Purification and Characterization of Triacylglycerols in Natural Oils

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Purification and Characterization of Triacylglycerols in Natural Oils

Purification and Characterization of Triacylglycerols in Natural Oils

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Part A. Isolation and Purification of the Unknown Lipids

Weight of nutmeg = 5.05559g

Weight of 250ml round bottomed flask with solid after evaporation is 142.2445g

Weight of an empty 250ml round bottomed flask is 139.7451g

Therefore, weight of crude product = 142.2445 – 139.7451

= 2.4994g

Weight of purified lipid is the difference in the weight of the flasks.

Weight of an empty beaker is 30.487

Weight of beaker with product is 30.8827g

Therefore, weight of purified product is the difference in the weight of the flasks

Weight of purified product = 30.8827 – 30.4873

= 0.3954g

Recovery yield % = g of purified lipid/g of crude lipid 100

= 0.3954/2.4994 100

= 14.3795%

Yield of product in g/g of nutmeg used in the isolation

= yield of purified product in grams / weight of nutmeg used

= 0.3594 / 5.05559g

= 0.0711 g/g of nutmeg used in isolation

Part B. Characterization of the Unknown Lipid

Lipids Distance Travelled Rf – Correct Results

Rf = Distance from the origin migrated by a compound / Distance from origin migrated by solvent

Separation was done on a thin layer plate that was 20 * 20cm in dimensions. Separation was done using a solvent that consisted of hexane: diethyl : acetic acid in the ration 80:20:1. The Rf value was determined by taking the quotient of the distance travelled from the origin by the compound over the distance travelled from the origin by the solvent.

For the nutmeg, the retention factor = Distance travelled by solvent/Distance travelled by the compound. The distance travelled way 100mm

For Nutmeg Rf = 31/100

= o.31

The retention factor of the other lipids as calculated from the chromatogram are:

56/100 = 0.56

60/100 = 0.60

22/100 = 0.22

78/100 = 0.78

53/100 = 0.53

The figure in bold is the one which represents the darkest band with all the other bands representing non-significant lipids.


Compound Retention Factor

Nutmeg 0.23, 0.45, 0.31, 0.65

Triglyceride 0.60

Cholesterol ester 0.60

Oleic acid 0.22

Fatty acid methyl ester 0.30

Glycerol phosphatide 0.60

Diacylglycerol(Monoolein) 0.68

A vivid observation of the chromatogram reveals that the most significant component was the triglyceride. The purification step was effective to aa small extent due to a lack of accuracy in the measurement of the samples. Additionally, there may be pollutants which affected the nature of the results to a large extent.

Retention Time % of Total

3.001 0.725

5.883 0.418

6.086 0.231

6.188 0.147

6.506 0.124

6.576 0.167

6.904 5.887

7.059 0.882

7.152 5.839

7.234 2.144

7.292 1.556

7.589 0.214

7.799 70.791

8.457 5.857

8.693 0.178

9.050 4.692


Retention Time Fatty acids present

3.001 Safrole5.883 Geraniol6.086 Eugenol6.188 Myristin6.506 Methoxy Eugenol6.576 Iso-elemicin6.904 Dipentene7.059 i-Terpinol7.152 d-Borneol7.234 d-Linalool

7.292 Elemicin7.589 Menthone7.799 Menthyl Isovalerate8.457 Camphene

8.693 Methyl palmitate9.050 Methyl palmitoleate8. Gas chromatography encompasses vaporization of a sample and its subsequent injection onto the chromatographic head column. The flow of inert gases which are in a gaseous mobile phase transports the sample. The adsorption of the column takes place on the surface of the inert solid; this is enhanced by the liquid stationery phase. The thermal conductivity detector is preferred when analyzing hydrocarbons that are smaller in molecular mass as well as analysis of inorganic gases. Its efficiency is achieved when the thermal conductivity of two gas flows is compared; the carrier gases which is pure and the sample. The thermal conductivity of the gas determines the behavior of the sample under study.

9. Weight % = Peak area / Total peak area for all the FAMES

Total peak area = 2013600

For Trimyristin = 1.171 e + 6 / 2013600

= 58.15

For Oleic acid = 77640/2013600

= 3.8%

10. Table for the components identified in the Free acid methyl esters identified in the sample.

Component % in the nutmeg Retention Time Essential oil 12.5% 8.457 9.693e + 4

Unsaponifiable constituents 8.5% 7.059 1.460e + 4

Oleic acid 3.0% 9.050 7.764e + 4

Linoleic acid as glyceride 0.5% 5.883 6914

Formic, acetate and cerotic acids Insignificant amounts 9.208 2431

Trimyristin 73.0% 7.799 1.171e + 6

The fatty acid that is present in greatest amounts is Trimyristin. It is a saturated fatty acid of myristic acid. My results deviate to a small extent with the results. Here is the link http://www.fao.org/docrep/v4084e/v4084e04.htm


Nutmeg is a seed that is rich in essential and fatty oils. Extraction of essential and fatty oils was observed in the experiment. The two types of oils in existent are essential oils and fixed oils. One characteristic nature of essential oils is that they are very volatile. On the other hand, fixed oils as their name suggests are generally non-volatile. The solubility of different lipids varies significantly with the type of composition. They dissolve in organic compounds like hexane but not in water. One major reason for this is that they are non-polar compounds which cannot dissolve in non-polar water. They dissolve in ether because both the lipids and water are non-polar hence are able to mix with one another.

Saponification is the process by which alkaline hydrolysis of esters leads to the formation of esters. Soaps are salts of alkaline salts like sodium and potassium of long chain fatty acids. The process occurs when the triglycerides that are present in fats or oils react with sodium hydroxide of potassium hydroxide leading to the formation of soap and glycerol. The presence of fatty acids is shown by the solution turning cloudy. However, since the bubble formation was minimal, the fatty acid presence was very small.

The gas chromatography is an equally important biochemical technique that forms the basis for any scientific analytical work. For it to be successful; there are various components that make it up and are all essential to the reproducibility of the results. One of the major components is the carrier gas; the carrier gas should be chemically inert in order to avoid reaction that may arise from the contact with the sample under investigation. The preferred carrier gases are among them, helium. The carrier system is fitted with a molecular sieve that is responsible for water removal as well as other impurities. The choice of the carrier gas is dictated by the detector type that is used.

The injection part is another feature within the gas chromatography set up; it is the point at which the sample under study is introduced into the system. The sample size should be small in size in order to ensure optimal column efficiency. Large sized samples lead to loss of resolution; the sample is preferably introduced as a plague of vapor and a micro-syringe is used. The sample port temperature is usually at 50 degrees high above the least volatile component’s boiling point.

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