Alexandria University
Faculty Of Science
Practical Training Course In Applications Of Plant Physiology
Prepared By Dr . Mohamed Mohamed Ibrahim
Lecturer Of Plant Physiology
Faculty Of Science , Botany Dept.
Alexandria University
E.Mail: m_ibramim 2004@yahoo.com
LIPID
Soluble in non-polar solvents and insoluble in polar
solvents. Lipid is not polymers.
Lipids:
1.
Fatty acids
2.
Neutral fats and oils
3.
Waxes
4.
Phospholipid
5.
Sterols
6.
Fat soluble vitamins
Fatty Acids
O
R C OH
#1 Carbon
O
R C OH
Acid Group
Polar End - Hydrophilic End
Non-polar End - Hydrophobic End
(Fat-soluble tail)
Saturated Fatty Acids
8
7
CH3 CH2
O
5
3
4
6
2
1
CH2 CH2 CH2 CH2 CH2 C OH
Octanoic Acid
Unsaturated Fatty Acids
8
CH3
7
CH2
5
6
CH2 CH2
4
CH2
O
3
2
1
CH2 CH2 C
OH
3 - Octenoic Acid
8
7
CH3 CH2
O
5
3
4
6
2
1
CH2 CH2 CH2 CH2 CH2 C OH
3, 6 - Octadienoic Acid
Short hand:
8:1 (D3)
8:2 (D3,6)
Cis And Trans Fatty Acids
H
H
CH3 (CH2 )7 C C (CH2 )7
10 9
O
C OH
Cis 9 - Octadecenoic Acid (oleic)
H
CH3 (CH2 )7
O
C C
(CH2 )7
C OH
H
Trans 9 - Octadecenoic Acid (elaidic acid)
Polyunsaturated Fatty Acids
Linoleic acid:
Cis, cis, 9, 12 - Octadecadienoic acid
Linolenic acid:
Cis, cis, cis 9, 12, 15 - Octadecatrienoic acid
Arachidonic acid:
Cis, cis, cis, cis 5, 8, 11, 14 - Eicosatetraenoic
acid
Linoleic Acid
Linolenic Acid
Arachidonic Acid
Naturally-occurring fatty acids
O
R CH2
CH
7
CH CH 2
6
5
CH
4
CH CH 2
3
1.
Cis form
2.
Not conjugated --- isolated double bond.
3.
Even numbered fatty acids.
C OH
CLASSIFICATION OF FATTY ACIDS PRESENT
AS GLYCERIDES IN FOOD FATS
Common
Name
Systematic
Name
Formula
Common source
I. Saturated Fatty Acids
Butyric
Butanoic
CH3(CH2)2COOH
butterfat
Caproic
Hexanoic
CH3(CH2)4COOH
Caprylic
Octanoic
CH3(CH2)6COOH
Capric
Decanoic
CH3(CH2)8COOH
Lauric
Dodecanoic
CH3(CH2)10COOH
Myristic
Tetradecanoic CH3(CH2)12COOH
Palmitic
Hexadecanoic CH3(CH2)14COOH
Stearic
Octadecanoic
CH3(CH2)16COOH
Arachidic
Eicosanoic
CH3(CH2)18COOH
butterfat, coconut
and palm nut oils
coconut and palm
nut oils, butterfat
coconut and palm
nut oils, butterfat
coconut and palm
nut oils, butterfat
coconut and Palm nut oil, most
animal and plant fats
practically all animal and
plant fats
animal fats and minor
component of plant fats
peanut oil
Common
Name
Systematic
Name
Formula
Common source
II. Unsaturated Fatty Acids
A. Monoethenoic Acids
Oleic
Cis 9-octadecenoic
C17H33COOH
plant and animal fats
Elaidic
Trans 9-Octadecenoic
C17H33COOH
animal fats
C17H31COOH
peanut, linseed, and
cottonseed oils
C17H29COOH
linseed and other seed
oils
peanut seed fats
B. Diethenoic Acids
Linoleic
9,12-Octadecadienoic
C. Triethenoid Acids
Linolenic
9,12,15-Octadecatrienoic
Eleostearic 9,11,13-Octadecatrienoic
C17H29COOH
D. Tetraethenoid Acids
Moroctic
Arachidonic
4,8,12,15Octadecatetraenoic
5,8,11,14-
C17H27COOH
fish oils
C19H31COOH
traces in animal fats
Common and Systematic Names of Fatty Acids
Common
Name
Systematic
Name
Formula
Common source
A. Monoethenoic Acids
Oleic
Cis 9-octadecenoic
C17H33COOH
plant and animal fats
Elaidic
Trans 9-Octadecenoic
C17H33COOH
animal fats
C17H31COOH
peanut, linseed, and
cottonseed oils
C17H29COOH
linseed and other seed
oils
peanut seed fats
B. Diethenoic Acids
Linoleic
9,12-Octadecadienoic
C. Triethenoid Acids
Linolenic
9,12,15-Octadecatrienoic
Eleostearic 9,11,13-Octadecatrienoic
C17H29COOH
D. Tetraethenoid Acids
Moroctic
Arachidonic
4,8,12,15Octadecatetraenoic
5,8,11,14Eicosatetraenoic
C17H27COOH
fish oils
C19H31COOH
traces in animal fats
Melting Points and Solubility in Water of Fatty Acids
Melting Point
Solubility in H O
Chain Length
2
CHARACTERISTICS OF FATTY ACIDS
Fatty Acids
M.P.(0C)
