysis of food depends upon proper sampling and preservation of sample before analysis. An ideal sample should be as close as possible in all of its intrinsic properties to the material from which it is taken. The sample preparation for lipid analysis depends on Type of food Type and nature of lipids in the food Drying sample: (Vacuum oven drying at low temperature or lyophilization ) Particle size reduction: (grinding at low temperature to minimize lipid oxidation. Finished products may best be ground after freezing with liquid nitrogen.) Acid hydrolysis: The sample can be predigested by refluxing for 1 h with 3N hydrochloric acid. For example, the acid hydrolysis of two eggs requires 10 ml of HCl and heating in a water bath at 65◦C for 15–25 min or until the solution is clear.   Dried egg: 42.39%F (AH) 36.74%F (NAH) Flour: 1.73% (AH) 1.20% (NAH)

14. Solvent selection: Ideal solvents for fat extraction should have a high solvent power for lipids and low or no solvent power for proteins, amino acids, and carbohydrates. They should evaporate readily and leave no residue, Have a relatively low boiling point, Nonflammable and nontoxic in both liquid and vapor states. The ideal solvent should penetrate sample particles readily, be in single component form to avoid fractionation, Inexpensive Nonhygroscopic Ethyl ether has a boiling point of 34.6 ◦C and is a better solvent for fat than petroleum ether.

15. Continuous Solvent Extraction Method: Goldfish Method Principle: For continuous solvent extraction, solvent from a boiling flask continuously flows over the sample held in a ceramic thimble. Fat content is measured by weight loss of the sample or by weight of the fat removed. Advantage: The continuous methods give faster and more efficient extraction than semicontinuous extraction methods. Disadvantage: However, they may cause channeling which results in incomplete extraction Calculations: Weight of fat in sample=( beaker+fat ) −beaker % Fat on dry weight basis = (g of fat in sample/g of dried sample) × 100

16. Semicontinuous Solvent Extraction Method: Soxhlet Method Principle: For semicontinuous solvent extraction, the solvent builds up in the extraction chamber for 5–10 min and completely surrounds the sample and then siphons back to the boiling flask. Fat content is measured by weight loss of the sample or by weight of the fat removed. Advantage: This method provides a soaking effect of the sample and does not cause channeling. Disadvantage : However, this method requires more time than the continuous method. Calculation: % Fat on dry weight basis = (g of fat in sample/g of dried sample)×100

17. Discontinuous Solvent Extraction Methods: Mojonnier Method Principle: Fat is extracted with a mixture of ethyl ether and petroleum ether in a Mojonnier flask, and the extracted fat is dried to a constant weight and expressed as percent fat by weight. Calculations % Fat = 100 × {[(wt dish + fat) − (wt dish)] −( avg wt blank residue)}/wt sample The advantages  itself, it more precise because  MOJONNIER method  use extraction for three times thus it can reduce the rate of contamination. The disadvantages  is it use many chemical as the solvent which some of the solvent is harm to human skin and hazard

18. Procedure: (a) Weigh, to the nearest 0.1mg, 10 g of milk into a Mojonnier fat extraction flask. (b) Add 1.5 ml of NH 4 OH and shake vigorously. Add 2 ml if the sample is sour. NH 4 OH neutralizes the acidic sample and dissolves protein. (c) Add 10ml of 95% ethanol and shake for 90 s. The alcohol prevents possible gel formation. (d) Add 25ml of ethyl ether and shake for 90 s. The ether dissolves the lipid. (e) Cool if necessary, and add 25ml of petroleum ether and shake for 90 s. The petroleum ether removes moisture from the ethyl ether extract and dissolves more nonpolar lipid. (f) Centrifuge for 30 s at 600 rpm. (g) Decant ether solution from the Mojonnier flask into the previously weighed Mojonnier fat dish. (h) Perform second and third extractions in the same manner as for the first extraction described previously (ethanol, ethyl ether, petroleum ether, centrifugation, decant). ( i ) Evaporate the solvent in the dish on the electric hot plate at ≤100◦C in a hood. (j) Dry the dish and fat to a constant weight in a forced air oven at 100 ◦C ± 1◦C. (k) Cool the dish to room temperature and weigh.

19. Nonsolvent wet extraction methods: A: Babcock Method for Milk Fat: Principle: In the Babcock method, H 2 SO4 is added to a known amount of milk in the Babcock bottle. The sulfuric acid digests protein, generates heat, and releases the fat. Centrifugation and hot water addition isolate fat for quantification in the graduated portion of the test bottle. The fat is measured volumetrically, but the result is expressed as percent fat by weight. Applications: The Babcock method, which is a common official method for the determination of fat in milk, takes about 45min and duplicate tests should agree within 0.1%. The Babcock method does not determine the phospholipids in the milk products. It is not applicable to products containing chocolate or added sugar without modification because of charring of chocolate and sugars by sulfuric acid. Babcock milk test bottles for milk

20. B: Gerber Method for Milk Fat Principle: The principle of the Gerber method is similar to that of the Babcock method, but it uses sulfuric acid and amyl alcohol. The sulfuric acid digests proteins and carbohydrates, releases fat, and maintains the fat in a liquid state by generating heat. Applications The Gerber method is comparable to the Babcock method but is simpler and faster and has wider application to a variety of dairy products. The isoamyl alcohol generally prevents the charring of sugar found with the regular Babcock method. Butyrometer Dr. Niklaus  Gerber  of Switzerland in 1891

21. Total Fat by GC for Nutrition Labeling: Sample preparation for GC:

22. Total Fat by GC for Nutrition Labeling Example of chromatogram from gas chromatography analysis