A. TITLE : Quantitative Analysis of Lipid

B. DATE OF EXPERIMENT : 28th November 2013

C. PURPOSE : Determining the number of peroxide and free

fatty acid

D. BASIC THEORY :

Lipids are one of the major constituents of foods, and are important in our diet

for a number of reasons. They are a major source of energy and provide essential

lipid nutrients. Nevertheless, over-consumption of certain lipid components can

be detrimental to our health, e.g. cholesterol and saturated fats. In many foods the

lipid component plays a major role in determining the overall physical

characteristics, such as flavor, texture, mouthfeel and appearance. For this reason,

it is difficult to develop low-fat alternatives of many foods, because once the fat is

removed some of the most important physical characteristics are lost. Finally,

many fats are prone to lipid oxidation, which leads to the formation of off-flavors

and potentially harmful products. Some of the most important properties of

concern to the food analyst are:

Total lipid concentration

Type of lipids present

Physicochemical properties of lipids, e.g., crystallization, melting point,

smoke point, rheology, density and color

Structural organization of lipids within a food

Properties of Lipids in Foods

Lipids are usually defined as those components that are soluble in organic

solvents (such as ether, hexane or chloroform), but are insoluble in water. This

group of substances includes triacylglycercols, 

diacylglycercols,monoacylglycercols, free fatty acids, phospholipids,

sterols, caretonoids and vitamins A and D. The lipid fraction of a fatty food

therefore contains a complex mixture of different types of molecule. Even

so, triacylglycercolsare the major component of most foods, typically making up

more than 95 to 99% of the total lipids present. Triacylglycerols are esters of three

fatty acids and a glycerol molecule. The fatty acids normally found in foods vary

in chain length, degree of unsaturation and position on the glycerol molecule.

Consequently, the triacylglycerol fraction itself consists of a complex mixture of

different types of molecules. Each type of fat has a different profile of lipids

present which determines the precise nature of its nutritional and physiochemical

properties. The terms fat, oil and lipid are often used interchangeably by food

scientists. Although sometimes the term fat is used to describe those lipids that are

solid at the specified temperature, whereas the term oil is used to describe those

lipids that are liquid at the specified temperature.

Iodine Value

The iodine value (IV) gives a measure of the average degree

of unsaturation of a lipid: the higher the iodine value, the greater the number of

C=C double bonds. By definition the iodine value is expressed as the grams of

iodine absorbed per 100g of lipid. One of the most commonly used methods for

determining the iodine value of lipids is "Wijs method". The lipid to be analyzed

is weighed and dissolved in a suitable organic solvent, to which a known excess

of iodine chloride is added. Some of the ICl reacts with the double bonds in the

unsaturated lipids, while the rest remains:

R-CH=CH-R + IClexcess →R-CHI-CHCl-R + IClremaining

The amount of ICl that has reacted is determined by measuring the amount

of ICl remaining after the reaction has gone to completion

(IClreacted =IClexcess - IClremaining). The amount of ICl remaining is determined by

adding excess potassium iodide to the solution to liberate iodine, and then titrating

with a sodium thiosulfate (Na2S2O3) solution in the presence of starch to

determine the concentration of iodine released:

IClremaining + 2KI → KCl + KI + I2

I2 + starch + 2Na2S2O3 (blue) → 2NaI + starch + Na2S4O6(colorless)

Iodine itself has a reddish brown color, but this is often not intense enough to be

used as a good indication of the end-point of the reaction. For this reason, starch is

usually used as an indicator because it forms a molecular complex with the iodine

that has a deep blue color. Initially, starch is added to the solution that contains

the iodine and the solution goes a dark blue. Then, the solution is titrated with a

sodium thiosulfate solution of known molarity. While there is any I2 remaining in

the solution it stays blue, but once all of the I2 has been converted to I- it turns

colorless. Thus, a change in solution appearance from blue to colorless can be

used as the end-point of the titration.

The concentration of C=C in the original sample can therefore be calculated by

measuring the amount of sodium thiosulfate needed to complete the titration. The

higher the degree of unsaturation, the more iodine absorbed, and the higher the

iodine value. The iodine value is used to obtain a measure of the average degree

of unsaturation of oils, and to follow processes such as hydrogenation and

oxidation that involve changes in the degree ofunsaturation.

