Lab 3

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CHEM 282 Experiment #3: The Extraction of Green-Leaf Pigments and their Chromatographic Separation Sophie Wolkoff (20107258) & Kaelen Partridge (20127197) TA: Bily Deng February 6, 2020 Experimental:

Step 1 of this experiment included a brief introduction to the classes of compounds that provide plants with their colour. These groups included chlorophylls, carotenoids and flavonoids. In step 2, the extraction of chlorophyll and carotenoid pigments was performed by grinding spinach leaves in a mortar with a 1:1 hexanes- methanol solution. This mixture was filtered into a separatory funnel where it was rinsed with hexanes and washed repeatedly with water. The resulting organic extract was collected to be dried with sodium sulfate and decanted in order to collect a dry liquid. This liquid was subsequently evaporated on a steam bath until about 1-2 mL remained. Step 3 involved the thin-layer chromatographic (TLC) analysis of the plant extract. Three jars containing varying ratios of hexanes:ethyl acetate solution, 9:1, 7:3 and 5:5 by volume, were prepared about 10-15 minutes before their use. A TLC plate was then placed in each of the jars to develop, each plate containing a spot of the green extract obtained from the lab and a spot of the provided β-carotene sample. The solvent rose to within 0.5cm from the top of the plate before they were removed, dried, and measured for the final positions of their pigments. Rf values were calculated for each plate using their measurement values, and β-carotene was identified within the extract on the plates using a side-by-side comparison with the authentic β-carotene sample. The column chromatographic analysis of the extract in step 4 was performed in groups of four, combining the organic extracts from both pairs. A slurry of silica gel in hexanes was prepared and added to the column in portions in order to form a chromatographic column. After the column was completely set up, being careful not to involve water, the green hexanes extract was added and drained to enter the silica. The column was then eluted slowly with a 9:1 mixtures of hexanes:ethyl acetate as the

pigments separated within the column. The fastest moving pigment was collected into a pre-weighed flask to be analyzed again by TLC, using β-carotene as the reference, and evaporated to render a final mass of residue. Results Table 1: R f value for varying solvents Solvent Ratio of Hexanes:Ethyl-Acetate Solvent height (cm) Sample height (cm) Reference β-carotene height (cm) R f value 5:5 4.00 3.95 3.95 0.988 7:3 3.70 3.65 3.65 0.987 9:1 3.75 2.50 2.50 0.667 R f value calculations R f = sample height (cm)/solvent height (cm) 5:5 R f = 3.95/4.00 = 0.988 7:3 R f = 3.65/3.70 = 0.987 9:1 R f = 2.50/3.75 = 0.667 Table 2: R f values for after column chromatography Solvent ratio of Hexanes:Ethyl-Acetate Solvent height (cm) Sample height (cm) Reference β-carotene R f value

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height (cm) 9:1 3.35 2.50 2.35 0.746 R f value calculation for 9:1 solvent R f = 2.50/3.35 = 0.746 Mass of β-carotene residue in sample Mass = mass of flask and residue (g) - mass of flask (g) Mass = 23.954 g - 23.887 g = 0.067 g % of β-carotene in spinach % = (mass of β-carotene residue/mass of spinach) x 100% = (0.746 g / 5.022 g) x 100% = 14.85% β-carotene TLC plates 5:5 7:3 9:1 9:1 after column chromatography B- carotene from extract Referenc e B- carotene B- carotene from extract B- carotene from extract B- carotene from extract Referenc e B- carotene 3.75

Discussion: Part A Biological compounds within a plant can be extracted through a variety of methods. Most of these methods rely on the extracting power of different solvents and the application of heat or mixing.¹ Extraction techniques play a significant role in the final result and success of a collected bioactive compound. In order to extract material, plants can be ground up, washed, dried, or freeze dried in order to increase the surface area for sufficient mixing with the solvent present. The most common factors that impact the process of extraction are the matrix properties of the plant part, the solvent, the temperature, pressure and time.¹ In order to understand the extraction selectivity from various natural sources, different extraction techniques should be used in diverse conditions. Given the immense variations among bioactive compounds and plant species, it is essential to understand the integrated approaches built to screen out these various compounds of which carry a number of human health benefits.¹ Thin-layer chromatography is a process used to separate different components within a certain mixture using a thin, uniform layer of silica gel or alumina coated onto a small plate of glass, metal or plastic.² A small drop of a solution mixture is placed on the baseline of a plate and dried, and the plate is then placed in a shallow layer of a suitable solvent. The layer of silica gel acts as the stationary phase and the liquid solvent acts as the mobile phase in the chromatography. The mobile phase flows through the stationary phase carrying the components of the mixture with it, and the different components present in the mixture separate as they travel up at different rates.² The solvent rises Referenc e B- carotene Referenc e B- carotene 3.70 cm 4.00 cm 3.95 cm cm 3.65 cm 2.50 cm 2.50 cm 3.35 cm

until it almost reaches the top of the plate in order to display maximum separation of the components. Measurements of the total distance travelled by the solvent and the distance travelled by individual spots can be recorded in order to calculate an Rf value for each dye. Column chromatography works on a larger scale by placing mixtures containing different components in various-sized vertical glass columns in order to separate them into their individual constituents. A concentrated solution of the mixture being tested is firstly developed, preferably in the solvent that is used in the column.³ The column is then prepared by mixing silica with the suitable solvent and pouring it into the column. The solvent within the column is drained, and then the concentrated solution is added to the top of the column to be subsequently drained in order to absorb into the column. New solvent is then continuously added to the top of the column and the tap is opened so it can flow through the column and collect in a beaker or flask. When the tap is on, the compounds in the mixture move depending on the polarity of the sample molecules.⁴ The chromatography consists of two phases; the mobile phase, the liquid solvent or solvent mixture, and the stationary phase, the adsorbent in the column. The compound mixture travels along with the mobile phase through the stationary phase, and separates depending on the degree of adhesion to the silica in the column of each component in the sample.⁴ The extraction efficiency of any conventional method predominantly depends on the selection of solvents.¹ As well in chromatography, the solvent determines the success of the separation of components in a mixture. The solubility of the mixture within the chosen solvent can play an important role as constituents should have a

