Experiment 7: Qualitative Test of lipids and preparation of Carboxylic Acid Salts from Lipids
Procedure
Refer to the procedure in biochemistry laboratory manual from pages 41-42.
Results
Sample
Results (positive or negative)
Ester Assay
Phosphate Assay
Cholesterol Assay
Triglyceride
Positive
Negative
Negative
Cholesterol
Negative
Negative
Positive
Phospholipids
Negative
Negative
Negative
Unknown
Positive
Negative
Negative
Why soap is referred as a 'salt'?
This is because soap is produced by a saponification reaction of a fat or oil. During the production sodium hydroxide is used to neutralize the fatty acid and convert it to the salt.
What kind of molecular properties enable the substances to dissolve in water?
The attractive forces known as dipole-dipole forces between the water and solute molecules allow the substances to mix nicely.
Show the structures of reaction for saponification of ethyl acetate.
Discussion
The objectives of this experiment is to determine the qualitative test of lipids and preparation of carboxylic acid salts from lipids. In the qualitative test of lipids experiment, there are divided into three different assay which is ester assay, phosphate assay and cholesterol assay whereby they are used to determine the presence of functional group in triglyceride, cholesterol, phospholipids and unknown sample. The unknown sample will be one of the other three samples. The positive result of each test tells us that the chemical compound is found within the structure of the sample being tested
Based on the result obtained, the result indicates that, ester assay only gives a positive result for triglyceride and unknown samples. The positive indication of an ester bond is a color change to magenta. The phospholipid and cholesterol both exhibited this qualitative change. Based on the reaction, the colour change due to the chemical reaction of the hydroxyl groups of the glycerol form ester bonds with the carboxyl groups. The ester is converted to hydroxamic acid by reaction with hydroxylamine. The formed hydroxamic acid reacts with FeCl3 to produce magenta colored complex.
Next, analysis for phosphate assay is conducted. Phosphate assay is carried on in order to determine the presence of phospholipid detection. A small amount of the sample is acidified with 4 M nitric acid and 6 % ammonium molybdate. The presence of phosphate ions is indicated by the formation of a bright yellow precipitate layer of ammonium phosphomolybdate. Formation of yellow colour may be followed by slow formation of a fine yellow precipitate indicating the presence of phospholipid biosurfactant. Based on the result obtained all the sample are not reacted with the test. It is expected for the phospholipid sample to test positive, but they did not. This is maybe because of several error that occur during conducting this experiment which is the phospholipid sample are not homogenize properly thus resulting in suspension at the bottom of the bottle. This cause the concentration of sample is low therefore difficult to identify the colour change.
While for cholesterol assay, based on the result, it indicates that only cholesterol sample reacted with the cholesterol test. The formation of greenish after a few minutes indicates that the result is positive. The colour reaction occurs when a mixture of concentrated sulphuric acid and acetic anhydride is added to a cholesterol sample in the methylene chloride. This is because steroid that containing either two double bond in the same ring or a double bond adjacent to the ring containing a double bond or hydroxyl group will resulting in positive result.
Next, for the preparation of carboxylic acid from lipids it is called saponification as soap can be made from the base hydrolysis of lipid. This is because lipid consist glycerol and three fatty acid. In this experiment, ester have been hydrolyzed to alcohol and carboxylic acid components in the presence of sodium hydroxide, NaOH. Fats, oils, and fatty acids are insoluble in water because their hydrophobic tails are so long. As NaOH have been used in this experiment, it provide a basic condition to be used for hydrolysis. Thus, fatty acids produced are deprotonated and are present as carboxylate salts. Because these product carboxylate salts are charged, they are much more soluble in water than the corresponding uncharged fatty acids. Since the carboxylate salts also each have a long nonpolar tail, they are also compatible with nonpolar greases and oils and can be used as soap.
Conclusion
In conclusion, triglyceride and unknown sample give a positive result for ester assay while cholesterol give a positive result for cholesterol assay but for phosphate assay all the sample give a negative result.
References
Fossum, C. (n.d.). 79 Experiment 13 – Preparation of Soap. Retrieved May 15, 2017, from http://webcache.googleusercontent.com/search?q=cache%3Ay0lQAVdyjXQJ%3Awww.laney.edu%2Fwp%2Fcheli-fossum%2Ffiles%2F2012%2F01%2F13-Saponification.pdf%2B&cd=13&hl=en&ct=clnk
LIPID. (n.d.). Retrieved May 15, 2017, from http://www.cyberlipid.org/fa/acid0004.html
Protien Biochemistry Tests. (n.d.). Retrieved May 15, 2017, from https://www.wattpad.com/100097-protien-biochemistry-tests
Result
Preparation of carboxylic acid from lipid (Saponification process) Preparation of carboxylic acid from lipid (Saponification process) Cholestrol Assay – (change to greenish) cholesterol sample give + result Cholestrol Assay – (change to greenish) cholesterol sample give + result Phosphate Assay – All the sample give - result Phosphate Assay – All the sample give - result Ester Assay – (change to magenta) Triglyceride and unknown give + result Ester Assay – (change to magenta) Triglyceride and unknown give + result
Preparation of carboxylic acid from lipid (Saponification process)
Preparation of carboxylic acid from lipid (Saponification process)
Cholestrol Assay – (change to greenish) cholesterol sample give + result
Cholestrol Assay – (change to greenish) cholesterol sample give + result
Phosphate Assay – All the sample give - result
Phosphate Assay – All the sample give - result
Ester Assay – (change to magenta) Triglyceride and unknown give + result
Ester Assay – (change to magenta) Triglyceride and unknown give + result
