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  1. ANALISIS LEMAK Abdul Rohman Faculty of Pharmacy, GadjahMada University, Yogyakarta, Indonesia

  2. Working definition: • Compounds that are soluble in organic solvents (usually ethers). They are derived from living organisms and usually contain fatty acids. • Most fats in foods exist as TAG’s (triacylglycerols), which are non-polar. • SIMPLE LIPIDS include fatty acid esters with glycerol (TAGs, DAG or MAGs), and long chain alcohols (waxes). FAT ANALYSIS

  3. Crude Fat Components • Fats/Oils- TAG’s • Waxes- long-chain alcohols and fatty acids • Phospholipids- phosphoric acid esterified to a fatty acid chain (phosphatides) • Glycolipids- simple sugar esterified to a fatty acid chain • Sterols- specialized ring structure, serving in biological functioning • Free Fatty Acids- carbon chain of various lengths.

  4. Kategorilipid (Carrasco-Pancorbodkk., 2009; Fahydkk., 2005).

  5. Lipid Analysis: trend in the future???

  6. Lipidomics: trend in the future???

  7. Fat Analysis • Analytical Methods generally rely on extraction of the fat from a food and weighing the extracted fat • FDA is interested in a method that is based on amount of fatty acids in 100g of food.

  8. SOLVENT SELECTION • Solvent selection is important since a solvent that is too polar will poorly extract nonpolar lipids and will extract non-lipid materials (like carbohydrates) • Too nonpolar will be inefficient for more polar lipids. • IDEAL SOLVENT FOR FAT EXTRACTION • High solvent power for lipids • Low solvent power for nonlipids • No residue • Evaporate easily (low heat of vaporization) • Low boiling point • Non flammable / not explosive • Nontoxic • Cheap • Non-hygroscopic

  9. Ethyl ether is used a lot but is • Very flammable, • Explosion hazard • Forms peroxides • Expensive. • Petroleum ether is not too expensive and is an excellent solvent for lipids • More selective for more hydrophobic lipids • Non hygroscopic • Less flammable • Cheaper Mixtures of ethyl etherandpetroleum ether are common Mixtures of chloroformandmethanol are also common (Bligh-Dyer) Solvent Selection

  10. SOLVENT SELECTION • Solvent selection is critical to fat extraction. • Solvents such as methanol, ethanol, and acetone will readily dissolve fats, but would also extract large amounts of moisture, CHO, and protein.

  11. Solvent Extraction: Solvent from a continuously boiling solvent source flows over the sample held in a sample thimble. Fat content is measured by weight loss of the sample or by weight of fat removed. • Ethyl ether, petroleum ether, hexane, or methylene chloride are common solvents • Extraction times range from 4-16 hrs • Sample is weighed, mixed with sand to increase surface area, and dried in a forced air oven. • Lipid is extracted by the solvent • Solvent is removed by evaporation or under reduced pressure, then dried at 100°C for 30 min. GOLDFISCH Extraction

  12. AlatpengekstraksilemakGoldfisch (Sumber: Labconco, 2011).

  13. Similar sample prep to Goldfisch method • Fat is extracted, semi-continuously, with an organic solvent • Sample is in contact with the solvent in the extraction chamber for 5-10 min (see diagram) • Extraction time: 5-6 drops per second (4 hr). 2-3 drops per second (16 hrs). • Fat content is measured by weigh loss of sample or weight of fat removed SOXHLET Extraction

  14. AlatSoxhlet


  16. Fats and Oils Characterization

  17. Acid value (bilanganasam) • Bilanganasamataunilaiasamdanjugadikenaldenganindekskeasaman. • Didefinisikansebagaibanyaknyamiligramkaliumhidroksida (KOH) yang dibutuhkanuntukmenetralkanasambebasdalam 1 gram minyak, lemak.

  18. Bilanganasam = Kadar asamlemakbebas (%) =

  19. Free Fatty Acids (FFA’s) • Degree of hydrolysis (hydrolytic rancidity) • Example: good frying oil should have 0.05% max. FFA’s (as oleic acid) • High level of FFA means a poorly refined fat or fat breakdown after storage or use.

