Laporan Lipid
-
Upload
linda-wirianty -
Category
Documents
-
view
60 -
download
0
description
Transcript of Laporan Lipid
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