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Thin layer Chromatography lab report Introduction
Thin layer Chromatography lab report Introduction
Thin layer Chromatography lab report Introduction
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This lab used thin-layer chromatography to analyze the polarity of an Analtech dye mixture and the polarity of pigments isolated from a spinach leaf using liquid-liquid extraction. Chromatography is a method of separating mixtures into their pure compounds. The separation occurs because the intermolecular attractions in the mixture differ in their polarity. The stationary phase also has intermolecular attractions. Separation occurs as the mobile phase passes over the stationary phase. All types of chromatography have a stationary and mobile phase. A stationary phase is a substance that has different levels of attraction to the mixture. The stationary phase in this lab was the silica gel on the TLC plate. The movie phase is the solvent to carry …show more content…
Additionally, chromatography can be used to test the purity of a compound. If a sample is run with several solvent systems, and there is only spot each time, the sample is probably pure. Additionally, TLC can be used to identify an unknown molecule. If an unknown and know are run with several different solvent systems, and have the Rf every time, they are probably identical. Rf stands for retention factor and is equal to the distance the spot travels divided by the distance the solvent travels. TLC can also be used to monitor the progress of a …show more content…
Rf values have a range from 0 to 1. An Rf value of 0 indicates the mixture doesn't move while a value of 1 indicates the mixture traveled the complete length of the plate. For a specific solvent each compound has a specific Rf value. This is also true of the solvent concentration. Rf values are useful in identifying unknowns. In this lab, the lower the Rf value the more polar the compound was. This lab also isolated spinach pigments by liquid-liquid extraction. Liquid-liquid extraction separates compounds based on their differing solubilities into different immiscible liquids. Hexane and ethanol were added to the broken up spinach. This created two different layers. The two layers formed because hexane and ethanol are immiscible meaning the do not mix. The ethanol layer was removed and discarded. Water was then added creating two new layers. Water and hexane are also immiscible leading to the formation of the layers. The hexane layer was removed and concentrated in a rotavap. It was then put on a TLC plate and thin-layer chromatography was performed. All of the pigments isolated were non-polar which is why they were able to be extracted in the hexane
There is a technique called polarimetry that uses polarized light and asymmetric carbons like those found in glucose. The amount of polaritization can be used to determine the amount of active molecules present in solution.3 The equation used is measured rotation of angle = (a)lc, (a) is optical activity, c is the concentration, and l is the pathlenght. The concept seems similar to Beers
The spots moved 3.8cm, 2.3cm, 2.1cm, 1.8cm, and 2.5 cm, for the methyl benzoate, crude product, mother liquor, recrystallized product, and isomeric mixture, respectively. The Rf values were determined to be.475,.2875,.2625,.225, and.3125, for the methyl benzoate, crude product, mother liquor, recrystallized product, and isomeric mixture, respectively. Electron releasing groups (ERG) activate electrophilic substitution, and make the ortho and para positions negative, and are called ortho para directors. In these reactions, the ortho and para products will be created in a much greater abundance. Electron Withdrawing groups (EWG) make the ortho and para positions positive.
A spectrum is a group of light wavelengths that are ordered in relation to their wavelength length. The electromagnetic spectrum consists radio waves, microwaves, infrared, visible, ultraviolet, X-rays and gamma rays. (1)Specifically, this lab looks at the visible light part of the spectrum because one of the colors in the visible light spectrum is shine through the sample. The visible light spectrum consists of colors of red, orange, yellow, green, blue, indigo, and violet. The color chosen to be shine through the sample is affected by the color of sample when mixed with the indicator Ammonium Vanadomolybdate (AMV). The color on the color wheel that is opposite of the solution’s color is the color that is shined through the
The objective of this experiment was to perform extraction. This is a separation and purification technique, based on different solubility of compounds in immiscible solvent mixtures. Extraction is conducted by shaking the solution with the solvent, until two layers are formed. One layer can then be separated from the other. If the separation does not happen in one try, multiple attempts may be needed.
Experiment #3: The purpose of this experiment to test the chromatography of plant pigments the alcohol test strip test will be used.
... samples before the incubation of 108 seconds. Then the 100 µL of colour reagent was put to the sample, merged and incubated for further 10 minutes. The absorbance at 615nm and 700nm wavelengths was calculated on the samples in the Cobas analyser and the sample concentration was measure according to :
No Fresh Aqueous Solution Yellow No Solution 4 Yellow No Methanol Orange Yes. In plate I, three of the four solutions had high Rf values compared to the fourth one. Solution 3 has the lowest Rf value because of the polarity of the solution.
