ANALISIS LEMAK

ANALISIS LEMAK

Abdul RohmanFaculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesiahttp://acadstaff.ugm.ac.id/abdulrohman

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).

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.

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]

Lipid Analysis: trend in the future???

Lipidomics: trend in the future???

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.

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

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)

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.

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.

condenser

sample

solvent

fat in solvent

GOLDFISCH

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

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

Alat Soxhlet

PHYSICAL PROPERTIES ACID VALUE/FREE FATTY ACIDS SAPONIFICATION NUMBER IODINE VALUE OXIDATION HYDROLYSIS PEROXIDE VALUE OXIDATION TESTS

FAT CHARACTERIZATION

Fats and Oils Characterization

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.

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

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)

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

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

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

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.

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)

Bilangan Iodium = gI2/100 g lipid

Bilangan iodium =

(g)sampelberat 12,69 x tio N x sampel) tioV blanko tio(V

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

FTIR spectroscopy for IV determination

Iodine ValueUsed to characterize oils:

Following hydrogenationDegree of oxidation (unsaturation decreases during oxidation)

Comparison of oilsQuality control

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

Reaksi oksidasi minyak

Inisiasi

Propagasi

Terminasi

Pembentukan produk oksidasi primer

Pembentukan produk oksidasi sekunder

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

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

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

PV is expressed as milliequivalents of peroxide per kg of sample

Determination for Peroxide Value

(g)sampelberatNxml tiosulfattiosulfat

x 1000PV =

Uji produk oksidasi sekunderTBARSAnisidin value

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

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

Anisidin valueOCH3

NH2

+ R1

R2

CH

H

OOCH3

NCH

HC

CR1

R2

H+

p-anisidin

alk-2-enal

Produk konjugasi

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).

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)

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