Transesterification of EPO

Transcription

1 Transesterification of EPO Background: In the normal human diet, about 25 to 50% of the caloric intake consist of fats and oils. These substances are the most concentrated forms of food energy in our diet. When metabolized, fats produce about 9.5 kcal of energy per gram. When fats and oils were hydrolyzed, they gave rise to several "fatty acids" and the trihydroxylic alcohol, glycerol. Thus, fats and oils are esters of glycerol, called glycerides or acylglycerols. Because glycerol has three hydroxyl groups, it is possible to have mono-, di-, and triglycerides. Fats and oils are predominantly triglycerides (triacylglycerols), constituted as follows: Most fats and oils are esters of glycerol, and their differences result from the differences in the fatty acids with which glycerol may be combined. The most common fatty acids have 12, 14, 16, or 18 carbons, although acids with both lesser and greater numbers of carbons are found in several fats and oils. These common fatty acids are listed in Table below along with their structures. These acids are both saturated and unsaturated. The saturated acids tend to be solids, while the unsaturated acids are usually liquids. Fats are made up of fatty acids that are mostly saturated, while oils are primarily composed of fatty acid portions that have greater numbers of double bonds. Unsaturation lowers the melting point. Fats (solids) are usually obtained from animal

2 sources, while oils (liquids) are commonly obtained from vegetable sources. Therefore, vegetable oils usually have a higher degree of unsaturation. About 20 to 30 different fatty acids are found in fats and oils, and it is not uncommon for a given fat or oil to be composed of as many as 10 to 12 (or more) different fatty acids. Omega-6-fatty acid, omega-3-fatty acids Omega-6-fatty acids are one of two groups of essential fatty acid. Linoleic acid (LA) and gamma linolenic acid (GLA) are essential fatty acids. LA cannot be manufactured in the body and must be consumed in the diet. LA is an 18 carbon chain with the first double bond on the sixth carbon from the omega end (Carbon #1). LA is absorbed and converted into GLA. The GLA is the precursor to prostaglandins, PG1, and PG2. Omega-3-fatty acids are the second group of essential fatty acid. Alpha-linolenic acid (LNA) is an essential fatty acid that falls in the omega-3-fatty acid group. Linoleic Acid (LA) Delta-6-desaturase Gamma Linolenic Acid (GLA) Alpha LinolenicAcid (LNA) PGE1 di -homo-gamma linolenic acid (DHGA) Arachidonic Acid (AA) Cyclooxygenase Ecosapentaenoic Acid (EPA) Cyclooxygenase PGE2 PGE3 Both PGE1 and PGE3 are anti-inflammatory agents, while PGE2 is inflammatory. An example of the mechanism of how the three prostaglandins work in the coronary system. PGE1 and PGE3 keep platelets slippery and flowing, preventing blood clotting. PGE2 is inflammatory and increases platelet stickiness and blood clotting. All three forms of prostaglandins must be present to ensure a functioning clotting system. PGE1 appears to act as a diuretic, while PGE2 aids in the retention of water and salts in the kidneys. Evening primrose oil (EPO) is an excellent source of both linoleic acid and gamma linolenic acid. For those that lack the enzyme to convert LA to GLA, EPO is a good source of gamma linolenic acid to produce PGE1 and PGE2. Experiment: The purpose of this experiment is to determine the fatty acid composition of Evening Primrose Oil (EPO).

3 Triglycerides are triesters of glycerol, but they rarely have the same acid groups attached to glycerol. Typical oil may have some molecules of a triglyceride with all of the same fatty acids attached. You will also find molecules in oil that have different fatty acids attached to glycerol. Since there are as many as ten different fatty acids represented in fats and oils, there are many different combinations of triglyceride molecules present in the soybean oil. To solve the problems inherent in analyzing a complex mixture of different triglyceride molecules present, it is necessary to break apart the triglycerides into their individual fatty acids. After this is done, the composition of the sample can be determined. The classical procedure for obtaining the fatty acids from a fat is saponification (soapmaking), or hydrolysis with alkali to give the sodium salts of the fatty carboxylic acids mixed with glycerol. Such a mixture constitutes soap. Acidifying the soap mixture gives the fatty acids: Unfortunately, saponification is slow at room temperature; and at higher temperatures, the unsaturated acids show a tendency to isomerize the positions of their double bonds. An additional complication is that although some of the acids are solids, others are liquids, and all are high boiling. Instead of saponification, we shall use the transesterification reaction, in which one alcohol group of an ester is exchanged for another. If we use a monohydroxylic alcohol like methanol, three different methyl esters and one molecule of glycerol will be produced. The methyl esters have lower boiling points than the acids, and can easily be analyzed by gas chromatography. If sodium methoxide in methanol is used, the triglycerides are converted cleanly into the methyl esters. Reaction in which the nucleophile is an alcohol can be promoted by the conjugate base of the alcohol. R 1 COOCH 2 R 1 COOCH 3 HO CH 2 R 2 COOCH 2 CH 3 O - Na + R 2 COOCH 3 + HO CH 2 CH 3 OH R 3 COOCH 2 R 2 COOCH 3 HO CH 2 Triglyceride methyl esters glycerol Acetic acid is added after the reaction to acidify the mixture, and the methyl esters are extracted into hexane and transferred to a vial for gas chromatography analysis. Data Analysis Sample will be analyzed by Gas Chromatography. Your chromatogram will be available on the following Friday. This means that lab report will not be due until the last lab.

4 Gas Chromatography: Injection Port The sample is introduced in a heated injection port where the compound is vaporized and introduced to the carrier gas. Most commonly the carrier gas is helium. The helium is the mobile phase. Column The compounds are separated in the column which contains the stationary phase The stationary phase is housed in a tubing which contains a support material which could be a variety of materials such as crushed firebrick, silica, alumina or glass beads. The solid support holds the stationary phase which is a liquid, a wax or a low melting solid. The stationary phase varies in polarity. Hydrocarbons grease is a non polar stationary phase, silicone oil is medium polarity, and diethylene glycol succinate is polar. Which type of stationary phase one uses depends on the type of compounds being separated. In the heated column, the compounds equilibrate between gas and liquid phase. The length of time that a compound stays in the column is dependent on it s solubility in the liquid phase. Remember that likes dissolve in likes. Separation is determined by: 1. The compounds boiling point, the lower the boiling point, and the faster it will travel through the GC. In general, compounds with the same functional group will be separated by molecular weight. 2. The rate of flow of the carrier gas. The flow rate must set optimally so the samples 3. The choice of liquid phase. Detector The detector is a mass spectrometer. The molecules in a gas stream goes through an ionizing chamber where it is ionized. The positively ionized fragments are sorted by mass and detected. Functional groups give a general signature fragment patterns. The quantity of ions determines the size of the signal. The larger the sample the more ions, the larger the signal. Qualitative determination (identification) What is present? a. Identification by retention time b. Retention time is the time from injection to detection. If the parameters are not changed, such as the helium flow and temperature, the same compound will have the same retention time. d. By running a standard mixture with known components, an unknown mixture can be identified by comparing retention times. e. The standards are identified by comparing the mass spectra to a library. Quantitative determination. How much of the components are present? a. The computer determines the % component by calculation the area of the peak. b. The ratio of the individual peak of the component over the total area of all the component gives the relative percent of each of the component. c. The standards we will use for this experiment are: Methyl linoleate Gamma methyl linolenate Methyl stearate Methyl palmitate

5 Methyl oleate d. The fatty acid used as standard is the major components of EPO; however there could be minor fatty acid that could show up as a peak. You will not be able to identify any of the peak that are not standard but you will be able to quantify an unknown fatty acid.