Foods which contain high concentrations of unsaturated lipids are particularly

susceptible to lipid oxidation. Lipid oxidation is one of the major forms of

spoilage in foods, because it leads to the formation of off-flavors and potentially

toxic compounds. Lipid oxidation is an extremely complex process involving

numerous reactions that give rise to a variety of chemical and physical changes in

lipids. Food scientists have developed a number of methods to characterize the

extent of lipid oxidation in foods, and to determine whether or not a particular

lipid is susceptible to oxidation.

Peroxide value

Peroxides (R-OOH) are primary reaction products formed in the initial stages

of oxidation, and therefore give an indication of the progress of lipid oxidation.

One of the most commonly used methods to determine peroxide value utilizes the

ability of peroxides to liberate iodine from potatssium iodide. The lipid is

dissolved in a suitable organic solvent and an excess of KI is added:

ROOH + KIexcess → ROH + KOH + I2

Once the reaction has gone to completion, the amount of ROOH that has reacted

can be determined by measuring the amount of iodine formed. This is done by

titration with sodium thiosulfate and a starch indicator:

 I2 + starch + 2Na2S2O3 (blue) → 2NaI + starch + Na2S4O6(colorless)

The amount of sodium thiosulfate required to titrate the reaction is related to the

concentration of peroxides in the original sample (as described earlier for the

iodine value). There are a number of problems with the use of peroxide value as

an indication of lipid oxidation. Firstly, peroxides are primary products that are

broken down in the latter stages of lipid oxidation. Thus, a low value of PV may

represent either the initial or final stages of oxidation. Secondly, the results of the

procedure are highly sensitive to the conditions used to carry out the experiment,

and so the test must always be standardized. This technique is an example of a

measurement of the increase in concentration of primary reaction products.

Acid value

The acid value is a measure of the amount of free acids present in a given

amount of fat. The lipids are extracted from the food sample and then dissolved in

an ethanol solution containing an indicator. This solution is then titrated with

alkali (KOH) until a pinkish color appears. The acid value is defined as the mg of

KOH necessary to neutralize the fatty acids present in 1g of lipid. The acid value

may be overestimated if other acid components are present in the

system, e.g. amino acids or acid phosphates. The acid value is often a good

measure of the break down of the triacylglycrols into free fatty acids, which has

an adverse effect on the quality of many lipids.

The levels of free fatty acids can be determined through a process called

acid/base titration. A titration is a way of determining the concentration of a

substance by adding a known concentration of a reagent to it until we see an

effect. In our case, we will add a known concentration of sodium hydroxide to the

free fatty acids contained in vegetable oil until we see a color change (this color

change is due to a change in pH which we can observe by adding a pH indicator).

When the mixture’s color has been changed (from yellow to bright pink), then it

has reached its equivalence point and the free fatty acids have been neutralized.

2NaOH + [free fatty acid] → Na2[neutralized free fatty acid] + 2H2O

E. TOOLS AND MATERIALS

Tools :

Beaker Glass

Volumetric pipette

Pro pipette

Buret

Satif and clamp

Erlenmeyer

Materials :

Palm oil

Acetic acid : chloroform (3:2)

KI saturated

Na2S2O3 0,1 N

Starch solution 1%

NaOH 1%

Oxalate solution 0,1 N

PP indicator 1%

Ethanol 96%

F. PROCEDURE

1. Determining the number of peroxide

Peroxide number

Placed in erlenmeyer Added 30 ml of acetic acid-chloroform (3:2) Mixed until oil dissolve perfectly Added 0,5 ml of KI saturated Let it 20 minutes Added 30 ml of aquades Titrated with Na2S2O3 until the yellow color

almost disappear Added 0,5 ml of starch solution 1 % Titrated with Na2S2O3 until the solution colorless Calculated peroxide number

5 gram of sample

Peroxide number

Placed in erlenmeyer Added 30 ml of acetic acid-chloroform (3:2) Mixed until oil dissolve perfectly Added 0,5 ml of KI saturated Let it 20 minutes Added 30 ml of aquades Titrated with Na2S2O3 until the yellow color

almost disappear Added 0,5 ml of starch solution 1 % Titrated with Na2S2O3 until the solution colorless Calculated peroxide number