mg/100 ml Soluble in H2O
C4
-8
-
C6
-4
970
C8
16
75
C10
31
6
C12
44
0.55
C14
54
0.18
C16
63
0.08
C18
70
0.04
Effects of Double Bonds on the Melting Points
F. A.
M. P. (0C)
16:0
16:1
18:0
18:1
18:2
18:3
20:0
20:4
60
1
63
16
-5
-11
75
-50
M.P.
# Double bonds
FAT AND OILS
Mostly Triglycerides:
O
HO C R
O
H2 C OH
HC OH
H2 C OH
Glycerol
+
HO C R
O
HO C R
3 Fatty Acids
O
H2 C O C R
O
HC O C R
O
H2 C O C R
+ 3 H2O
GLYCERIDES
O
H2 C O C (CH2 )16 CH3
H2 C OH
HC
OH O
HC
H2 C O C (CH2 )16 CH3
OH O
H2 C O C (CH2 )16 CH3
Monoglyceride (a - monostearin)
Diglyceride (a, a' - distearin)
O
H2 C
HC
H2 C
O C (CH2 )16 CH3
O
O C (CH2 )14 CH3
O
( C18 )
O C
(C18 )
(CH2 )16 CH3
(C16 )
Triglyceride (b - palmityl distearin)
Oleic
Palmitic
OPP
Palmitic
a - oleodipalmitin
1 - oleodipalmitin
Linoleic
Oleic
LOO
Oleic
a - Linoleyldiolein
1 - Linoleyldiolein
FATS AND OILS ARE PRIMARILY TRIGLYCERIDES (97-99%)
Vegetable oil - world supply - 68%
Cocoa butter - solid fat
Oil seeds - liquid oil
Animal fat - 28% (from Hogs and Cattle)
Marine oil - 4%
Whale oil
cod liver oil
Fatty Acids (%) of Fats and Oils
Fatty Acids
4
6
8
10
12
14
16
16:1
18:0
18:1
18:2
18:3
Butter
3
3
2
3
3
10
26
7
15
29
2
2
Coconut Cottonseed
6
6
44
18
11
6
7
2
1
4
1
3
18
53
Soybean
12
2
24
54
8
MELTING POINTS OF TRIGLYCERIDES
Triglyceride
Melting Point (°C)
C6
-15
C12
15
C14
33
C16
45
C18
55
C18:1 (cis)
-32
C18:1 (trans)
15
WAXES
Fatty acids + Long chain alcohol
Important in fruits:
1. Natural protective layer in fruits, vegetables, etc.
2. Added in some cases for appearance and protection.
Beeswax (myricyl palmitate), Spermaceti (cetyl palmitate)
O
C30 H61
O
C C15 H31
C16 H33
O
O C C15 H31
PHOSPHOLIPID
Lecithin (phosphatidyl choline)
O
O
H2 C O
R C O
C
R
CH
O
H2 C O P O
O_
Phosphatidic Acid
CH3
CH2
CH2
+
N CH3
CH3
Choline
STEROLS
Male & female sex hormones
Bile acids
Vitamin D
Adrenal corticosteroids
Cholesterol
21
H3C
18
H3C
HO
22
CH3
20
12
19
17
H3C 11 13 14
10
1
9
2
8
3
6 7
4
5
16
15
CH3
FAT SOLUBLE VITAMINS
Vitamin A:
H3 C
CH3
CH3
5
8
9
CH3
CH3
7
6
4
3
CH2 OH
2
1
CH 3
H 3C
H 3C
CH 3
CH 3
Vitamin D2:
H
H
CH 2
HO
Vitamin E:
R1
R2
HO
R3
CH3
CH
3
O
(CH2 CH2 CH2 CH2 )2 CH2 CH2CH2 CH(CH 3 )2
ANALYTICAL METHODS TO MEASURE THE
CONSTANTS OF FATS AND OILS
1.
