Fat analysis

Dr. Arfaa Sajid

Assistant Professor
Department of Chemistry,UOL
Lipids, proteins, and carbohydrates constitute the
principal structural components of foods.

Lipids are a group of substances that, in general, are

soluble in ether, chloroform, or other organic solvents
but are sparingly soluble in water.
Some lipids, such as triacylglycerols, are very hydrophobic. (body fat &
vegetable fat)

Other lipids, such as di- and monoacylglycerols, have both hydrophobic

and hydrophilic moieties in their molecules and are soluble in relatively
polar solvents. (Ice cream, peanut butter, shortening)
Short-chain fatty acids such as C1–C4 are completely miscible in
water and insoluble in nonpolar solvents.

Formic acid Acetic acid

Propanoic acid Butyric acid

Lipids comprise a broad group of substances that have some common
properties and compositional similarities.

Triacylglycerols are fats and oils that represent the most prevalent
category of the group of compounds known as lipids.

“Lipid” commonly refers to the broad term, total collection of food

molecules that are insoluble in water.
Fats generally refer to those lipids that are solid at room temperature.
Oils generally refer to those lipids that are liquid at room temperature.

Oil Fat
Liquid at room temperature Solid at room temperature
Plant source Animal source
Unsaturated Fatty acids Saturated at room temperature
 Classification of lipids:
1) Simple Lipids:

Ester of fatty acids with alcohol:

 Fats: Esters of fatty acids with glycerol – triacylglycerols

 Waxes: Esters of fatty acids with long-chain alcohols other than

glycerols (e.g., myricyl palmitate, cetyl palmitate, vitamin A esters, and
vitamin D esters)

Mericyl alcohol: C30H62O

Cetyl alcohol: C10H34O
Mericyl palmitate
2) Compound Lipids:

Compounds containing groups in addition to an ester of a fatty acid with

an alcohol:

 Phospholipids: Glycerol esters of fatty acids, phosphoric acids, and

other groups containing nitrogen (e.g., phosphatidyl choline,
phosphatidyl serine, phosphatidyl ethanolamine, and phosphatidyl
inositol) It has glycerol as a backbone.

phosphatidyl choline
 Cerebrosides: Compounds containing fatty acids, a carbohydrate,
and a nitrogen moiety (e.g., galactocerebroside and glucocerebroside)

 Sphingolipids: Compounds containing fatty acids, a nitrogen

moiety, and phosphoryl group (e.g., sphingomyelins). It has ceramide
backbone (sphingosine and a fatty acid).
3) Derived Lipids
Derived lipids are substances derived from neutral lipids or compound
lipids. They have the general properties of lipids – examples are fatty
acids, long chain alcohols, sterols, fat-soluble vitamins, and
hydrocarbons.

Sterol
 Importance of Analysis:

An accurate and precise quantitative and qualitative analysis of lipids

in foods is important for

 Accurate nutritional labeling,

 Determination of whether the food meets the standard of

identity,

 To ensure that the product meets manufacturing specifications.

Inaccuracies in analysis may prove costly for manufacturers and

could result in a product of undesirable quality and functionality.
 General considerations:
 Lipids are soluble in organic solvents and insoluble in water. Therefore,
water insolubility is the essential analytical property used as the basis for
the separation of lipids from proteins, water, and carbohydrates in foods.

 The wide range of relative hydrophobicity of different lipids makes the

selection of a single universal solvent impossible for lipid extraction of
foods. For example Glycolipids are soluble in alcohols and have a low
solubility in hexane.
In contrast, triacylglycerols are soluble in hexane and petroleum ether,
which are nonpolar solvents.

 Some lipids in foods are components of complex lipoproteins and

liposaccharides; therefore, successful extraction requires that bonds
between lipids and proteins or carbohydrates be broken so that the lipids
can be freed and solubilized in the extracting organic solvents.
Methods for fat analysis:

 Solvent extraction methods:

1. Continuous Solvent Extraction Method: Goldfish Method
2. Semicontinuous Solvent Extraction Method: Soxhlet Method
3. Discontinuous Solvent Extraction Methods: Mojonnier Method

 Non solvent wet extraction methods:

1. Babcock Method
2. Gerber Method

 Instrumental methods:
1. Infrared Method
2. Specific Gravity (Foss-Let Method)
3. Nuclear Magnetic Resonance
4. Gas Chromatography
Solvent extraction method:
Sample preparation:
The validity of fat analysis of food depends upon proper sampling and
preservation of sample before analysis.
An ideal sample should be as close as possible in all of its intrinsic properties
to the material from which it is taken.
The sample preparation for lipid analysis depends on
1. Type of food
2. Type and nature of lipids in the food

 Drying sample: (Vacuum oven drying at low temperature or lyophilization)

 Particle size reduction: (grinding at low temperature to minimize lipid
oxidation. Finished products may best be ground after freezing with liquid
nitrogen.)
 Acid hydrolysis: The sample can be predigested by refluxing for 1 h with
3N hydrochloric acid. For example, the acid hydrolysis of two eggs requires 10
ml of HCl and heating in a water bath at 65◦C for 15–25 min or until the solution
is clear.  Dried egg: 42.39%F (AH) 36.74%F (NAH)
Flour: 1.73% (AH) 1.20% (NAH)
 Solvent selection:

 Ideal solvents for fat extraction should have a high solvent power for lipids
and low or no solvent power for proteins, amino acids, and carbohydrates.

