CHROMATOGRAPHIC METHODS: After successful extraction of phospholipids from their source analysis can be performed for the detection of specific phospholipids. This section will discuss chromatographic methods used for the analysis of phospholipids. All systems of chromatography consist of a stationary and mobile phase. A monster placed on a stationary phase, i.e., a solid or a liquid, and the mobile phase, a gas or a liquid, is allowed by modifying the system. The components of the sample will be separated on the basis of their ranging physical and chemical properties, imparting different affinities for the two phases. Thin layer chromatography (TLC) was the first chromatographic method for assessing phospholipids, and is commonly used today. …show more content…
Most of the systems vary to include ratios of chloroform, methanol and water. triethylamine, ethanol, hexane, and isopropanol are also common solvents in the mobile phase. Phospholipids migrate to the stationary phase a certain distance on the basis of the composition and affinity for the mobile phase. Identification is based on the delay (Rf), wherein the ratio of the distance moved by the analyte (i.e., the phospholipids) from the origin of the distance moved by the flowing solvent from the origin. Each of the analyte will have its own Rf value under certain circumstances. The separation of the phospholipid classes can be improved by two-dimensional chromatography. This technique requires developing the TLC plate in a direction, then dried, and developed in a solvent mixture at a 90 ° the first development (Singh and Jiang, …show more content…
One progress on TLC called high performance TLC (HPTLC; Sherma and Jain, 2000).HPTLC makes use of gel qualities that are finer, so that thinner plates and smaller. This allows faster separation times and better separation efficiency. HPTLC has improved reduced resolution and detection limits, so that the to walk two dimensions. To phospholipids visible on the TLC plates are used detection reagents. spots corresponding phospholipids may be carbonized by the addition of phosphomolybdic acid, sulfuric acid or copper sulfate in phosphoric acid, and then heating of the sample. The result charred spots can be scraped off the plate and measured by densitometry. In order to visualize the spots without charring, solutions such as rhodamine 6G or berberine can be used for UV-detection. iodine vapor is commonly used; but it does not work well with lipids which do not contain double bonds (Hoving,
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The investigation was carried out to identify the presence or absence of biological molecules in serum 2216. If the concentration in each test tube of the dilutions carried out will be more concentrated then the concentration of the test tube before it, then the color will be at an equal concentration with the other dilutions performed. The hypothesis was wrong because of the difference in concentrations due to the different measurements within the dilutions done. The test for starch was to add a drop of iodine solution to the pipette in the spotting tile. A reducing sugar solutions is add inside a test tube with 3 drops to then add 3 drops of benedicts and plane in a water bath.
Organic modifiers are used to change the retention time of different analytes. Organic modifiers lower mobile phase polarity. By increasing the amount of water lead to the repulsion of hydrophobic analytes out of the mobile phase. The hydrophobic analytes are pushed onto the stationary phase where they reside for duration up to the partitioning into the mobile phase. When ionic analytes exist in the sample, the addition of ion and buffer to the mobile phase are necessary.
Leah Romero 10/30/2017 Conclusion Lab 3 Chem 102L In lab 3, fundamentals of chromatography, the purpose was to examine how components of mixtures can be separated by taking advantage of different in physical properties. A huge process in this lab was paper chromatography, which was used to isolate food dyes that are found in different drink mixes. The different chromatograms of FD&C dyes were compared to identify which dyes are present in each of the mixes.
3.) Use the pipette to gather a small amount of the stomach contents. 4.) Lipid Test: Apply the stomach solution onto the paper bag tear. If a translucent spot appears, it is a positive result and there are lipids present in the stomach contents.
The mobile phase used was a mixture of ammonium acetate buffer and acetonitrile at a ratio of 400:600. A flow rate of 1 mL/min was maintained, and the detection wavelength was 292 nm (22). The required studies were carried out to estimate the precision and accuracy of the HPLC method and were found to be within limits [percent coefficient of variation was less than 15%]. Sample preparation briefly involved 0.4 μ membrane filter through which the sample was filtered, diluted with mobile phase, and 10 μL was spiked into
If there is a color change, then it is known that protein is present in the solution. Finally, lipids are tested. 5 mL of water are added to 5 mL of oil. 5 drops of Sudan 3 are added, and if the color changes, then lipids are present. Next, the McMush is tested.
