BIOCHEMISTRY: Lipid Isolation and Analysis Practical Experiment Report

This practical assignment focuses on the isolation and analysis of lipids using thin-layer chromatography (TLC). The experiment involves extracting lipids from horse blood, egg yolk, and butter using chloroform:methanol. The extracted lipids are then separated on a TLC plate, and the migration distances of the lipid components are measured and compared to standards, including sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, cholesterol, and palmitic acid. The results are presented in tables showing the relative migration (Rf) values for each sample and standard. The analysis includes identifying unknown lipids and discussing prelab questions regarding lipid behavior and the chromatography process. The experiment aims to demonstrate the principles of lipid separation based on solubility and adsorption, emphasizing the importance of TLC in identifying and understanding lipid compounds. The student also provides calculations for the relative migration (Rf) values to identify the unknown lipids.

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INTRODUCTION
Structurally diverse group of amphipathic molecules is called lipids. Lipids have variety of
functions e.g. signaling, structure, energy and energy storage. In mammalian system the bulk of
mass of lipid molecules are found structurally in cellular membranes. The major component of
cellular membranes is phospholipids and can be put into classes based on their head groups and
further into species based on the acyl group composition. Proteins are separated from lipids
through organic solvents, carbohydrates and water soluble metabolites lipids are often separated
into groups through chromatography (Chen, Hai and Wang, 2016). Chromatography of choice
has been always the thin layer chromatography. Lipid composition of cells and membranes can
significantly differ with respect to lipid group, acyl group identity and phospholipid class
METHODOLOGY
Part (I); Isolation of lipids from red blood cells of a horse
NOTE: The pellet in the following steps should not be disturbed. A better result is more
important to this experiment than high purity. You should ask your demonstrator of any stage
you not sure of.
1) Take cells that have sediment through resuspension.
2) Move 250 μL of a horse blood which has been defibrinated into a micro centrifuge tube.
3) Rotate the blood sample for 2 min at 5000 rpm.
4) Use a pipette to remove and dispose the colorless and clear supernatant.
5) Annex 500 μL of 10 mm sodium phosphate buffer ( pH 7.0) and resuspend the pellet by
vortexing.
6) Mix membrane storage buffer with 800 μL.

7) Cool the sample on ice for like 2 min.
8) Try to centrifuge the sample for like 10 min at a maximum speed of (=16,000 g) in a micro
centrifuge.
NOTE: It is very difficult to see the resulting pellet, you to hold it up against a bright
light. And also it has a slightly more intense red color than the clear red supernatant.
9) Do away with and carefully dismiss the supernatant using a pipette (Vander et al., 2018). Do
not disturb the pellet.
10) Mix vigorously with 100 μL of chloroform: methanol (9:1) to dissolve the membrane liquid.
11) Rotate the liquid extract (=16,000g) for 2 min in a micro centrifuge.
12) By using micro pipette transfer the lower clear chloroform: methanol to a clean micro
centrifuge tube, and label it sample 1.
Part 2: Liquids from egg yolk’s isolation
1. Use a pipette to remove 2 μLof a egg yolk and add it a tube carrying 55 μL chloroform:
methanol.
2. Vigorously mix it well.
3. At a maximum speed (=16,000), centrifuge the sample for 1 min on top of a table.
Transfer 20 μL of the liquid from the bottom to a new tube and label it sample 2.
Part 3: Isolate liquids from butter
1) By using pipette remove 2 μL of butter (it is not possible to pipette it; so just scoop it
with pipette tip) and add to a tube containing 98 μL of chloroform: methanol (9:1).
2) Vigorously mix it well.
3) Take a new tube and transfer 20 μL of the liquid and label it sample 3.

