lecture 9 lipid analysis

n Saturated Fatty Acids Butyric Butanoic CH3CH22COOH butterfat Caproic Hexanoic CH3CH24COOH butterfat, coconut, palm nut oils Caprylic Octanoic CH3CH26COOH Coconut, palm, nut oils, butte

Lipid analysis

Pham Van Hung, PhD

Roles of Food Lipids

z Energy Source

z Carrier of Fat Soluble Vitamins

z Main Flavor Source of Foods

z Hormone and Cell Structure

z Nerve System

z Thermal Insulation of Body

z Mouth Feeling

z Texture of Foods

z Emulsifying Agents

z Mold Releasing and Anti-spattering Agent

Definition

Any of a group of substances that in

general are soluble in organic solvents,

but are not soluble in water

2 Neutral fats and oils

6 Fat soluble vitamins lipids:

Types of Lipids

Fatty Acids

O

R C OH

#1 Carbon

Acid Group

O

R C OH

Non-polar End - Hydrophobic End

(Fat-soluble tail)

Polar End - Hydrophilic End

Saturated Fatty Acids

CH3 CH2 CH2 CH2 CH2 CH2 CH2 C OH

O 1 2 4

5 6 7

Octanoic Acid

Unsaturated Fatty Acids

CH3 CH2 CH2 CH2 CH2 CH2 CH2 C OH

O 1 2 4

5 6

7

CH3 CH2 CH2 CH2 CH2 CH2 CH2 C OH

O 1 2 4

5 6

7

3 - Octenoic Acid

3, 6 - Octadienoic Acid

8:2 (Δ3,6)

Cis 9 - Octadecenoic Acid (oleic)

Trans 9 - Octadecenoic Acid (elaidic acid)

O

CH3(CH2)7 C C (CH2)7 C OH

H H 9 10

O

H H

Cis and Trans 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

R CH2 [ CH CH CH2 CH CH ] CH2 C OH

O

2 Not conjugated - isolated double bond

3 Even numbered fatty acids

n

Saturated Fatty Acids

Butyric Butanoic CH3(CH2)2COOH butterfat Caproic Hexanoic CH3(CH2)4COOH butterfat, coconut, palm nut oils Caprylic Octanoic CH3(CH2)6COOH Coconut, palm, nut oils, butterfat Capric Decanoic CH3(CH2)8COOH Coconut, palm, nut oils, butterfat Lauric Dodecanoic CH3(CH2)10COOH Coconut, palm, nut oils, butterfat Myristic Tetradecanoic CH3(CH2)12COOH Coconut, palm, nut oil, animal fats Palmitic Hexadecanoic CH3(CH2)14COOH practically all animal, plant fats Stearic Octadecanoic CH3(CH2)16COOH animal fat, plant fats Arachidic Eicosanoic CH3(CH2)18COOH peanut oil

Common Name Systematic Name Formula Common source

Unsaturated 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

B Diethenoic Acids

Linoleic 9,12-Octadecadienoic C17H31COOH peanut, linseed, and

cottonseed oils

C Triethenoid Acids

Linolenic 9,12,15-Octadecatrienoic C17H29COOH linseed and other seed

oils Eleostearic 9,11,13-Octadecatrienoic C17H29COOH peanut seed fats

D Tetraethenoid Acids

Moroctic 4,8,12,15-Octadecatetraenoic C17H27COOH fish oils

Arachidonic

5,8,11,14-Eicosatetraenoic

C19H31COOH traces in animal fats

Important Fatty Acids for Nutrition

• EPA (Eicosapentaenoic acid) 20:5(n-3) Omega-3, all-cis –eicosa-5,8,11,14,17 pentaenoic acid

• DHA (Docosahexaenoic acid) 22:6 (n-3) omega-3, all-cis -docosa-4,7,10,13,16,19-hexaenoic acid

Fatty Acids Melting Points and Solubility in Water

Solubility in H O

Fatty Acid Chain Length

2

Melting Point

z

x

x

x

x x

x x x

Fatty Acids M.P.(C) mg/100 ml in H2O*

Characteristics of Fatty Acids

* Solubility

Effects of Double Bonds on the Melting Points

F A M P (C)

M.P.

# Double bonds

x

x

x x

FAT AND OILS

Triacylglycerol (Triglycerides )

O

H 2 C O

HC O

H 2 C O

HO C R

HO C R

HO C R

O O

O

H 2 C O C R

O O

H 2 C O C R

HC O C R

Glycerol 3 Fatty Acids

Glycerides

Monoacylglycerol (Monoglyceride) Diacylglycerol (Diglyceride)

H2C OH

HC OH

H2C O O

C (CH2)16CH3

H2C O

HC OH

H2C O

O

C (CH2)16CH3

C (CH2)16CH3

O

Triacylglycerol (Triglyceride) (β - palmityl distearin)

H2C O

HC O

H2C O O

C (CH2)16CH3

C (CH2)16CH3 O

O

C (CH2)14CH3

( C18) (C16) (C18)

