Biochemistry Lab


Biochemistry Lab Essay, Research Paper

I. Introduction

In this experiment, lipids from ground nutmeg are extracted using a combination of solvents and identify the lipids through chromatography. The purpose of using solvent combinations is to elute the lipids based on their polarity to binding of the silica gel. The chromatography is performed on a silica gel plate and the use of iodine to visualize the lipids. By calculating the Rf values for each compound and comparing them to the known lipids, we are able to distinguish the lipids within the grounded nutmeg.

II. Procedures

Add 5 g crushed nutmeg and 50 mL hexane-isopropanol into a flask and warm for 15 minutes.

Remove the extra solvent on a steam bath under a hood while flushing the flask with N2 gas, leaving the crude extract. Weigh extract.

Prepare silica gel column. Add 6 g of silica gel in 20 mL of hexane to make a slurry. Block column with small piece of glass wool, add 5 mL of hexane and then add the silica slurry up to the 10 cm mark.

Prepare .05 to .075 g of crude lipid by dissolving it in hexane. Add to the silica gel slurry in the column.

Begin collecting samples with the pure hexane. Keep adding hexane so that the silica gel column does not run dry. Collect one 20 ml sample. Repeat with 90:10 hexane and collect 4 20-mL bottles. Repeat with 80:20 hexane and collect 2 20-mL samples.

Analyze each fraction by spotting 10 times with capillary tubes on a TLC plate, which is exposed to iodine vapor for 15 minutes.

III. Results

Lipids Distance Traveled Rf – Correct Results

Rf = Distance Solute Traveled / Distance Solvent Traveled (mm)

Hexane- 0

Hexane 90:10- 123/140 = .879

Hexane 90:10- 39/141= .277

Hexane 90:10- 38/141= .270

Hexane 90:10- 0

Hexane 80:20- 20/139= .863

Hexane 80:20- 19/139= .137

Triolein- 107/137= .78

Oleic Acid- 44/143= .309

Cholesteryl Linolet- 141/142= .99

Crude Lipid- (10 spots)

0, .035, .063, .092, .155, .204, .303, .45, .669, .99

Lipids Distance Traveled Rf – Incorrect Results

Hexane- 0

Hexane 90:10 (All 4 samples)- 0

Hexane 80:20 (Both Samples)- 0

Triolene- 85/149= .570

Oleic Acid- 55/149= .369

Cholesteryl Linolet- 150/150= 1

Crude Lipid- 15 spots

IV. Discussion

In this experiment, extracted lipids are compared to three known lipids listed above. The results obtained for our group were classified as incorrect due to unknown experimental error. Therefore, results from another group were attained for discussion purposes. In looking at the results it cannot be clearly identified what the unknown lipid is. The Rf values from the three standard are triolene: .78, oleic acid: .309, and cholesteryl linolet: .99. The crude lipid produced 10 different spots. Spot 9: .669 is fairly close to the standard triolene. On the crude results, spot 10: .99 is close to the standard cholesteryl linolet. The rest of the hexane 90:10 and 80:20 samples did not match up to the standards. The hexane was supposed to produce a 0 Rf values since it is nonpolar and would not be expected to travel on the TLC plate. Theoretically, the hexane extract, a very nonpolar compound, should have traveled further than the 90:10 and 80:20. In looking at the results this is exactly what happened since the final value of the lipid was .99. The Rf value of the hexane extract should be near one as those of cholesteryl linolet or oleic acid because the silica gel is polar. The 80:20 extract is the most polar extract as far as this experiment is concerned and it should have the lowest Rf value since it will bind to the silica gel plate and will not travel with the solvent front as readily as nonpolar molecules. The 80:20 should have Rf values near the oleic acid, since both are polar. As for the 90:10, the Rf value does not suggest that it is any of the standard lipids, however, during the visual inspection, its location appeared to be in the same position as triolein. The Rf value of triolein and that of the 90:10 extract are the nearest to each other with a marginal amount of error. The error may be because improper measurements were made because the solute has migrated horizontally from its original spot, giving it a smaller value then would be expected.

In comparing these results with our group’s results, one can see many discreprencies. First of all, the hexane samples in all three concentrations did not produce any results. The oleic acid and cholesteryl linolet had correct values matching up to the other group’s results. But the triolene had a value of .57 and the triolene sample from the other group was .78, showing a difference. The major reason that the results may have been different is experimental error. While collecting samples, the column went dry at one occasion. This may have produced samples with incorrect amounts of lipid in it. The silica gel column has to be kept running with fluids at all times. Otherwise, the results will be incorrect, as our results were.

V. Conclusion

The experiment served its purpose by demonstrating the characteristics of lipids. The useful techniques of adsorption chromatography on a silica gel column and analysis by thin layer chromatography proved to be useful techniques. In my opinion these techniques should be learned by anyone that wants to go in a biology/biochemistry career. This experiment also proved how important experimental technique and procedure is. A little mistake or experimental error, such as the column drying, can produce highly incorrect results. Overall, the experiment was very enjoyable and fatty acids can be concluded as a nonpolar with slight polarity substance.

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