Part 4: By using thin layer chromatography separate the liquids
1) Cut your TLC plate to fit in tanks for both solvent systems, and then check the solvent
level in the TLC tanks in your staining bath.
2) Do not remove the silica gel coating while performing it.
3) The position of eight equidistant application points on the line should be indicated.
4) Indicating is performed by removing 2.5 μL of your sample with a pipette and touching
the surface gently with the opening of the micropipette.
RESULTS
Figure 1: TLC Plate
SM* PC* PE* C* PA*
08 10 52 6.7 82
Dye front 132 132 132 132 132
Table 1: Migration distance of standards and SS2 dye front in mm
Horse Blood Egg Yolk Butter
22 09 60
50 50 80
67 69

85 90
Table 2: Migration distance of sample and SS2 dye front in mm
Sample Calculation
Relative migration, Rf =Distance of spot migration/Distance of solvent migration
Rf (SM standard) =08/132=0.0606
Rf (PC standard) =10/132=0.7575
Rf (PE standard) =52/132=0.3939
Rf (C standard) =67/132=0.5075
Rf (PA standard) =82/132=0.6212121
Rf (Horse Blood) =22/132=0.1667
Rf (Egg Yolk) =9/132=0.681681
Rf (Butter) =60/132=0.4545
Standard Rf
Sphingomyelin 0.0606
Phosphatidylcholine 0.7575
Phosphtidyethanolamine 0.3939
Cholesterol 0.5075
Paimitic acid 0.6212
Blood
Rf Identity
0.1667 Unidentified
0.37878 Unidentified
0.507 Cholesterol
0.6439 Unidentified
Egg Yolk
Rf Identity
0.681 Unidentified
0.37878 Unidentified
0.5227 Unidentified
0.681 Unidentified
Butter
Rf Identity
0.4545 Unidentified
0.6061 Unidentified

ANALYSIS
The identity of the other lipids which have not been identified with any of the standards used and
may include free fatty acids UGA among other lipids that were not included as part of the
standard for this study. The Unidentified lipids tend to have a feature of relative low migration
values that are less than those of the standards that are used (Heinrich, 2015). These lipids move
a significantly larger distance which is in most cases more than half the distance of the spot.
PRELAB QUESTIONS
What kinds of lipid do you expect in erythrocyte membrane?
Cholesterol and phospholipids
What happens in step 5 of the lipid isolation protocol?
Removal or extraction of the lipid in blood cell takes place in which it is separated from the
membrane (Wang et al., 2015).
What happens in step 10 of the lipid isolation protocol? What would happen if you add water
instead of chloroform/methanol?
In this step, dissolution of the lipids in the organic solvents takes place. Water does not dissolve
oil hence the isolation process would not be completed in case water is added instead of
methanol or any other organic solvent.
Order each of the six components of the chromatography solvents in terms of their polarity
Stationary phase>>Mobile phase
Which solvent system is more polar?

The stationary phase is more polar as compared to the mobile phase
Why is acid added to the solvents?
Acids are added to correct streaking which would otherwise make it difficult to calculate the Rf
CONCLUSION
Chromatography is based on the principle that various compounds have various solubility as well
as adsorption to the two phases where they are to be divided. The experimental objectives of this
study which was to demonstrate isolation and separation of various lipids was attained as thin
layer chromatography is a crucial technique when attempting to establish the identity of
compounds and understand how they separate.

References
Chen, X., Hai, X. and Wang, J., 2016. Graphene/graphene oxide and their derivatives in the
separation/isolation and preconcentration of protein species: a review. Analytica chimica
acta, 922, pp.1-10
Heinrich, H.W., Pluriselect GmbH, 2015. Device and method for isolating cells, bioparticles
and/or molecules from liquids. U.S. Patent 8,969,073
Kolmakov, A., 2016. Membrane Based Environmental Cells for SEM in Liquids (No. Liquid Cell
Electron Microscopy). Cambridge University Press
Vander Hoff, M., Benson, T., Pugh, M. and Bell, J., SMARTFLOW TECHNOLOGIES Inc,
2018. Method and systems for isolation and/or separation of target products from animal
produced waste streams. U.S. Patent Application 10/005,697
Wang, C., Hu, X., Guan, P., Wu, D., Qian, L., Li, J. and Song, R., 2015. Separation and
purification of thymopentin with molecular imprinting membrane by solid phase extraction
disks. Journal of pharmaceutical and biomedical analysis, 102, pp.137-143