ANALISIS LEMAK
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ANALISIS LEMAK
Abdul RohmanFaculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesiahttp://acadstaff.ugm.ac.id/abdulrohman
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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).
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Crude Fat ComponentsFats/Oils- TAG’s Waxes- long-chain alcohols and fatty
acidsPhospholipids- 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.
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Kategori lipid (Carrasco-Pancorbo dkk., 2009; Fahy dkk., 2005).
Kateori Sing
Sub-kategori
Asil lemak FA Asam lemak [FA01];
Gliserolipid
GL MAG [GL01]; DAG [FA02]; TAG [FA03]
Gliserofosfolipid
GP Asam fosfatidat [GP10]; Fosfatidilkolin [GP01]; Fosfatidilserin [GP03]; Fosfatidilgliserol [GP04]
Spingolipid
SP Spingoid basa [SP01]; Ceramida [SP 02]; Fosfospingolipid [SP03]; Fosfonospingolipid [SP04];
Sterol lipid ST Sterol [ST01]; Steroid [ST02]; Sekosteroid [ST03]
Prenol lipid
PR Isoprenoid [PR01]; Kuinon dan hidrokuinon [PR02]
Sakarolipid
SL Gula asilamino [SL01]; Gula asilamino glikan [SL02]
Poliketida PK Poliketida makrolida [PK01]; Poliketida aromatis [PK02]
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Lipid Analysis: trend in the future???
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Lipidomics: trend in the future???
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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.
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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
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Solvent SelectionEthyl ether is used a lot but is
Very flammable,Explosion hazard Forms peroxidesExpensive.
Petroleum ether is not too expensive and is an excellent solvent for lipids
More selective for more hydrophobic lipidsNon hygroscopic Less flammableCheaper
Mixtures of ethyl ether and petroleum ether are commonMixtures of chloroform and methanol are also common (Bligh-Dyer)
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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.
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GOLDFISCH Extraction
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 hrsSample is weighed, mixed with sand to increase surface
area, and dried in a forced air oven.Lipid is extracted by the solventSolvent is removed by evaporation or under reduced
pressure, then dried at 100°C for 30 min.
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condenser
sample
solvent
fat in solvent
GOLDFISCH
Alat pengekstraksi lemak Goldfisch (Sumber: Labconco, 2011).
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SOXHLET ExtractionSimilar sample prep to Goldfisch methodFat is extracted, semi-continuously, with an organic
solventSample 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
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Alat Soxhlet
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PHYSICAL PROPERTIES ACID VALUE/FREE FATTY ACIDS SAPONIFICATION NUMBER IODINE VALUE OXIDATION HYDROLYSIS PEROXIDE VALUE OXIDATION TESTS
FAT CHARACTERIZATION
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Fats and Oils Characterization
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Acid value (bilangan asam)Bilangan asam atau nilai asam dan
juga dikenal dengan indeks keasaman.
Didefinisikan sebagai banyaknya miligram kalium hidroksida (KOH) yang dibutuhkan untuk menetralkan asam bebas dalam 1 gram minyak, lemak.
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Bilangan asam = (g)sampel berat 56,1 x N x ml KOHKOH
Kadar asam lemak bebas (%) =)sampel (mgberat
100xBM x x N ml KOHKOH %
Sumber minyak Asam lemak terbanyak
BM asam lemak terbanyak
Kelapa sawit Palmitat (C16H32O2) 256Kelapa, inti sawit
Laurat (C12H24O2) 200
Susu Oleat (C18H34O2) 282Jagung, kedelai Linoleat (C18H32O2) 278
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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.
Free Fatty Acids (FFA’s)
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Saponification is the process of breaking down or degrading a neutral fat into glycerol and fatty acids by treating the sample with alkali.
HeatTriacylglyceride ---> Fatty acids + Glycerol KOH
Saponification Value
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Bilangan penyabunanBilangan penyabunan atau nilai
penyabunan atau bilangan Koettsdorfer.
Didefinisikan sebagai banyaknya miligram KOH yang dibutuhkan untuk menyabunkan lemak secara sempurna dari 1 gram lemak atau minyak. Bilangan penyabunan =
(g)sampel berat 56,1 x HCl N x sampel) V HClblanko(V HCl
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Bilangan iodium Bilangan iodium atau angka iodium didefinisikan
sebagai banyaknya iodium yang diserap oleh 100 gram minyak, lemak.
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
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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.