5 gram of aquades

2. Determining Free Fatty Acid (%FFA)

% FFA

Placed in Erlenmeyer Added 10 ml of alcohol 96% and 5-8 drops of PP

indicator Titrated with NaOH 0,1 N until the color change

be pink and stable for 30 seconds Calculated % FFA

6 gram of sample

G. TABLE OF DATA

No Procedure Result Hypothesis Conclusion

1. Determining the number of peroxide SampleMass of sample :

I : 5.2928 gramII : 5.5295 gramIII : 5.4715 gram

Sample + acetic acid-chloroform : colorless orange

+ KI : turbid orange

+ aquades : 2 layer (turbid(+++) and yellow layer)

+ Na2S2O3 : 2 layer (turbid(+) and yellow layer)

+ amylum : 2 layer (turbid(++) and yellow layer

+ Na2S2O3 : 2 layer (colorless and yellow layer)

ROOH + KIexcess

→ ROH + KOH + I2

 I2 + starch + 2Na2S2O3

→ 2NaI + starch +

Na2S4O6(colorless)

The peroxide number of oil is 24,63

5 gram of sample

Placed in erlenmeyerAdded 30 ml of acetic acid-chloroform (3:2)Mixed until oil dissolve perfectlyAdded 0,5 ml of KI saturatedLet it 20 minutesAdded 30 ml of aquadesTitrated with Na2S2O3 until the yellow color almost disappearAdded 0,5 ml of starch solution 1 %Titrated with Na2S2O3 until the solution colorlessCalculated peroxide number

Peroxide number

V Na2S2O3 :I : 1.5 mlII : 1.6 mlIII : 1.2 ml

BlancoBlanco solution : colorless

+ KI + starch : turbid

+ Na2S2O3 : 2 layer (colorless and turbid layer).V Na2S2O3 : 0,1 ml

2. Determining Free Fatty Acid (%FFA) Mass of sample :I : 6.0587 gramII : 6.0718 gramIII : 6.0014 gram

Sample + ethanol : 2 layer ( dark yellow and colorless layer)

+ NaOH : 2 layer (red brick and brown layer)

V NaOH :I : 0.9 mlII : 0.8 mlIII : 0.8 ml

Blanco + ethanol : colorless

+ PP : colorless

+ NaOH : pinkVNaOH : 0.15 ml

2NaOH + [free fatty acid] → Na2[neutralized free fatty acid] + 2H2O

% FFA is 0.288 %6 gram of sample

Placed in ErlenmeyerAdded 10 ml of alcohol 96% and 5-8 drops of PP indicatorTitrated with NaOH 0,1 N until the color change be pink and stable for 30 secondsCalculated % FFA

% FFA

H. ANALYSIS DATA

The aims of first experiment is determining the peroxide number of

palm oil. Peroxides (R-OOH) are primary reaction products formed in the

initial stages of oxidation, and therefore give an indication of the progress

of lipid oxidation. One of the most commonly used methods to determine

peroxide value utilizes the ability of peroxides to liberate iodine

from potassium iodide. Firstly, sample was prepared 5 grams. In this

experiment there are repeating steps, so 3 sample was prepared. The mass

of sample are :

I = 5.2928 gram

II = 5.5295 gram

III = 5.4715 gram

This samples than dissolve in 30 ml of mixture of acetic acid and

choloroform (3 : 2). The color of this solution is clear orange. Mixture of

acetic acid and cholorform was chosen as media of reaction because it is

nonpolar. Lipid, although has “polar head”, its tail (which is very long) is

nonpolar. So lipid can dissolve perfectly in mixture of acetic acid and

choloroform. The color of solution change to be turbid orange when 0.5 ml

of KI saturated poured in it. The reaction is :

ROOH + KIexcess → ROH + KOH + I2

This solution then closed with aluminium foil and let 20 minutes.

