INRODUCTION

Lipid is major constituent of foods. That is important in our diet. There are source of
energy, essential nutrients, flavour, texture, mouth feel and appearance. But when take it over
consumption certain lipids can be detrimental to health. For example cholesterol and
saturated fats. The components of lipid are soluble in organic solvents such as ether, hexane,
acetone or chloroform. Lipid exist as free fatty acid, phospholipids, sterols,
monoacylglycerol, diacylglycerol and triglycerol, waxes and fat soluble vitamins such as
vitamin A&D and pigment (carotenoids). There are many method to distinguish them to other
components in food. Three types of analytical techniques is solvent extraction, non-solvent
extraction and instrumental methods. Solvent extraction methods are lipid separation based
on its solubility in organic solvents and insolubility in water. Commonly used for isolating
lipid from other food matrix for total lipid content determination of foods.
Batch solvent or discontinuous extraction methods are based on mixing sample and
solvent in a suitable flask for example using separotary funnel. Flask shaken vigorously and
allow solvent and aqueous phase to separate (by gravity or centrifugation). Aqueous phase
decanted off. The concentration of lipid in solvent was determined by evaporating solvent
and measure weight of the remaining lipid after evaporation/drying (Cooke,1986). For
example Werner-Schmid method.For semi-continuous extraction method, the efficient and
rapid extraction method for lipids in foods. The most commonly method is Soxhlet method.










TITLE
Soxhlet extraction method
OBJECTIVE
To determine the total fat content of Snack by using Soxhlet method

MATERIAL
Petroleum ether, analytical balance (Sartorius), dessicator, Soxhlet apparatus, filter paper,
cotton wool, round bottom flask, heating mantle, water bath graduated cylinder, beaker,
mortar and pestle, spatula and oven (memmert).


Methods
In the first week, clean and dry round bottom flask were obtained and labelled clearly.
Then, about 2g of dried sample were weighed into a piece of filter paper. The filter paper was
folded. The paper and content were placed into a Soxhlet extractor. Round bottom flask was
labelled and was weighed accurately. 150ml of petroleum ether was added into the round
bottom flask by using measuring cylinder. Apparatus and condenser was connected, water
and extract were turned on for a minimum of 8 hours on an electrothermal extraction unit.
In the second week, flask containing the petroleum ether extract was removed after
the extraction completed. The round bottom flask was placed on the stream bath to remove
the residual petroleum ether. When all solvent had been removed, the outside of flask was
dried with tissue paper before placing in oven set at 105c for one hour of drying. The flask
was transferred immediately into a dessicator to be cooled and weighed.



TITLE
Werner Schmid method
OBJECTIVE
To determine the fat content of fresh milk using Werner Schmid Method

MATERIAL
Concentrated hydrochloric acid, ethanol and diethyl ether, test tube, tongs, boiling water bath
(memmert), separatory flask, evaporating dish, analytical balance (Sartorius), oven
(memmert), dessicator and pipette.

Methods
First, 10g of sample was weighed and poured into the test tube. Then, 10ml
concentrated of hydrochloric acid was added. After that, the boiling tube was immersed in
boiling water until all the casein has dissolved. At this stage the mixture should be brown or
violet in colour and the fat can be seen on the surface. The tube was cooled in running water.
All the content was transferred into the separatory flask. The fat extracted by shaking it with
30 ml diethyl ether. The layers were allowed to separate. The solution was decanted into
evaporating dish. The extraction was repeated three times and the solvent was distilled into
the same evaporating dish. The evaporating dish contains fat was dried at 105c for 1 hours
was cooled and weighed.
Calculation
% Fat in sample = Weight of fat in sample (g) 100
Weight of sample taken (g)
RESULT

Sample Group Weight of
sample (g)
Weight of round
bottom flask (g)
Weight of round
bottom flask + fat
(g)
Weight of fat
in sample (g)
% crude fat
content
Snack 1 2.0043 121.39 121.8460 0.456 22.70
2 2.0129 122.8643 123.3158 0.4515 22.43
3 2.0063 126.2014 126.6574 0.456 22.75
Average + SD 22.63+ 0.172
Coconut
milk
powder
4 2.0114 127.4054 128.5356 1.1302 56.19
5 2.0052 126.3700 127.5408 1.1708 58.39
6 2.0014 129.8800 131.0098 1.1298 56.45
Average + SD 57.01 + 1.20
Tabl 3.1 :The result obtained from Soxhlet method of Snack and Coconut milk powder

