Isolation, Purification, and Partial Characterization of a Lipid Extract from Nutmeg Beatriz E. Saldana Farias∗ Biochemistry 1 Laboratory Experiment 8 BIO 4010 { 3 Report submission date: November 20, 2015 Instructor: Wade Dauberman E-mail: bsaldana2012@my.fit.edu Abstract The purpose of the laboratory experiment was to identify the molecular structure and lipid class of the most abundant lipid in Nutmeg through lipid extraction and thin layer chromatography (TLC). First the lipids were extracted from nutmeg, which resulted in a 46% yield, and then the extract was placed on a glass silica plate, along with Palmitic acid, Phosphatidylcholine, Triolein, Tripalmitin, and Myristic acid, for analysis though TLC. Different samples move at different rates up the glass silica plate due to their differences in polarity and solubility. The TLC depicted seven different lipids in the nutmeg extract and a variety of Rf values for all of the lipids. According to various sources, Myristic acid is derived from nutmeg, and due to the fact that none of the Rf values calculated for the nutmeg extract matched that of myristic acid, it can be determined that the TLC was not entirely successful. ∗To whom correspondence should be addressed 1 INTRODUCTION There are four major biomolecule groups, one of which consists of lipids, a general category for triacylglycerols, glycerophosphatides, sphingolipids, and sterols. Lipids are used for energy storage, signaling, vitamin absorbency, and structural components of the cellular membrane. These biomolecules are most frequently found bound to another compound, thus making their extraction very difficult. In 1879 Soxhlet Franz first developed a method for lipid extraction, by developing a solid-liquid extraction apparatus that was first used to separate fats from food (Soxhlet 1879). To this date scientists continue to develop new methods for lipid extraction, due to the fact that the environment from which the lipid needs to be extracted from affects the methodology of the extraction. The purpose of lipid extractions varies, but it remains a common research technique for a number of different analysis. Lipid extractions are a widely method for research in a variety of different scientific fields. For example, in geology there is a whole field dealing with geochemical biomarker extraction which aims to investigate the molecular and isotopic composition of the matter stored in geological samples (Jansen et al. 2006). The samples are typically processed in order to extract lipids, and then the extracted material undergoes various methods of analysis from isotopic ratio mass spectrometry to elemental analysis; the ultimate goal of most geochemical lipid extractions is to understand the sample composition and its history (Schwark 2013). Lipid extractions are also commonly used in the medical field, food science, and of course, in the field of biochemistry. In this laboratory session, the students used lipid extraction to collect the lipids in nutmeg, and then used the extract to preform thin layer chromatography in order to compare the lipids in nutmeg to five other lipids available in the laboratory. Nutmeg is a seed originating from a Myristica fragrans plant, an evergreen tree indigenous to Indonesia. Nutmeg contains a large ammount of a single type of lipid that is easily extractable, thus it is widely used for experimentation in lipid extraction (Frank et al. 1971)s. 2 The purpose of the laboratory was to identify the molecular structure and category of the lipid mentioned above. There are about eight different categories of lipids, all of which have a different range of Rf values when analyzed using thin layer chromatography; these categories are fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and poyketides. The lipids used to make the comparison were Palmitic acid, Phosphatidylcholine, Triolein, Tripalmitin, and Myristic acid. Palmitic acid is a saturated fatty acid, and is the first fatty acid produced during fatty acid synthesis when there is an excess of carbohydrates; palmitic acid is a carboxylic acid connected to a fifteen carbon straight chain (Cox 2013). Phosphatidylcholine is glecerophospholipid that contains a choline as a head group and a glycerophosphoric tail, which consists of several fatty acids; one of the fatty acids is saturated and the other unsaturated. Triolein is a member of the glycerolipid category; Triolein is a triglyceride with a symmetrical structure, thus the three fatty acid chains are composed of the same fatty acid. Tripalmitin is triglyceride derived from palmitic acid, ergo is also a member of the glycerolipid family. Finally, Myristic acid is a saturated fatty acid consisting of a chain of thirteen carbons and a carboxylic acid head typically derived from nutmeg in its triglyceride form, Trimyristin (Spricigo et al. 1999). Thin Layer chromatography (TLC) is an analytic technique developed by Nikolai A. Izmailov in 1937 when the necessity of a faster analytical method arose in order to obtain results in a short