Fats and Oils

World Supply

Fatty Acids (%) of Fats and Oils

Fatty Acids Butter Coconut Cottonseed Soybean

Melting Points of Triglycerides

C18:1 (cis) -32

Triglyceride Melting Point (°C)

C18:1 (trans) 15

Waxes Fatty acid + 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

C30H61 O C C15H31

O

C16H33 O C C15H31

Lecithin (phosphatidyl choline)

O

O

O

R C O CH

O_

Sterols Male & female sex hormones Bile acids

Vitamin D Adrenal corticosteroids Cholesterol

HO

H3C

H3C

H3C CH3

CH3

1 3

5 6 7 8 10

11 13

14 15 16 17 18

21 22 19

20

Cholesterol

Fat Soluble Vitamins Vitamin A

CH2OH

CH3

CH3

H3C

1 2 3 4 5 6 7 8 9

Vitamin D2

Vitamin E

HO

CH2 H H

H 3C

CH 3

CH 3

O

R1

R2

HO

R3

CH3

(CH2CH2CH2CH2)2CH2CH2CH2CH(CH3)2

CH3

Analytical Methods for The Determination of Characteristics

of Fats and Oils

1 Acid Value

2 Saponification Value

4 Gas Chromatographic Analysis for Fatty Acids

6 Cholesterol Determination

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

RCOOH + KOH ROO

-K+

+ H2O

The free fatty acid content in a good soybean oil should be

less than or equal to 0.05 % The average molecular

weight of free fatty acids of the oil is 280 which is the

molecular weight of linoleic aid

What is the maximum acid value of the good soybean oil?

What is the content (%) of free fatty acids of a soybean

oil if the acid value is 0.3? The average molecular weight

of free fatty acids of the oil is 280

0.05 % in 1gram is 0.5 mg fatty acid in 1 gram of oil

56 mg of KOH reacts with 280 mg of RCOOH

280 mg of RCOOH / 56 mg of KOH = 5:1 The 0.1 mg KOH reacts with 0.5 mg RCOOH Acid Value is 0.1

• What is the content (%) of free fatty acids of

soybean oil if the acid value is 0.3?

• Acid value 0.3 means that 0.3 mg KOH is

required to react with the free fatty acid in1g

(1000mg) of oil

• The 56mg KOH reacts with 280 mg free fatty

acid, 56 : 280 =1 : 5

• 1mg KOH reacts with 5 mg free fatty acid

• The 0.3 mg KOH reacts with 1.5 mg of free

fatty acid in 1 gram oil

• 1.5 mg free fatty acid /1000 mg oil x100(%)

=0.15 %

Saponification Value

Saponification - Hydrolysis of ester (triglycerider) under alkaline condition

O

C R O

O

C R

C R O

H 2 C O

HC O

H 2 C O

KOH

H H H

H 2 C O

HC O

H 2 C O

R C O - K +

Definition : mgs of KOH required to saponify 1 g of fat

Saponification Value

CH2 O C

O (CH2)6 CH3

CH O C

O (CH2)6 CH3

CH2 O C

O (CH2)6 CH3

CH2 O C

O (CH2)16 CH3

CH O C

O (CH2)16 CH3

CH2 O C

O (CH2)16 CH3

Tricaprylin (MW= 450)

Tristearin (MW= 890)

1Gram of Oils A and B

A

B

Saponification Value

Definition : mgs of KOH required to saponify 1 g of fat

:Large molecular triacylglycerols : Small molecular triacylglycerols

• The molecular weights of tributyrin and tristearin

are 300 and 900, respectively If the numbers of

tributyrin in 1 gram is 21 x 1020, what are the

approximate numbers of tristearin in 1 gram?

• If the saponification value is tristearin is 190,

what is the approximate saponification value of

tributyrin?

Sample A has large molecular weight triglyceride (e.g MW.890)

Sample B has small molecular weight triglyceride(e.g MW.450)

In one gram of sample, number of triglyceride in B is about two times more than number of triglyceride in A

Less mg of KOH is needed to saponify sample A than sample B

Therefore, saponification value of A is about half of that of sample B

Avogadro’s Number (N) = 6.02 x 1023/ mol

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.

Saponification number =[ (B – S) x N of HCl x 56] /gram of sample

B - ml of HCl required to titrate KOH in Blank.

S - ml of HCl required to titrate excess KOH by Sample.

