ANALISIS LEMAK

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 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.
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]

Gliserofosfol GP Asam fosfatidat [GP10]; Fosfatidilkolin [GP01];
ipid 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 Selection
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 ether and petroleum ether are common

Mixtures 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 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.
Alat pengekstraksi lemak Goldfisch
(Sumber: Labconco, 2011).
 SOXHLET Extraction
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
Alat Soxhlet
 FAT CHARACTERIZATION

 PHYSICAL PROPERTIES
 ACID VALUE/FREE FATTY ACIDS
 SAPONIFICATION NUMBER
 IODINE VALUE
 OXIDATION
 HYDROLYSIS
 PEROXIDE VALUE
 OXIDATION TESTS
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.
 ml KOH x N KOH x 56,1
Bilangan asam = berat sampel (g)

x N KOH x BM x100%
Kadar asam lemak bebas (%) = ml KOH

berat sampel (mg )

Sumber minyak Asam lemak BM asam lemak
terbanyak terbanyak
Kelapa sawit Palmitat (C16H32O2) 256
Kelapa, inti sawit Laurat (C12H24O2) 200
Susu Oleat (C18H34O2) 282
Jagung, kedelai Linoleat (C18H32O2) 278
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.
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
Bilangan penyabunan

Bilangan 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 =
(V HCl blanko  V HCl sampel) x N HCl x 56,1
berat sampel (g)
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 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 starch indicator
I2 + Starch + thiosulfate = colorless endpoint
(Blue colored)
Bilangan Iodium = gI2/100 g lipid

Bilangan iodium =
(V tio blanko  V tio sampel) x N tio x 12,69
berat sampel (g)
Bilangan iodium beberapa lipid
No Senyawa Bilangan Iodium
1 Minyak jarak (castor oil) 83 sampai 88
2 Minyak jagung ( corn oil) 102 sampai 128
3 Minyak kapas (cottonseed oil) 109 sampai 116
4 Lanolin hidrous 18 sampai 36
5 Lanolin anhidrous 18 sampai 36
6 Asam oleat 85 sampai 95
7 Minyak zaitun 79 sampai 88
8 Minyak sesami 103 sampai 116
9 Asam stearat t.l.d. 4
10 Setyl alkohol t.l.d. 2
FTIR spectroscopy for IV
determination
Iodine Value

Used to characterize oils:

Following hydrogenation
Degree of oxidation (unsaturation decreases
during oxidation)
Comparison of oils
Quality control
 LIPID OXIDATION
35
Lipid System Under
Reactants and Products

30
Oxidizing Conditions
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10

1 2 3 4 5 6 7 8 9

Time
Reaksi oksidasi minyak

Inisiasi

Propagasi

Terminasi
Pembentukan produk oksidasi primer
Pembentukan produk oksidasi sekunder
 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.
 LIPID OXIDATION
35
Lipid System Under
Reactants and Products

30
Oxidizing Conditions
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10

1 2 3 4 5 6 7 8 9

Time
 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
 Determination for Peroxide Value
 PV is expressed as milliequivalents of
peroxide per kg of sample

ml tiosulfat x N tiosulfat
PV = x 1000
berat sampel (g)
Uji produk oksidasi sekunder

TBARS
Anisidin value
 Secondary product:
Pembentukan malonaldehid

a) dari hidroperoksida asam linolenat b) dari radikal bebas 2-nonenal

OH
O
O
H H H
C CH C C
R1 C C C R2 2-nonenal
H H H H
O2

OH
O
O O
H H O
C CH C C
R1 C C C R2 2-nonenal
H H H H

O O H H
C C H
R1 + HC CH + HC C R2 C5H11 O HO C O
H
C C
H2 + H
Malonaldehid H H

O O

HC CH
C
H2
Malonaldehid
Reaksi antara TBA dan MDA
O O HS N OH

HC CH + 2
C N
H2
Malonaldehid
OH
Asam tiobarbiturat (TBA)

H+

SH
HO N
S N OH
N
H + 2H2O
N C
C C OH
H H

Produk kondensasi
 Anisidin value

OCH3
OCH3
O

+
R1 H+
C H
H
R2
alk-2-enal H R1
NH2 C
N C
p-anisidin
CH
Produk konjugasi R2
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 GC
ID Component (Acid Methyl ID Component (Acid Methyl Esters)
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)
Cholesterol
 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 toluene
3. Derivatize to make trimethylsilylethers
 4. Injected into a GC