Acid Value
2.
Saponification Value
3.
Iodine Value
4.
Gas Chromatographic Analysis for Fatty Acids
5.
Liquid Chromatography
6.
Cholesterol Determination
1. Acid Value
Number of mgs of KOH required to neutralize the Free
Fatty Acids in 1 g of fat.
AV =
ml of KOH x N x 56
Weight of Sample
= mg of KOH
2. Saponification Value
Saponification - hydrolysis of ester under alkaline
condition.
O
H2 C O C R
O
HC O C R
O
H2 C O C R
H2 C OH
O
+
3 KOH
HC
OH
H2 C OH
+
3 R C OK
Saponification Value of Fats and Oils
Fat
Saponification #
Milk Fat
210-233
Coconut Oil
250-264
Cotton Seed Oil
189-198
Soybean Oil
189-195
Lard
190-202
2. Saponification Value Determination
Saponification # --mgs of KOH required to saponify 1 g of fat.
1.
5 g in 250 ml Erlenmeyer.
2.
50 ml KOH in Erlenmeyer.
3.
Boil for saponification.
4.
Titrate with HCl using phenolphthalein.
5.
Conduct blank determination.
SP# =
56.1(B - S) x N of HCl
Gram of Sample
B - ml of HCl required by Blank.
S - ml of HCl required by Sample.
3. Iodine Number
Number of iodine (g) absorbed by 100 g of oil.
Molecular weight and iodine number can calculate the
number of double bonds. 1 g of fat adsorbed 1.5 g of
iodine value = 150.
Iodine Value Determination
Iodine Value = (ml of Na2S2O3 volume for blank - ml of Na2S2O3
volume for sample)  N of Na2S2O3  0.127g/meq  100
Weight of Sample (g)
CH
CH
CH
Cl
+ ICl
Iodine chloride
CH
I
Excess unreacted ICl
ICl
I2 +
+
KI
2 Na2 S2 O3
KCl
+
Na2 S4 O6
I2
+ 2 NaI
Iodine Numbers of Triglycerides
Fatty Acids
# of Double-bonds
Iodine #
Palmitoleic Acid
1
95
Oleic Acid
1
86
Linoleic Acid
2
173
Linolenic Acid
3
261
Arachidonic Acid
4
320
Compositions (%) of Fatty Acids of Fats
Fat
C4
C6 C10 C16 C18 C18:1 C18:2 C18:3 C20:4
1
5
5
20
40
2
20
35
3
10
50
4
20
40
40
5
10
20
20
6
30
40
5
40
10
20
20
100
4. GC Analysis for Fatty Acids
1.
Extract fat.
2.
Saponify (hydrolysis under basic condition).
3.
Prepare methyl ester (CH3ONa).
4.
Chromatography methyl ester.
5.
Determine peak areas of fatty acids.
Fatty acids are identified by retention time.
6.
Compare with response curve of standard.
Fatty Acids Methyl Esters:
Response
18:1
14
16
18:2
18
18:3
20
21:1
22
Time
GC condition: 10% DEGS Column (from supelco)
Column temperature 200C.
24
5.
TRIGLYCERIDE ANALYSIS BY LIQUID
CHROMATOGRAPHY
Soybean Oil
Solvent CH3CN/HF
Column 84346 (Waters Associates)
RESPONSE
RETENTION TIME
Oleate-containing triglycerides in olive oil
Fatty Acid
Composition
Total Acyl Carbons:
Unsaturation
Equivalent Carbon
Number
OL2
54:5
44
O2L
54:4
46
OPL
52:3
46
O3
54:3
48
OSL
54:3
48
O2P
52:2
48
O2S
54:2
50
OPS
52:1
50
OS2
54:1
52
6. CHOLESTEROL DETERMINATION
Enzymatic Determination: Cholesterol Oxidase
Cholesterol Oxidase
etc. +
HO
H2 O2
O
CH3O
H2 O2 + H2N
OCH3
NH2
CH3O
Peroxidase
HN
OCH3
NH
+ H2 O
0-Dianisidine
Oxidized 0-Dianisidine
(Colorless)
(Brown color)At 440 nm
Absorption
at 440 nm
 g/ml Cholesterol
Cholesterol by GLC
1.