After extracting, filtering, and condensing the blue and pink dyes off of the candies, it can be determined that the Blue candy is FD & C Dye Blue 1 and the Pink candy is a mix of Yellow 5 and Red 40. During the solvent evaporation for the blue dye, it was observed that when concentrated, the transparency of the dye was similar to that of Blue 1. During the paper chromatography of Project 2 Session 1, the blue dye showed on the chromatogram to have the highest spot height. Similar to the paper chromatography today which the blue dye was observed having the higher spot height as well. Figure 3 of the Project 2 In-Lab Report displays Blue 1 of having an Rf value of .729. The blue dye off of the candy, as seen in Figure 2, has an Rf value of .657.
Purple cabbage leaves were ground into a pulp, and were boiled. After the water turned deep purple, the anthocyanin extract liquid was filtered into the Ehrlenmeyer. The graduated cylinder was filled to the top of the scaled area with the extract. Four pellets of KOH were added. 20 drops of HCL were added. The mixture was swirled gently until color bands appeared. The sections of the graduate and the colors were recorded. The pH for each section was
Begin collecting samples with the pure hexane. Keep adding hexane so that the silica gel column does not run dry. Collect one 20 ml sample. Repeat with 90:10 hexane and collect 4 20-mL bottles. Repeat with 80:20 hexane and collect 2 20-mL samples.
Pipet chill 0.5M sucrose onto the chloroplast pellet. Use a wooden stick to mix the pellet with the sucrose until it is completely resuspended. Pipet the resuspended pellet into microcentrifuge tube labeled “P2. and keep the tube on ice. For the next steps, pipet DCPIP mix into cuvettes 1- 6. And pipet deionized water into cuvette Pre-warm the cuvettes for 5 minutes in a 37C water bath and set the spectrophotometer to 620 nm. Measure the initial absorbance of cuvettes 1-6 using cuvette 7 as the blank. Place cuvettes 2, 4, and 6 in a dark, 37C incubator. Place cuvettes 1, 3 and 5 in a test tube rack with a 100W light bulb. Read the absorbance of cuvettes 3 and 5 EVERY MINUTE for the next 10. Measure the absorbance of the dark incubated cuvettes 2, 4 and 6 after 15 minutes. After conducting the isolation of chloroplast and the photosynthesis assay of both plant sources, out of red cabbage and spinach, the spinach had a constant decrease of absorbance compared to red cabbage were it varied as the minutes passed by. Also as a result, for the red cabbage there was not a noticeable pellet like their was in the
Overall, the use of SEC runs into the problems of manipulation of the phase being rather difficult without compromising the experiment, but it has its valid applications especially when used in tandem with other techniques. 2.2 Affinity Chromatography In contrast with size exclusion chromatography, affinity chromatography relies very much on the interactions between the solute and the stationary phase. In an affinity chromatography experiment, the separation of the sample is based on the binding of the solute to binding sites present within the matrix of the stationary phase. SEC has the major drawback of not being a very manipulatable process when compared to the specificity that can be achieved with affinity chromatography.
This experiment demonstrated the ability of agarose gel electrophoresis to separate the mixture of dyes into their individual components by the application of a combination of dyes to the same sample well. The experiment effectively demonstrated that the dyes where different in structure, energy, and composition. Most of the dyes where negatively charged at neutral pHs and only one with positive charge. The positive charge one moved an opposite direction compared to the other dyes.
The extraction procedure isolates the pigments in spinach that will be used in the TLC analysis by leaving the insoluble properties behind. Each step serves a purpose in doing so, for example, the spinach is blended into a puree in order facilitate the centrifuge process. 75/25 hexane/acetone solution is used in the blending process because hexane alone is not enough and acetone alone is miscible in water. Anhydrous sodium sulfate is used to dry the organic solution after it has been through the centrifuge tube and separated into a test tube. The purpose of the alumina column is to filter unwanted inorganic chemicals while letting the desired organic chemicals to pass through.
Stationary phase is of extreme importance in an HPLC analysis, as the chemical nature of the same and its compatibility with the analyte of interest is extremely significant for efficient separation. The most commonly used stationary phase is silica packed column which acts as a adsorbent. Each component in the sample interacts with these silica particles and gets eluted out in different time intervals. These silica columns may be of C14 or C18 type depending on the component of interest and also the columns themselves come in various dimensions each with a specific purpose of analysis.