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:

Part I: Extraction and isolation of lipids from egg yolks, begin gravimetric analysis.
Part II: Continue gravimetric analysis, calculate total amount of lipids, separate lipids using silica column chromatography, concentrate neutral and polar lipids. Make fatty acid methyl esters (consult text by Rodney Boyer) and analyze by GC/MS.
Part III: 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.

Questions that you should be considering before, during and after the lab:

Chromatography
What makes a molecule polar?
What interactions are occurring 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?

For your introduction, you will need to search the literature to answer the preceding questions and fully describe the importance of studying lipids.    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 recommended 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 primary sources from the literature.