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Iodine ValueA known solution of KI is used to reduce
excess ICl (or IBr) to free iodineR-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 starch indicator
I2 + Starch + thiosulfate = colorless endpoint (Blue colored)
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Bilangan Iodium = gI2/100 g lipid
Bilangan iodium =
(g)sampelberat 12,69 x tio N x sampel) tioV blanko tio(V
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Bilangan iodium beberapa lipid
No Senyawa Bilangan Iodium12345678910
Minyak jarak (castor oil)Minyak jagung ( corn oil)Minyak kapas (cottonseed oil)Lanolin hidrousLanolin anhidrous Asam oleat Minyak zaitunMinyak sesamiAsam stearatSetyl alkohol
83 sampai 88102 sampai 128109 sampai 11618 sampai 3618 sampai 3685 sampai 9579 sampai 88103 sampai 116t.l.d. 4t.l.d. 2
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FTIR spectroscopy for IV determination
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Iodine ValueUsed to characterize oils:
Following hydrogenationDegree of oxidation (unsaturation decreases during oxidation)
Comparison of oilsQuality control
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LIPID OXIDATION
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9
Time
Reac
tant
s an
d Pr
oduc
ts
Oxygen UptakePeroxidesSecondary Products
Lipid System Under Oxidizing Conditions
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Reaksi oksidasi minyak
Inisiasi
Propagasi
Terminasi
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Pembentukan produk oksidasi primer
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Pembentukan produk oksidasi sekunder
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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.
Peroxide Value
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LIPID OXIDATION
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9
Time
Reac
tant
s an
d Pr
oduc
ts
Oxygen UptakePeroxidesSecondary Products
Lipid System Under Oxidizing Conditions
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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
Peroxide Value
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PV is expressed as milliequivalents of peroxide per kg of sample
Determination for Peroxide Value
(g)sampelberatNxml tiosulfattiosulfat
x 1000PV =
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Uji produk oksidasi sekunderTBARSAnisidin value
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Secondary product:Pembentukan malonaldehid
a) dari hidroperoksida asam linolenat b) dari radikal bebas 2-nonenal
R1
HC
CH
CHCH
HC
CH
HC
R2
OOH
R1
CCH
CHCH
HC
CH
HC
R2
OO
HCCH2
CH
OO
HC
HC
CH
HC
R2R1 +
Malonaldehid
+
O
2-nonenalH
O2
O
2-nonenalH
OOH
HC
CH
HO O
H
C5H11 O
H+
HCCH2
CH
OO
Malonaldehid
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Reaksi antara TBA dan MDA
HCCH2
CH
OO
Malonaldehid
+ 2N
NHS OH
OHAsam tiobarbiturat (TBA)
H+
N
NS
CH
HC
CH
N
NOH
OH
SH
HO
+ 2H2O
Produk kondensasi
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Anisidin valueOCH3
NH2
+ R1
R2
CH
H
OOCH3
NCH
HC
CR1
R2
H+
p-anisidin
alk-2-enal
Produk konjugasi
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FA composition: Gas Chromatography
Kondisi: kolom, SPTM-2560 (100 m x 0,25 mm i.d; ketebalan lapisan 0,20 µm). Suhu oven: 140 oC (5 menit), dinaikkan sampai 240 oC dengan kecepatan 4 oC/menit. Gas pembawa, helium 20 cm/detik; detektor, FID 260 oC; injector 260 oC dengan colume injeksi 1 µL dan nisbah pemecahan injeksi 100: 1 (Sigma, Aldrich, USA).
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Peak ID of FAMEs by GCID Component (Acid Methyl
Esters) ID Component (Acid Methyl Esters)
1 C4:0 (Butyric) 20 C18:2n6t(Linolelaidic) 2 C6:0 (Caproic) 21 C18:3n6 ( -Linolenic) 3 C8:0 (Caprylic) 22 C 1 8:3n3 ( -Linolenic) 4 C 10:0 (Capric) 23 C20:0 (Arachidic) 5 C11:0 (Undecanoic) 24 C20:1n9 (cis-11-Eicosenoic) 6 C12:0 (Lauric) 25 C20:2 (cis-11;14-Eicosadienoic) 7 C13:0 (Tridecanoic) 26 C20:3n6 (cis-8;11;14-
Eicosatrienoic) 8 C14:0 (Myristic) 27 C20:3n3 (cis-11;14;17-
Eicosatrienoic) 9 C14:1 (Myristoleic) 28 C20:4n6 (Arachidonic) 10 C 15:0 (Pentadecanoic) 29 C20:5n3 (cis-5;8;11;14;17-
Eicosapentaenoic) 11 C15:1 (cis- 10-Pentadecenoic) 30 C21:0 (Henicosanoic) 12 C 16:0 (Palmitic) 31 C22:0 (Behenic)
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Many methods available: TLC, GC, HPLC, enzymatic, etc.
GC is most common approach: 1. Saponify fat with potassium hydroxide
(cholesterol is in the unsaponifiable fraction).2. Extract fraction with benzene or toluene3. Derivatize to make trimethylsilylethers
4. Injected into a GC
Cholesterol