ANALISIS LEMAK Abdul Rohman Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia

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2 ANALISIS LEMAK Abdul Rohman Faculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia http://acadstaff.ugm.ac.id/abdulrohman

3 FAT ANALYSIS 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).

4 Crude Fat Components zFats/Oils- TAG’s zWaxes- long-chain alcohols and fatty acids zPhospholipids- phosphoric acid esterified to a fatty acid chain (phosphatides) zGlycolipids- simple sugar esterified to a fatty acid chain zSterols- specialized ring structure, serving in biological functioning zFree Fatty Acids- carbon chain of various lengths.

5 Kategori lipid (Carrasco-Pancorbo dkk., 2009; Fahy dkk., 2005). KateoriSingSub-kategori Asil lemakFAAsam lemak [FA01]; GliserolipidGLMAG [GL01]; DAG [FA02]; TAG [FA03] Gliserofosfol ipid GPAsam fosfatidat [GP10]; Fosfatidilkolin [GP01]; Fosfatidilserin [GP03]; Fosfatidilgliserol [GP04] SpingolipidSPSpingoid basa [SP01]; Ceramida [SP 02]; Fosfospingolipid [SP03]; Fosfonospingolipid [SP04]; Sterol lipidSTSterol [ST01]; Steroid [ST02]; Sekosteroid [ST03] Prenol lipidPRIsoprenoid [PR01]; Kuinon dan hidrokuinon [PR02] SakarolipidSLGula asilamino [SL01]; Gula asilamino glikan [SL02] PoliketidaPKPoliketida makrolida [PK01]; Poliketida aromatis [PK02]

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7 Lipid Analysis: trend in the future???

8 Lipidomics: trend in the future???

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

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

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

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

13 GOLDFISCH Extraction è è 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. è è Ethyl ether, petroleum ether, hexane, or methylene chloride are common solvents è è Extraction times range from 4-16 hrs è è Sample is weighed, mixed with sand to increase surface area, and dried in a forced air oven. è è Lipid is extracted by the solvent è è Solvent is removed by evaporation or under reduced pressure, then dried at 100°C for 30 min.

14 Alat pengekstraksi lemak Goldfisch (Sumber: Labconco, 2011).

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

16 Alat Soxhlet

17 l PHYSICAL PROPERTIES l ACID VALUE/FREE FATTY ACIDS l SAPONIFICATION NUMBER l IODINE VALUE l OXIDATION l HYDROLYSIS l PEROXIDE VALUE l OXIDATION TESTS FAT CHARACTERIZATION

18 Fats and Oils Characterization

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20 Acid value (bilangan asam) zBilangan asam atau nilai asam dan juga dikenal dengan indeks keasaman. z Didefinisikan sebagai banyaknya miligram kalium hidroksida (KOH) yang dibutuhkan untuk menetralkan asam bebas dalam 1 gram minyak, lemak.

21 Bilangan asam = Kadar asam lemak bebas (%) =

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

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24 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 Saponification Value

25 Bilangan penyabunan zBilangan penyabunan atau nilai penyabunan atau bilangan Koettsdorfer. zDidefinisikan sebagai banyaknya miligram KOH yang dibutuhkan untuk menyabunkan lemak secara sempurna dari 1 gram lemak atau minyak. Bilangan penyabunan =

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27 Bilangan iodium zBilangan iodium atau angka iodium didefinisikan sebagai banyaknya iodium yang diserap oleh 100 gram minyak, lemak. z Bilangan ini merupakan pengukuran kuantitatif yang menyatakan banyaknya asam-asam lemak tidak jenuh, baik dalam bentuk bebas atau dalam bentuk ester, yang terdapat dalam minyak atau lemak karena asam lemak ini mempunyai sifat yang mampu menyerap iodium

28 Iodine Value What does it tell us about the oil? zThe higher the amount of unsaturation, the more iodine is absorbed. zTherefore the higher the iodine value, the greater the degree of unsaturation.

29 Iodine Value zA 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) zReaction scheme: ICl + 2KI  KCl + KI + I 2 zThe liberated iodine is then titrated with a standardized solution of sodium thiosulfate using a starch indicator zI 2 + Starch + thiosulfate = colorless endpoint (Blue colored)

30 Bilangan Iodium = gI 2 /100 g lipid Bilangan iodium =

31 Bilangan iodium beberapa lipid

32 FTIR spectroscopy for IV determination

33 Iodine Value Used to characterize oils: zFollowing hydrogenation zDegree of oxidation (unsaturation decreases during oxidation) zComparison of oils zQuality control

34 LIPID OXIDATION Lipid System Under Oxidizing Conditions

35 Reaksi oksidasi minyak Inisiasi Propagasi Terminasi

36 Pembentukan produk oksidasi primer

37 Pembentukan produk oksidasi sekunder

38 l Measures peroxides and hydroperoxides in an oil which are the primary oxidation products (usually the first things formed). l 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. l Plot of PV vs. storage time shows that PV will peak during oxidation. Peroxide Value

39 LIPID OXIDATION Lipid System Under Oxidizing Conditions

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

41 l PV is expressed as milliequivalents of peroxide per kg of sample Determination for Peroxide Value x 1000 PV =

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43 Uji produk oksidasi sekunder zTBARS zAnisidin value

44 Secondary product: Pembentukan malonaldehid

45 Reaksi antara TBA dan MDA

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48 Anisidin value

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50 FA composition: Gas Chromatography Kondisi: kolom, SPTM-2560 (100 m x 0,25 mm i.d; ketebalan lapisan 0,20 µm). Suhu oven: 140 o C (5 menit), dinaikkan sampai 240 o C dengan kecepatan 4 o C/menit. Gas pembawa, helium 20 cm/detik; detektor, FID 260 o C; injector 260 o C dengan colume injeksi 1 µL dan nisbah pemecahan injeksi 100: 1 (Sigma, Aldrich, USA).

51 Peak ID of FAMEs by GC

52 l Many methods available: TLC, GC, HPLC, enzymatic, etc. zGC is most common approach: z1. Saponify fat with potassium hydroxide (cholesterol is in the unsaponifiable fraction). 2. Extract fraction with benzene or toluene 3. Derivatize to make trimethylsilylethers z4. Injected into a GC Cholesterol