  20. Saponification Value Saponification is the process of breaking down or degrading a neutral fat into glycerol and fatty acids by treating the sample with alkali. Heat Triacylglyceride ---> Fatty acids + Glycerol KOH

  21. Bilanganpenyabunan • Bilangan penyabunan atau nilai penyabunan atau bilangan Koettsdorfer. • Didefinisikansebagaibanyaknyamiligram KOH yang dibutuhkanuntukmenyabunkanlemaksecarasempurnadari 1 gram lemakatauminyak. Bilanganpenyabunan =

  22. Bilanganiodium • Bilanganiodiumatauangkaiodiumdidefinisikansebagaibanyaknyaiodium yang diserapoleh 100 gram minyak, lemak. • Bilanganinimerupakanpengukurankuantitatif yang menyatakanbanyaknyaasam-asamlemaktidakjenuh, baikdalambentukbebasataudalambentuk ester, yang terdapatdalamminyakataulemakkarenaasamlemakinimempunyaisifat yang mampumenyerapiodium

  23. Iodine Value What does it tell us about the oil? • The higher the amount of unsaturation, the more iodine is absorbed. • Therefore the higher the iodine value, the greater the degree of unsaturation.

  24. Iodine Value • A known solution of KI is used to reduce excess ICl (or IBr) to free iodine R-C-C = C-C-R + ICl R-C-CI-CCl-C-R + ICl [Excess] (remaining) • Reaction scheme:ICl + 2KIKCl + KI + I2 • The liberated iodine is then titrated with a standardized solution of sodium thiosulfate using a starchindicator • I2 + Starch + thiosulfate = colorless endpoint (Blue colored)

  25. BilanganIodium = gI2/100 g lipid Bilanganiodium =

  26. Bilanganiodiumbeberapa lipid

  27. FTIR spectroscopy for IV determination

  28. Iodine Value Used to characterize oils: • Following hydrogenation • Degree of oxidation (unsaturationdecreases during oxidation) • Comparison of oils • Quality control

  29. LIPID OXIDATION Lipid System Under Oxidizing Conditions

  30. Reaksioksidasiminyak Inisiasi Propagasi Terminasi

  31. Pembentukanprodukoksidasi primer

  32. Pembentukanprodukoksidasisekunder

  33. Peroxide Value • Measures peroxides and hydroperoxides in an oil which are the primary oxidation products (usually the first things formed). • The peroxide value measures the “present status of the oil”. Since peroxides are destroyed by heat and other oxidative reactions, a seriously degraded oil could have a low PV. • Plot of PV vs. storage time shows that PV will peak during oxidation.

  34. LIPID OXIDATION Lipid System Under Oxidizing Conditions

  35. Peroxide Value • The chemistry is simple. KI + peroxyl radical yields free Iodine (I2) • The iodine released from the reaction is measured in the same way as an iodine value. • I2 in the presence of amylose is blue. • I2 is reduced to KI and the endpoint determined by loss of blue color. • Oxygen error occurs when O2 present in the solution. 4I + O2 + 4H 2I2 + 2H2O

  36. Determination for Peroxide Value • PV is expressed as milliequivalents of peroxide per kg of sample x 1000 PV =

  37. Ujiprodukoksidasisekunder • TBARS • Anisidin value

  38. Secondary product:Pembentukanmalonaldehid

  39. Reaksiantara TBA dan MDA

  40. Anisidin value

  41. FA composition: Gas Chromatography Kondisi: kolom, SPTM-2560 (100 m x 0,25 mm i.d; ketebalanlapisan 0,20 µm). Suhu oven: 140 oC (5 menit), dinaikkansampai 240 oCdengankecepatan 4 oC/menit. Gas pembawa, helium 20 cm/detik; detektor, FID 260 oC; injector 260 oCdengancolumeinjeksi 1 µL dannisbahpemecahaninjeksi 100: 1 (Sigma, Aldrich, USA).

  42. Peak ID of FAMEs by GC