This exercise will investigate the properties of lipids present in chicken egg yolks. Fatty acids, cholesterol, and triglycerides are all examples of lipids. You will separate the lipids based upon their polarity using column chromatography and determine the amounts of each that are present using gravimetric analysis. You will also quantitate the amount of cholesterol present in a chicken egg yolk. These experiments have been organized into three weeks:
Week 1: Extraction and isolation of lipids from egg yolks, begin gravimetric analysis.
Week 2: Continue gravimetric analysis, calculate total amount of lipids, separate lipids using silica column chromatography, concentrate neutral and polar lipids.
Week 3: Determination of cholesterol concentration.
This experimental procedure has been adapted from: Basic Biochemical Methods by Renee R. Alexander and Joan M. Griffiths, 1993, Wiley-Liss, Inc. New York, NY.
Chromatography
What makes a molecule polar?
What interactions are occuring with the column packing? with the solvent?
Gravimetric analysis
Analytical techniques for sample handling, thorough drying, accurate weighing: How will
you ensure that your data are accurate?
Cholesterol determination
Complexation of cholesterol with ferric chloride: why is it colored?, what part of the
cholesterol molecule is involved? Quantitation by visual spectroscopy, standard curve: How
do you calculate how much cholesterol was present in the whole egg?
You will write one laboratory report that includes the introduction, methods, results and discussion of all three laboratory exercises. Dr. Stone will also consult your laboratory notebook, so please have it available on demand.
For your introduction, you will need to search the literature to answer the preceding questions and fully describe the importance of studying lipids. You will need to do the literature search prior to coming to class on November 6th, so that you will be able to fully participate in the discussion. In addition to the questions listed above, you should fully address the following:
1. Dietary lipids have been associated with an increased risk for several diseases. What are the diseases, what lipids are involved.
2. How do the above lipids cause the above disease(s)?
3. Lipids are beneficial, give several examples of this in mammals.
4. Lipids come in several sizes and shapes, describe them and list the normal function of each. For example, triglycerides consist of three fatty acids linked to one glycerol molecule and they are used as a source of energy. Other lipids to be described:
Cholesterol
Phospholipids
Sphingolipids
5. How much cholesterol does the average human synthesize each day? What is the reccommended daily dietary intake of cholesterol? Why should saturated fats be limited in a patient who has hypercholesteremia?
In the results section, report the amount of each lipid fraction present in your sample and calculate the amount that is present in a whole egg yolk. Also, report the amount of cholesterol present in your sample and calculate the amount present in a whole egg yolk. Be sure to show your standard curve for the cholesterol determination. What fraction contained the most cholesterol. Does the structure of cholesterol correlate to the fraction where it was primarily found?
Your discussion should compare your data for the gravimetric analysis and the cholesterol quantitation to the class averages for these values. (Each student will submit their data and this will be posted on the www.) Restate the order of the fractions that eluted from your silica column. Explain the interactions of the solute, solid support and solvent in terms of molecular structure and polarity and rationalize the elution order of the lipids.
Be sure to cite all of your literature references. You must have at least two
sources that are not text books, and encyclopedias should not be used.