amount of time (Berezkin et al. 2008). TLC is preformed on various surfaces, but for the purpose of this laboratory, it was preformed on a glass silica plate. The plate is first spotted and then placed in a chamber so that the solvent can move up the plate, smearing the spots originally created. TLC separates the components of a mixture by the different polarities. The solvent system used in the experiment had the capacity to separate triacylglycerols, cholesterol esters, fatty acid esters, and fatty acids. Different compounds that are spotted on the plate run at different rates, thus when a variety of fatty acids are spotted, they produce different length smears. After the whole procedure has been carried out, the spots become visible and the Retardation factor (Rf ) can be calculated. Lower Rf 3 values indicate slower motility of the sample, these discrepancies in travel rates are due to each substance's different polarities and solubility in the solvent (Sherma 1988). MATERIALS AND METHODS Nutmeg Extraction The first step in the experiment is to place 2.5 grams of nutmeg (crude liquid) into a 125ml flask and adding 25ml of a 3:2 mixture of hexane:isopropanol solvent into the flask; the mixture was then placed on a 37◦C water-bath for 15min. The solution was gently swirled while being heated to encourage appropriate lipid extraction and was covered with a loose fitting piece of foil to prevent evaporation of the solvents being used. After the 15min of heating, the solution was filtered through a large filter paper and into a flask using vacuum was used to expedite the filtration process. The original flask was cleaned with 20ml of the 3:2 mixture of hexane:isopropanol and the solution was filtered in order to extract the remaining nutmeg. A 125ml flask was pre-weighted and the extract was poured into it. The ◦ flask was then placed in a 90 C hot water bath and flushed with N2 gas in order to evaporate the remaining solvents. The flask was then placed into an ice-bath for 5min and once the oil had solidified, the flask was weighed again and the weight of the extract was calculated in order to obtain the crude product amount. Thin Layer Chromatography Thin layer chromatography was used to calculate the RF values of the examined samples, these samples were: Nutmeg extract, Palmitic acid, Phosphatidylcholine, Triolein, Tripalmitin, and Myristic acid. First 5ul of each sample was placed on a glass silica plate 2cm apart, then the plate was allowed to dry and it was spotted again with 5ul of each sam- 4 ple. The plate was then placed in a chamber containing no more than an inch of an 80:20:1 solvent system of hexane:diethyl ether:acetic acid for about 45min. After the solvent front had reached about 7cm from the original mark, the plate was removed from the solution, the liquid front was marked, and the plate was allowed to dry. Then the plate was placed in an iodine chamber in order to develop visible spots and smear marks. Once the plate was removed, the spots were marked promptly and the Rf value of each sample were calculated by dividing the distance from the origin migrated by the compound by the distance from the origin migrated by the solvent. RESULTS Nutmeg Extraction After the lipid was extracted from the nutmeg, the final recovery percent yield was calculated by dividing the grams of purified sample by the grams of crude liquid, the calculations revealed the extract weight to be 1.15grams. The weight of the purified sample was obtained by subtracting the final weight of the flask containing the purified substance by the original weight of the flask. The recovery yield percentage was 46% The calculations and results are depicted below: Initial weight of flask: 79.15grams Final weight of flask: 80.30grams Extract Weight = Final weight - Initial weight Extract Weight = 80.30 grams - 79.15 grams = 1.15 grams grams of purified liquid Recovery yield% = × 100% grams of crude liquid 1:15 grams Recovery yield% = × 100% = 46% 2:5 grams 5 Thin Layer Chromatography (TLC) After the thin layer chromatography was completed, the Rf values were calculated for each of the six samples. Table 2 depicts the calculated Rf values of each of the samples in ascending order, with Nutmeg Extract at the top, due to its abundant Rf values. Nutmeg extract contains a variety of lipids, so through TLC the lipids were separated and produced distinct smears, thus providing seven different Rf values for Nutmeg. Phosphatilylcholine had an Rf value of 0.16, Palmitic acid had an Rf value of 0.32, Tripalmitin had an Rf value of 0.38, and both Triolein and Myristic acid had Rf values of 0.46. Table 2: Rf values of each sample resulting from the thin layer chromatography Lipid Rf value(s) Nutmeg Extract 0.07, 0.11, 0.18, 0.37, 0.51, 0.62, 0.83 Phosphatidylcholine 0.16 Palmitic acid 0.32 Tripalmitin 0.38 Triolein 0.46 Myristic acid 0.46 DISCUSSION After meticulous execution of the methodologies described in the Materials and Methods section of the report, concrete results were obtained for further interpretation.