Every 5 minutes, this mixture was shake to increase the reaction in

solution. Once the reaction has gone to completion, the amount of ROOH

that has reacted can be determined by measuring the amount of iodine

formed. This is done by titration with sodium thiosulfate and a starch

indicator:

I2 + starch + 2Na2S2O3 (blue) → 2NaI + starch + Na2S4O6(colorless)

After 20 minutes, the sample solution added by 30 ml of aqudes. This

treatment cause formation of 2 layer in sample solution. The upper layer was

turbid (+++) while the bottom layer was yellow. After that the solution titrated

with Na2S2O3 until the turbidity of solution was decrease. To know the decreasing

of turbidity, after added by some drops of sodium thiosulphate, the solution was

let 20 seconds. When form 2 layer, where upper layer was turbid (+) and bottom

layer was yellow, starch solution was added and titration was continued until the

turbid layer become colorless. The volume Na2S2O3 until the turbid layer become

colorless are :

VI = 1,5 ml

VII = 1,6 ml

VIII = 1,2 ml

The amount of sodium thiosulfate required to titrate the reaction is

related to the concentration of peroxides in the original sample (as

described earlier for the iodine value). The equation in determining

peroxide numer as follow :

Peroxide number = ml Na2 S 2O 3 x N Na2 S 2O 3x 1000

mass of sample

The peroxide number in every sample are :

I = 26.45

II = 27.13

III = 20.31

From those result the average peroxide number of our experiment is 24.63

For blanco solution, the sample are aquades, the same steps was done. The color

of blanco solution is colorless. When this solution added by KI and starch, the

color change to be turbid. After titration of Na2S2O3, form 2 layer where upper

layer is colorless while the bottom layer is turbid. The volume of Na2S2O3 until

the upper layer change be turbid is 0.1 ml.

The aims of second experiment is determining the percentage of free fatty

acid in palm oil. The acid value is a measure of the amount of free acids present in

a given amount of fat. The acid value is often a good measure of the break down

of the triacylglycrols into free fatty acids, which has an adverse effect on the

quality of many lipids. 6 grams of sample is prepared. This experiment has 3 times

repetition so the mass of of sample is each repetition are :

I = 6.0587 gram

II = 6.0718 gram

III = 6.0014 gram

Then all samples mixed with ethanol. There are 2 layer in this

mixture that are dark yellow in bottom layer and colorless in upper layer.

This solution then added by PP indicator and titrated with NaOH until

there are color change. the color of solution change to be red brick in

upper layer and brown in bottom layer. The volume of NaOH until the

color change are :

I = 0.9 ml

II = 0.8 ml

III = 0.8 ml

When the mixture’s color has been changed (from yellow to bright pink), then it

has reached its equivalence point and the free fatty acids have been neutralized.

The reaction are :

2NaOH + [free fatty acid] → Na2[neutralized free fatty acid] + 2H2O

For blanco solution, the sample are aquades, the same steps was

done. The color of blanco solution is colorless. After titrated with NaOH,

the color change to be pink. The volume of NaOH needed until the color

change is 0.15 ml. the aquation used to determne the percentage of free

fatty acid (%FFA) as follow :

I = 0.317 %

II = 0.274 %

III = 0.274 %

From those result, the average percentage of free fatty acid (%FFA) in this

experiment is 0.288%.

I. CONCLUTION

1. The average peroxide number of the experiment is 24.63

2. The average percentage of free fatty acid (%FFA) of the experiment is

0.288%.

REFERENCES

Anonymous. Analysis of Lipids.(online)

http://people.umass.edu/~mcclemen/581Lipids.html (accessed on

November, 29th 2013)

Anonymous. Determining Molarity Through the Use of Titration.(online) http://www.techcouncilmd.com/mdbio/mdbiolab/pdf/Biodiesel%20Student%20Handout%202012.pdf (accessed on November, 29th 2013)

TIM. 2013. Petunjuk Praktikum Biokimia. Surabaya : Unipress

ATTACHMENT

No. Exp Picture Explanation

1. Determining the peroxide Number

The sample and blanco solution when let for

20 minutes

The sample solution when added by

aquades (right) and after first titration (left)

The sample solution after first titration (left)

and second titration (right)

Sample solution after second titration

Blanco solution after second titration

2 Determining Free Fatty Acid (%FFA)

Blanco solution after titrated with NaOH

Sample solution after titated with NaOH

All sample solution after titrated by NaOH