Fresh Milk
Group Replicate Weight
of sample
(g)
Weight of
evaporating
dish (g)
Weight of
evaporating
dish + fat (g)
% crude fat
content
Average + SD
(each group)
G1 1 10.0725 66.3068 66.4070 1.00 % 1.34 + 0.47
2 10.0931 62.4289 62.4289 1.67 %
G2 1 10.00 97.0880 97.1783 0.903 % 0.902 + 1.41
10
3
2 10.00 65.3900 65.4811 0.901 %
G3 1 10.0239 60.5124 60.6447 1.32 % 1.34 + 0.021
2 10.0666 61.8699 62.0060 1.35 %
G4 1 10.0741 60.0655 60.1567 0.91 % 0.98 + 0.099
2 10.0204 66.4223 66.5274 1.05 %
G5 1 10.0223 61.1670 61.2614 0.942 % 0.996 + 0.076
2 10.0000 62.2329 62.3380 1.05 %
G6 1 10.0178 86.7525 86.8779 1.25 % 1.21 + 0.057
2 10.0124 62.6524 62.7696 1.17 %
Average + SD 1.128 + 0.19
Table 3.2 : The results obtained from Werner-Schmid method


Graph 3.1 : Graph of crude fat content of snack and coconut milk powder by using Soxhlet
method

Graph 3.2 : Graph of crude fat content of fresh milk powder by using Werner-Schmid method







0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2
C
r
u
d
e

f
a
t

c
o
n
t
e
n
t

(
%
)

Type of food sample
Series 1
Crude fat content
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6
C
r
u
d
e

f
a
t

c
o
n
t
e
n
t

(
%
)

Group
Series 2
Crude fat content
Calculation

Snack (Table 3.1)

% Fat in sample = Weight of fat in sample (g) 100
Weight of sample taken (g)

Weight of fat in sample (g) = (weight of flask+fat) weight of flask

Weight of fat in sample = 123.3158g 122.8643g
= 0.4515g

% crude fat content of snack = (0.4515 / 2.0129) x 100%
= 22.43%

From table 3.1

Average = (22.70 + 22.43 + 22.75) / 3
= 22.63

Standart deviation =

= 0.172

Average SD = 22.63 0.172 % of crude fat content of snack
















From table 3.1, coconut milk powder

Average = (56.19 + 58.39 + 56.45) / 3
= 57.01 %

Standart deviation =

= 1.20

Average SD = 57.01 1.20 % crude fat content of coconut milk powder

Table 3.2, milk sample

% fat content = weight of fat in sample (g) x100%
Weight of sample taken (g)


