Iodine Value - Farmasi Carbon 2012

Report
ANALISIS LEMAK
Abdul Rohman
Faculty of Pharmacy, Gadjah Mada University,
Yogyakarta, Indonesia
http://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 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
Asil lemak
Sing Sub-kategori
FA Asam lemak [FA01];
Gliserolipid
GL
MAG [GL01]; DAG [FA02]; TAG [FA03]
Gliserofosfol GP
ipid
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
[PK02]
makrolida
[PK01];
Poliketida
aromatis
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.
Bilangan asam =
ml
KOH
xN
KOH
berat sampel (g)
Kadar asam lemak bebas (%) =
Sumber minyak
Kelapa sawit
x 56,1
ml
KOH
xN
berat
KOH
x BM x100 %
sampel (mg
Asam lemak
BM asam lemak
terbanyak
terbanyak
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
1
2
3
4
5
6
7
8
9
10
Senyawa
Minyak jarak (castor oil)
Minyak jagung ( corn oil)
Minyak kapas (cottonseed oil)
Lanolin hidrous
Lanolin anhidrous
Asam oleat
Minyak zaitun
Minyak sesami
Asam stearat
Setyl alkohol
Bilangan Iodium
83 sampai 88
102 sampai 128
109 sampai 116
18 sampai 36
18 sampai 36
85 sampai 95
79 sampai 88
103 sampai 116
t.l.d. 4
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
Reactants and Products
35
Lipid System Under
Oxidizing Conditions
30
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10
5
0
1
2
3
4
5
Time
6
7
8
9
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
Reactants and Products
35
Lipid System Under
Oxidizing Conditions
30
25
Oxygen Uptake
20
Peroxides
15
Secondary Products
10
5
0
1
2
3
4
5
Time
6
7
8
9
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
PV =
ml
tiosulfat
xN
tiosulfat
berat sampel (g)
x 1000
Uji produk oksidasi sekunder
TBARS
Anisidin value
Secondary product:
Pembentukan malonaldehid
b) dari radikal bebas 2-nonenal
a) dari hidroperoksida asam linolenat
OH
O
H
C
R1
H
C
CH
C
H
C
H
O
H
C
C
H
2-nonenal
R2
H
O2
OH
O
O
O
C
R1
CH
C
H
O
H
C
C
H
O
R1 + HC
CH
C
H2
Malonaldehid
C
H
R2
H
C
+
HC
O
H
C
2-nonenal
H
H
C
C
H
R2
C5H11
H
C
HO
O
O
C
H
+
H
H
O
O
HC
CH
C
H2
Malonaldehid
Reaksi antara TBA dan MDA
O
HS
O
HC
CH
+
2
C
H2
Malonaldehid
N
OH
N
OH
Asam tiobarbiturat (TBA)
H+
SH
HO
S
N
N
OH
N
+ 2H2O
H
C
N
C
H
C
H
Produk kondensasi
OH
Anisidin value
OCH3
OCH3
O
+
R1
H+
C
H
R2
NH2
p-anisidin
H
H
C
alk-2-enal
N
CH
Produk konjugasi
R1
C
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
ID
Component (Acid Methyl Esters)
1
2
Component (Acid Methyl
Esters)
C4:0 (Butyric)
C6:0 (Caproic)
20
21
C18:2n6t(Linolelaidic)
C18:3n6 ( -Linolenic)
3
4
5
6
7
C8:0 (Caprylic)
C 10:0 (Capric)
C11:0 (Undecanoic)
C12:0 (Lauric)
C13:0 (Tridecanoic)
22
23
24
25
26
8
C14:0 (Myristic)
27
9
10
C14:1 (Myristoleic)
C 15:0 (Pentadecanoic)
28
29
11
12
C15:1 (cis- 10-Pentadecenoic)
C 16:0 (Palmitic)
30
31
C 1 8:3n3 ( -Linolenic)
C20:0 (Arachidic)
C20:1n9 (cis-11-Eicosenoic)
C20:2 (cis-11;14-Eicosadienoic)
C20:3n6 (cis-8;11;14Eicosatrienoic)
C20:3n3 (cis-11;14;17Eicosatrienoic)
C20:4n6 (Arachidonic)
C20:5n3 (cis-5;8;11;14;17Eicosapentaenoic)
C21:0 (Henicosanoic)
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

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