Prepare cholesterol butyrate.
2.
Analyze by GLC.
time in GC - 15 min.
sensitivity - 10-7 g.
Spectromertic Absorption Standard Curve of Cholesterol
Absorption
at 440 nm
 g/ml Cholesterol
Cholesterol by GLC
1.
Prepare cholesterol butyrate.
2.
Analyze by GLC.
time in GC - 15 min.
sensitivity - 10-7 g.
LIPID CONTENT ANALYSES
1.
Gravimetric Method
(1) Wet extraction - Roese Gottliegb & Mojonnier.
(2) Dry extraction - Soxhlet Method.
2.
Volumetric Methods (Babcock, Gerber Methods)
1. Gravimetric Method
(1) Wet Extraction - Roese Gottlieb & Mojonnier.
For Milk:
1) 10 g milk + 1.25 ml NH4OH mix. solubilizes
protein and neutralizes.
2) + 10 ml EtOH - shake. Begins extraction, prevents
gelation of proteins.
3) + 25 ml Et2O - shake and mix.
4) + 25 ml petroleum ether, mix and shake.
(2) Dry Extraction - Soxhlet Method.
Sample in thimble is continuously extracted with ether
using Soxhlet condenser. After extraction, direct
measurement of fat
- evaporate ether and weigh the flask.
Indirect measurement - dry thimble and weigh thimble and
sample.
Soxhlet Method.
2. Volumetric Method (Babcock, Gerber Methods)
Theory:
1. Treat sample with H2SO4 or detergent.
2. Centrifuge to separate fat layer.
3. Measure the fat content using specially calibrated bottles.
Methods:
1. Known weight sample.
2. H2SO4 - digest protein, liquefy fat.
3. Add H2O so that fat will be in graduated part of bottle.
4. centrifuge to separate fat from other materials completely.
REACTIONS OF FATS
Hydrolytic Rancidity:
1. Triglyceride -> Fatty acids
Specially C4 butyric acid (or other short chain fatty
acids) are the real problem.
2. By lipase.
LIPID OXIDATION
Major flavor problems in food during storage are mainly
due to the oxidation of lipid.
Lipid Oxidation - free radical reactions.
1.
Initiation.
2.
Propagation.
3.
Termination.
Pentane Formation from Linolenic Acid
14
CH 3
(CH 2 )3
CH 2
13
CH
12
11
Initiation (metal)
CH3
(CH 2 )3
CH 2
12
.
CH
11
CH CH 2
10
CH CH
n
COOH
9
CH CH
CH 2 n COOH
+ O2
12
(CH 2 )3
CH
- H.
Propagation
CH 3
9
10
CH CH 2
CH 2
CH
11
10
CH CH
9
CH CH
CH 2 nCOOH
O
Propagation
+ H.
.
O
12
CH3
(CH 2 )3
CH 2
CH
O
_
H
12
(CH 2 )3
10
9
CH CH - CH 2 n COOH
O
Hydroperoxide
Decomposition
CH 3
11
CH CH
CH 2
CH
.OH
11
CH CH
10
9
CH CH
CH 2 n COOH
.
O
CH 3
(CH 2 )3
CH .
2
O
+ H C
12
11
CH CH
.
Termination
+ H
CH 3
(CH 2 )3
Pentane
CH 3
10
9
CH CH
CH 2 n COOH
ANALYSIS OF FLAVOR QUALITY & STABILITY OF OIL
1.
Peroxide Value
O
O
A.
KI + CH 3
B.
ROOH + 2 HI
I2 +
C.
I2 + 2 Na2 S 2 O3
2 NaI +
Peroxide Value =
C
OH
HI
+ CH 3
H2O +
Grams of Oil
OK
ROH
Na2 S4 O6
ml of Na2S2O3  N  1000
(milliequivalent peroxide/kg of sample)
C
2. Active Oxygen Method (AOM)
Determined the time required to obtain certain
peroxide value under specific experimental conditions.
The larger the AOM value, the better the flavor
stability of the oil.
3. TBA Test.
To determine the rancidity degree of meat or fish product.
HS
N
OH
O
+
N
O
C
CH2
C
H
H
OH
HS
N
OH
HO
N
N
SH
N
CH
CH
CH
OH
OH
Colored Pigment
+
2 H2 O