6.

0 ADDITIONAL QUESTIONS

1. In your own words, briefly explain the reactions that involve in saponification of
triglycerides.

Saponification can be defined as hydration reaction which triglyceride reacts with a

strong base i.e. sodium hydroxide to form glycerol and fatty acids.

Ester linkage that links the fatty acid and the glycerol backbone will be broken to produce
glycerol and soap. This process involves chemical degradation of lipids.It could be seen
from the reaction that every consumption of KOH by triacyglycerol will produce 3
molecules of soap.

2. Compare & discuss your results with theoretical saponification value for
each type of oil that has beenselected.

According to theory, the saponification value (SV) for corn is at a range of

187 to 196 whereas for SV for peanut is at 184 to 196. (Jan C.J.Bart, Natale Palmeri,
Stefano Cavallaro, 2010).The SV obtained for corn and peanut oil were 157.4 mg/g
and 151.28 mg/g respectively. The results of experiment were out of SV range for
peanut and oil. The inaccuracy of data might cause by errors during the
experiment. For instance, in back titration, hydrochloric acid might be added
excessively that caused overshooting the end point of titration.

3. Oil A has a saponification value of 100, whilst Oil B has a saponification

Value of 60. Based on the saponification values, determine the properties
oftheseoilsintermsofmolecularweightandfattyacidchain?

SVcan determine the average relative molecular mass of oils and is dependant
to fatty acid chains. The longer the carbon chain, the less acid required to liberate
per gram of 1 g of fat/oil. Oil A has a higher SV; it means that it has higher
percentage of ester bonds than longer chain of oil B. For having a longer chain,
 oil B has higher molecular weight(Jan C.J.Bart, Natale Palmeri, Stefano
Cavallaro, 2010).

4. Explain the significance of adding phenolphthalein as an indicator in

estimating the saponification value of fats.

Phenolphthalein is an indicator that used in acid-base titration. The excess KOH

is titrated with hydrochloric acid (HCl) to determine the volume of HCl needed to
neutralize the excess KOH. The colour of solution appeared pink (basic solution) when
phenolphthalein was added. During titration, the changed colour of pink to colourless
indicating the solution was neutralized. SV can be calculated by determining the volume
of titration.

5. Prepare the protocol for colorimetric or fluorometric method

for determining fatty acid concentrations/levels.

Flourometric method includes preparation of reagents, preparation of FFA

Standard, preparation of samples and assay protocol.Assay protocol fluorometric
are listed as below (Free Fatty Acid Assay Kit (Fluorometric)):

1. Add 10 µL of the free fatty acid (FFA) standards, samples or blanks to the 96-well
microtiter plate. h
2. Add 200 µL of 1X Enzyme Mixture A to each well.
3. Cover the plate wells to protect the reaction from light.
4. Incubate the plate wells at 37ºC for half an hour.
5. During the step 4 incubation, separately prepare the desired volume of Detection
Enzyme Mixture based on the number of tests to be performed. Maintaining all
components and mixtures at 4ºC
a. In a tube, add the appropriate volume of 1X Enzyme Mixture B.
b. To the 1X Enzyme Mixture B, add the corresponding volume of NEM
Reagent. Mix well.
c. Lastly, add the corresponding volume of Fluorometric Probe. Mix well.
6. Transfer 100 µL of the above Detection Enzyme Mixture to each well (from step
4).
7. Cover the plate wells to protect the reaction from light.
 8. Read the plate with a fluorescence microplate reader equipped for excitation in the
530-560 nm range and for emission in the 585-595 nm range.
9. Calculate the concentration of free fatty acid within samples by comparing the
sample fluorescence to the standard curve. Negative controls (without FFA)
should be subtracted.

CONCLUSION

The saponification value was estimated by reacting excess KOH with HCl. In this
experiment, two types of oils with ±1g mass were used. For better understanding of the
saponification value (SV), we collected data and analysis for other groups that conducting the
same experiment (four types of oils overall). SV (for the same oil) obtained for all groups
showed inconsistencies in data thus errors must occurred during the experiment, affecting the
data. SV obtained for corn oil and peanut oil also out of theoretical SV range. The SV
calculated from experiment for corn oil and peanut oil were 157.27 mg/g and 151.28 mg/g
respectively. The resulted data deviated much from theoretical SV of 187-195 (corn oil) and
187-196 for peanut oil. Error might occur during back titration i.e. HCl was added
excessively in the solution.

From this experiment, we learned that SV can be used to determine the relative
molecular mass and the chain length of the fatty acids of the fat or oil. Higher SV of oil
indicates lower molecular weight as it contains more short chains. Theoretically, SV of
peanut oil should be higher than corn oil (as molecular weight of peanut is higher that corn
oil), but the result was in contrary with the theory. The deviation in data might be contributed
by errors throughout the experiment.

The objective of this experiment was to estimate the saponification value of oils in
which four types of oil were prepared (2 oils for each group). In summation, the experiment
was considered a success as the objective of the experiment was achieved.

Bibliography
Free Fatty Acid Assay Kit (Fluorometric). (n.d.). Product Manual .

Jan C.J.Bart, Natale Palmeri, Stefano Cavallaro. (2010). 6 - Emerging new energy crops for biodiesel
production. From Soil to Oil Woodhead Publishing Series in Energy , 226-284.