Saponification Value Determination

A 5.00 grams of exotic tropical oil was saponified with excess KOH The unreacted KOH was then titrated with 1.00 N HCl The blank required 40 mL

of HCl and the sample required 20 mL

Please calculate the saponification value of the oil

Milk Fat 210-233

Coconut Oil 250-264

Cotton Seed Oil 189-198

Soybean Oil 189-195

Fat Saponification #

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

Trilinolein (MW= 878)

Triolein (MW= 884)

CH 2 O C O (CH 2 ) 7 CH CH (CH 2 ) 7 CH 3

CH 3

(CH 2 ) 7

CH CH (CH 2 ) 7

O C O CH

CH 3

(CH 2 ) 7

CH CH (CH 2 ) 7

O C O

CH 2

CH2 O C O (CH2)7CH CH CH 2 CH CH CH (CH2)7 O C O CH

CH CH (CH2)7 O C O

CH 2

CH (CH 2 ) 4 CH 3

CH3 (CH2)4 CH CH

CH2

CH 3

(CH2)4 CH CH

CH2

A

B

CH2 O C

O (CH2)7 CH CH

CH CH (CH2)7 O C O CH

(CH2)14 O C O

CH2

CH3 (CH2)4 CH CH CH2 (CH2)7 CH3

CH3

C H 2 O C

O ( C H 2)7 (C H 2)14 O

C O

C H

( C H 2)14 O

C O

C H 3

C H 3 ( C H 2)7

C H

C H

A

B

(ml of Na2S2O3volume for blank - ml of Na2S2O3

volume for sample) × N of Na2S2O3× 0.127g/meq × 100

Weight of Sample (g)

I 2

I 2

Na 2 S 2 O 3 Na2S4O6 NaI

+

+ 2

2 + + Excess unreacted ICl

Iodine Value Determination

Iodine Value =

ICl Iodine chloride +

• The chemical equation of iodine value determination shows that one double bond requires one I2

molecule which is 254 in molecular weight 100 comes from the 100 gram of sample for the definition

of iodine number

• Iodine value defines as the number of iodine in grams absorbed by 100 gram of sample

• Fatty acids A and B have only one double bond

per molecule The molecular weights of A and B

are 150 and 300, respectively The hypothetical

iodine value of Compound A is 150 What is the

Iodine value of compound B?

• Triglycerides A and B have the very similar

molecular weights of about 878 The compound

A has 6 double bonds per molecule and has

iodine value is 174 The compound B has 3

double bond per molecule What is the iodine

value of the compound B?

Iodine Values of Triglycerides

Palmitoleic Acid 1 95

Fatty Acids # of Double-bonds Iodine #

Arachidonic Acid 4 320

Number of Double bonds

Compound A and B have the same iodine values of

100 Compound A of molecular weight of 200 has one

double bond per molecule Determine the number of

double bonds of Compound B of molecular weight of

400

Double Bond Determination

The unknown compound has molecular weight

of 878 and iodine value of 173 Determine the number of double bonds in the unknown compound

Determination of Double per Molecule

Iodine Value xMolecule Weight

2 x 127 x 100

Number of Double

Bonds per Molecule =

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

β - palmityl distearin

H 2 C O

HC O

H 2 C O

O

C (CH 2 ) 16 CH 3

C (CH2)16CH3 O

O

C (CH 2 ) 14 CH 3

(C 18 ) (C 16 ) (C 18 )

Fatty Acids Methyl Esters

14

18:1

18:2 20 18:3

22 21:1 24

16 18

Time

GC condition: 10% DEGS Column (from Supelco)

Column temperature 200C

Triglyceride Analysis by Liquid Chromatography

Soybean Oil

Solvent CH3CN/HF

Column 84346 (Waters Associates)

RETENTION TIME

Triglycerides in Olive Oil

Cholestrol Determination

Enzymatic Methods

H2O2 Cholesterol Oxidase

etc +

H2O2

CH 3 O OCH 3

H 2 N NH 2 HN NH

OCH 3

CH 3 O

H2O Peroxidase

0-Dianisidine Oxidized 0-Dianisidine

(Colorless) (Brown color) at 440 nm

R

Absorption Standard Curve of Cholesterol

m g/ml Cholesterol

Cholesterol Analysis by GLC

1 Prepare cholesterol butyrate

Sensitivity - 10-7

g

CH3CH2CH2COOH

-H2O

HO

H3C

H3C

H3C CH3

CH3

1 3

5 6 7 8 10

11 13

14 15 16 17 18

21 22 19

20

Lipid Content Analysis

1 Gravimetric Method

(1) Wet extraction - Mojonnier

(2) Dry extraction - Soxhlet Method

2 Volumetric Methods

(1) Babcock Method

Gravimetric Method

(1) Wet Extraction - Mojonnier

For Milk:

1) 10 g milk + 1.25 ml NH4OH mix solubilizes protein and neutralizes

2) + 10 ml CH3CH2OH - shake Begins extraction, prevents gelation of proteins

3) + 25 ml CH3CH2OCH2CH3- shake and mix

4) + 25 ml petroleum ether, mix and shake

Dry Extraction - Soxhlet Method

Sample in thimble is continuously extracted with ether

using Soxhlet condenser

After the extraction of fat from the sample, evaporate

ether in the flask and weigh the flask The gain of the

weight of flask is the fat content

Soxhlet Method

Volumetric Method

Babcock Method

Theory:

1 Treat sample with H2SO4or 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 to make fat be in graduated part of bottle

4 Centrifuge to separate fat from other materials completely

The end!