INTRODUCTION A gas chromatograph (GC) can be utilized to analyze the contents of a sample quantitatively or in certain circumstances also qualitatively. In the case of preparative chromatography, a pure compound can be extracted from a mixture. The principle of gas chromatography can be explained as following: A micro syringe is used to inject a known volume of vaporous or liquid analyte into the head or entrance of a column whereby a stream of an inert gas acts a carrier (mobile phase). The column acts as a separator of individual or chemically similar components.
TLC was used to identify the actual unknown product as well as other products/reactants present in the filtered solution. The procedure was conducted by placing a TLC plate in a developing chamber that is filled with a small amount of solvent. The solvent cannot be too polar because it will cause spotted compounds on the TLC plate to rise up too fast, while a very non-polar solvent will not allow the spots to move. The polarity of the spots also determines how far it moves on the plate; non-polar spots are higher than polar ones. After spots on the TLC form, the Rf values are calculated and used to analyze the similarity of the compounds.
Introduction : Liposomes were discovered in the early 1960 by Bangham and colleagues (Bangham et al., 1965 ) and subsequently became the most extensively explored drug delivery system. Liposomes are concentric bilayered vesicle in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. A liposome can be formed at a variety of sizes as uni-lamellar or multi-lamellar construction, and its name relates to its structural building blocks, phospholipids, and not to its size. Liposomes are artificially prepared vesicles made of lipid bilayer.
The gel-clot method is the simplest and most basic form of detection. It is carried out by adding the reagent to a sample in a test tube and incubating said sample at 37°C for one hour. The tube is then inverted, if a clot is present then the result is positive. It is and endpoint method that can be made semi-quantitative by testing using serial dilutions. The results are expressed as endotoxin units (EU), which is a measure of endotoxin potency rather than the quantity.
Introduction The term chromatography actually means colour writing, and signifies a technique by which the substance to be examined is placed in a vertical glass tube containing an adsorbent, the different segments of the substance traveling through the adsorbent at distinctive rates of velocity, according to their degree of attraction to it, and producing bands of colour at different levels of the adsorption column. The substances least absorbed emerge earliest; those more strongly absorbed emerge later. (Wixom et al., 2011) In chromatography of all types, there is a mobile phase and a stationary phase.
DETERMINATION OF PERCENTAGE ETHANOL IN BEVERAGES 1. Introduction to Gas Chromatography Gas chromatography is a very powerful separation technique for compounds that are reasonably volatile. The components of a sample partitions into two phases, the 1st of these phases is a immobile bed with a great surface area, and the other is a gas phase that permeates through the immobile bed. The sample is evaporated and passed by the mobile gas phase or the carrier gas through the column. Samples separates into the stationary liquid phase, based on their solubilities at the given temperature.
Synopsis This experiment is the determination of Calcium Carbonate (CaCO3) content in toothpaste with the use of back titration while demonstrating quantitative transfer of solids and liquids. A accurately weighed quantity of toothpaste was dissolved in excess volumes of HCl. This solution is then titrated with NaOH to find the volume of the excess HCl. The volume of HCl reacted, which is found by substracting the volume of given HCl with the volume of excess HCl reacted, can be further manipulated with mole fractions to find the mass of CaCO3 and thus the CaCO3 content in toothpastes.
For TLC profiling, 4 TLC plates were prepared for the testing of each solvent. As shown in Figure 1, the green food dye was placed at the bottom center, specifically 0.5 cm away from the bottom of the plate, with the use of a capillary tube. Each one of the silica plates were then vertically placed in a small beaker with its inside surrounded by a filter paper saturated with the solvent to be tested and a small amount of the same solvent at the bottom. The TLC plate was then taken out when the rising solvent was about to reach the top of plate. The ammonia: 1-butanol solvent was tested 7 times due to some personal
In succinct terms, this method utilizes also an acid digestion and posterior extraction using a nonpolar solvent (like diethyl ether and/or petroleum ether) in a Mojonnier flask. Both ISO and AOAC methods are based on Soxhlet extraction and can be used as an arbitration or a reference method to determine the Total fat parameter in food samples in order to create the nutritional label. Actually, many countries have their reference and arbitration methods based on any of both methodologies cited. Nevertheless, in academia, the Folch method (Folch and others 1957) is commonly used in research and studies, especially in the field of nutrition, biochemistry and agriculture research. (Taha and others