 They should evaporate readily and leave no residue,

 Have a relatively low boiling point,
 Nonflammable and nontoxic in both liquid and vapor states.
 The ideal solvent should penetrate sample particles readily, be in single
component form to avoid fractionation,
 Inexpensive
 Nonhygroscopic

Ethyl ether has a boiling point of 34.6◦C and is a better solvent for fat than
petroleum ether.
Continuous Solvent Extraction Method: Goldfish Method

Principle:
For continuous solvent extraction, solvent
from a boiling flask continuously flows
over the sample held in a ceramic thimble.
Fat content is measured by weight loss of
the sample or by weight of the fat
removed.

Calculations:
Weight of fat in sample=(beaker+fat)−beaker

% Fat on dry weight basis = (g of fat in sample/g of dried sample) × 100

Advantage: The continuous methods give faster and more efficient extraction
than semicontinuous extraction methods.
Disadvantage: However, they may cause channeling which results in
incomplete extraction
Semicontinuous Solvent Extraction Method: Soxhlet
Method
Principle:
For semicontinuous solvent extraction,
the solvent builds up in the extraction
chamber for 5–10 min and completely
surrounds the sample and then siphons
back to the boiling flask. Fat content is
measured by weight loss of the sample
or by weight of the fat removed.
Advantage: This method provides a soaking
effect of the sample and does not cause
channeling.
Disadvantage: However, this method
requires more time than the continuous
method.
Calculation:
% Fat on dry weight basis = (g of fat in sample/g of dried sample)×100
 Discontinuous Solvent Extraction Methods: Mojonnier
Method

Principle:
Fat is extracted with a mixture of ethyl ether and
petroleum ether in a Mojonnier flask, and the
extracted fat is dried to a constant weight and
expressed as percent fat by weight.

The advantages itself, it more precise

because MOJONNIER method use extraction for three
times thus it can reduce the rate of contamination.
The disadvantages is it use many chemical as the
solvent which some of the solvent is harm to human
skin and hazard

Calculations
% Fat = 100 × {[(wt dish + fat) − (wt dish)] −(avg wt blank residue)}/wt sample
 Procedure:
(a) Weigh, to the nearest 0.1mg, 10 g of milk into a Mojonnier fat extraction
flask.
(b) Add 1.5 ml of NH4OH and shake vigorously. Add 2 ml if the sample is sour.
NH4OH neutralizes the acidic sample and dissolves protein.
(c) Add 10ml of 95% ethanol and shake for 90 s. The alcohol prevents possible
gel formation.
(d) Add 25ml of ethyl ether and shake for 90 s. The ether dissolves the lipid.
(e) Cool if necessary, and add 25ml of petroleum ether and shake for 90 s. The
petroleum ether removes moisture from the ethyl ether extract and dissolves
more nonpolar lipid.
(f) Centrifuge for 30 s at 600 rpm.
(g) Decant ether solution from the Mojonnier flask into the previously weighed
Mojonnier fat dish.
(h) Perform second and third extractions in the same manner as for the first
extraction described previously (ethanol, ethyl ether, petroleum ether,
centrifugation, decant).
(i) Evaporate the solvent in the dish on the electric hot plate at ≤100◦C in a hood.
(j) Dry the dish and fat to a constant weight in a forced air oven at 100◦C ± 1◦C.
(k) Cool the dish to room temperature and weigh.
Nonsolvent wet extraction methods:
A: Babcock Method for Milk Fat:

Principle:
In the Babcock method, H2SO4 is added
to a known amount of milk in the
Babcock bottle. The sulfuric acid digests
protein, generates heat, and releases the
fat. Centrifugation and hot water
addition isolate fat for quantification in
the graduated portion of the test bottle.
The fat is measured volumetrically, but
the result is expressed as percent fat by
weight.
Babcock milk test bottles for milk
Applications:
The Babcock method, which is a common official method for the determination of fat in
milk, takes about 45min and duplicate tests should agree within 0.1%.
The Babcock method does not determine the phospholipids in the milk products. It is
not applicable to products containing chocolate or added sugar without modification
because of charring of chocolate and sugars by sulfuric acid.
 B: Gerber Method for Milk Fat
Dr. Niklaus Gerber of Switzerland in 1891

Principle:
The principle of the Gerber method is
similar to that of the Babcock method,
but it uses sulfuric acid and amyl
alcohol. The sulfuric acid digests
proteins and carbohydrates, releases
fat, and maintains the fat in a liquid
state by generating heat.
Butyrometer

Applications
 The Gerber method is comparable to the Babcock method but is simpler and
faster and has wider application to a variety of dairy products.
 The isoamyl alcohol generally prevents the charring of sugar found with the
regular Babcock method.
Total Fat by GC for Nutrition Labeling:

Sample preparation for GC:

Total Fat by GC for Nutrition Labeling

Example of chromatogram from gas chromatography analysis