Weight of fat in sample (g) = (weight of flask+fat) weight of flask

Replicate 1

Weight of fat in sample, g = 97.1783g 97.088g
= 0.0903g

% crude fat content = 0.0903 x 100%
10.00
= 0.903%


Replicate 2

Weight of fat in sample, g= 65.4811g 65.39g
= 0.0911

% crude fat sample = 0.0911g x 100%
= 0.911 %
From table 3.2, fresh milk sample

Average = (1.34 + 0.902 + 1.34 + 0.98 + 0.996 + 1.21) / 6
= 1.128

Standard deviation
=

= 0.19

Average SD = 1.128 0.19% crude fat content of fresh milk

























DISCUSSION
To complete the investigate of fat content in food sample, two method were carried
out which are Soxhlet method and Werner Schmid method. The first experiment was
determination of fat in food samples by using Soxhlet method. The second experiment was
determination of fat content in food samples by using Werner Schmid method. Soxhlet
extraction is one of the most commonly used methods for determination of lipids in dried
foods. This is due to it is fairly simple to use. But there are afew of disadvantages of the
techniques which that a relatively dry sample is needed to allow the solvent to
penetrate,destructive, and it is time consuming.
Based on table 3.1, the coconut milk powder has more crude fat content compared to
the snacks. The crude fat content of coconut milk powder is 57.01 % of average and 1.20 %
of standard deviation while snack has 22.63 % crude fat content and 0.172 % of standard
deviation. It shows that the result of snack food sample is more reliable and more accurate to
the true value.
The second method that was used for fat extraction is Werner Shmid method. The
food sample is fresh milk. In this method, the diethyl ether and hydrochloric acid (HCl) had
been used. Neutral lipids or generally storage lipids are extracted with relatively non-polar
solvents such as diethyl ether but membrane-associated lipids are more polar and require
polar solvents such as ethanol or methanol to disrupt hydrogen bondings or electrostatic
forces. Alcohols are good solvents for most lipids, methanol and ethanol being the most
popular. Ethanol can be contaminated on storage by aldehydes.
Difficulties with low oil recovery from fat-filled milk powders and newly introduced
calcium salts of fatty acids as a rumen-inert fat supplement in the United Kingdom led to
regulatory changes in extraction procedures for oil from many compound feeds and feeds of
animal origin (Sanderson, 1986). These included boiling the feed sample in HCl to break
down the protein, and fat separated as a layer on the top of the acid liquid. Then the step
followed by washing with water before the ether extraction step. The protein dissolves in the
acid and the separated fat can be extracted by shaking at least three times with diethyl ether.
This modification yielded consistently higher oil values than ether extraction alone, thus
raising another difficulty,which value is correct and whether the same procedures were
being used for a specific feeding stuff both by analysts and sales people(Cooke, 1986).
Based on the result that has been carried out using Soxhlet extraction and Werner
Schmid method , snack has the highest fat contain, followed by coconut milk powder and
fresh milk. Fat contain for snack, coconut milk powder and fresh milk are 22.63%, 1.128%
and 0.907% .
During the conduction of experiments, safety precautions must be taken to avoid
unwanted event to occur. Ether has an extremely low flash point. Have no open flames
nearby. Avoid inhaling ether vapors. Store ether in metal containers. Handle open containers
(reagent cans and fat beakers) in a hood. Conduct the extractions in a well ventilated area.
Peroxides can accumulate in open containers of ether. These are explosive and shock
sensitive. Check each container opened for more than 30 days for peroxides. Ether-containing
peroxides must be disposed with special techniques. Electrical equipment is to be grounded.
Extractors should be spark-proof. Make sure all ether is evaporated from the beakers before
placing them in the oven to avoid a fire or explosion













QUESTION
1. Comment the suitability of the method with the supplied samples.

In currently used methods such as Soxhlet extraction, the fat content is
determined gravimetrically after extraction with organic solvents such as chloroform
or petroleum ether. The current methods require large volumes of solvents and time
periods from 2 to 16 hours. Many analysts want faster techniques that also require less
solvents. But, for dry samples, it is suitable to use the Soxhlet extraction to allow the
solvent to penetrate.
For fresh milk samples, it is suitable to use Werner-Schmid method, this is
because, this method use acid hydrolysis for lipid extraction. A significant portion of
the lipids in foods such as dairy, bread and flour products is bound to proteins and
carbohydrate, and direct extraction with nonpolar solvents is enefficient. So, such
food must be prepared for lipid extraction by acid hydrolysis.

2. Discuss the difference between one extraction with larger volume of solvent and
use the same amount of solvent total volume with several extraction in smaller
volumes.
Extracting several times with small volumes of organic solvent is more
efficient (to improve the efficiency of the extraction process )than one extraction with
a large volume. This is of particular significance when the value of K, the partition
coefficient is higher. In this case the aqueous phase would undergo further extractions
using fresh solvent, then all the solvent fractions would be collected together and the
lipid determined by weighing after evaporation of solvent. The efficiency of the
extraction of a particular type of lipid by a particular type of solvent can be quantified
by an equilibrium partition coefficient, K = c
solvent
/c
aqueous
, where c
solvent
and c
aqueous
are
the concentration of lipid in the solvent and aqueous phase, respectively. The higher
the partition coefficient the more efficient the extraction process.



CONCLUSION
There are vary in methods of fat determination. In this experiment, Soxhlet extraction
and Werner Schmid were used. The Soxhlet extraction was applicable to relatively dry food
sample compared to high moisture content food sample according to the average standard
deviation. While, for the Werner Schmid, it is applicable for determining of crude fat content
in fat milk sample.

REFERENCES
1. Cooke, B. C. 1986. The implications to research and the feed compounder of the new
oils and fats determination. Pages 8186 in Recent Advances in Animal Nutrition
1986. W. Haresign and D. J. A. Cole, ed. Butterworths, London.
2. Sanderson, P. 1986. A new method of analysis of feeding stuffs for the determination
of crude oils and fats. Pages 7780 in Recent Advances in Animal Nutrition1986. W.
Haresign and D. J. A. Cole, ed. Butterworths, London.
3. Retrieved october 5, 2014, from
https://www.chem.tamu.edu/rgroup/gladysz/documents/extractions.pdf
4. Retrieved october 5, 2014, from
http://www.journalofanimalscience.org/content/81/12/3250.full.pdf
5. Retrieved october 5, 2014, from
http://journalofanimalscience.org/content/81/12/3250.full