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small but definite solubility in their solvent.⁵ This is because high solubility will move constituents quickly with the solvent in chromatography, but low solubility will hardly cause any movement. Additionally, each compound or individual component in a sample mixture has a different polarity which can alter the movement in an extraction or chromatography. Non-polar components travel faster than polar components, and will have a higher tendency to move along with a mobile phase.⁴ This is because polar components will adhere more strongly to the present adsorbent or stationary phase. The polarity of the solvent can be modified by changing the proportion of mixtures within the solvent, which can affect the results of an extraction or chromatography. Part B Our results from the experiment coincide very well with the theory. This is shown in the Rf values collected from the 5:5, 7:3 and 9:1 mixtures of hexanes:ethyl acetate, which were 0.988, 0.987, and 0.667, respectively. Theory indicates that the two extremes of the Rf value are 0, where the solute remains fixed at its origin point, and 1, where the solute is especially soluble that it moves as far as the solvent. This corresponds with our results as the first two Rf values are 0.988 and 0.987 which are very close to 1, indicating that beta-carotene travelled extremely fast in the 5:5 and 7:3 solvent mixtures. It is evident in the Rf values collected from the 9:1 mixture that the beta-carotene did not move quickly, as its value was 0.667 initially and 0.746 after the column elution. It is also evident that the beta-carotene was the only component from the mixture that moved with the 9:1 solvent from its original spot on the TLC plate, as opposed to the many coloured spots appearing on the other TLC plates with 5:5 and 7:3 solvents.

These observations demonstrate that the 9:1 mixture of hexanes:ethyl acetate was the best proportioned solvent mixture for eluting the beta-carotene from the solution. When analyzing each of our TLC plates from the experiment, a yellow spot is consistently seen on each plate which aligns with the beta-carotene reference spot. This supports our accurate performance and data from the lab as beta-carotene was successfully extracted from the spinach leaves. Questions 1. Throughout this experiment, there were multiple occasions in which errors could have occurred. One error could have occurred when grinding the spinach leaves at the beginning. If they were not properly grinded up to the fullest extent, then it is possible that not all of the beta-carotene was properly extracted, decreasing the final percent recovery. Another error occurred when placing the TLC plates into the jars. As they were placed, one of them briefly tipped over, which could have affected the way that the spots travelled up the plate. Finally, another error could have occurred during the extraction. When draining the bottom, light green layer, it is possible that some of the dark green layer was drained along with it. This would have reduced our final yield for beta-carotene, as some of it would have been disposed of along with the light green solution. 2. The best mixture that we used for the separation of the components in our extract, was the 9:1 ratio mixture. This mixture was the best because after performing TLC with this mixture, only two dots were visible on the plate; the reference beta-carotene and the beta-carotene from the sample. This made it

very clear to tell which part of our sample was the beta-carotene, as opposed to the other plates that showed many different coloured dots along with the beta- carotene dot. The reason for the 9:1 mixture being the best solution, is due to the fact that it has an ideal ratio of non-polar to polar solvents. This ratio ensured that non-polar beta-carotene did not travel too quickly with the solvent, since there was enough non-polar solvent (hexanes) to enable it to stick to the plate, while still having polar solvent (ethyl acetate) to allow it to move along. 3. To move down the green pigments remaining at the top, I would suggest eluting the column with a solvent that is chemically similar to that of the green pigments, like chlorophyll. Chlorophyll is a more polar compound that beta-carotene is, and therefore it will help the pigments dissolve and move down the column faster due to the aid of the polar solvent. As well, increasing the amount of ethyl-acetate to hexanes in the elution solvent, would help to move the green pigment down the column. 4. The compound that would elute first is the compound that dissolves best in the mobile phase. In a mobile phase that contains more toluene than EtOAc, the mixture would be more non-polar than polar and therefore, a more non-polar compound will elute more readily within it, while a polar compound would likely remain in the solid phase. Resorcinol contains two hydroxyl groups that are able to form hydrogen bonds, making the compound quite polar, whereas 1-bromo-4- chlorobenzene has polarized bonds but is overall a non-polar compound. Therefore, due to these polarities, 1-bromo-4-chlorobenzene would elute first using 8:2 toluene-EtOAc.

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5. Column chromatography is generally used as a means to isolate specific compounds and to determine their polarity. It can be used for many different things such as: Food testing : Food composition can be tested with this method by obtaining a sample and using column chromatography to separate it into its components. Each part can then be analyzed to determine how pure the food is, and whether or not it is true to what manufacturers say. Drug testing : Column chromatography can be used to test a blood sample to determine if it contains certain drugs. This can be useful for finding out if an athlete is using performance enhancing drugs. Forensic testing: Samples from a crime scene can be analyzed using chromatography, to determine if any harmful substances were present at the scene.

References: 1. Azmir, J.; Zaidul, I. S. M.; Rahman, M. M.; Sharif, K. M.; Mohamed, A.; Sahena, F.; Jahurul, M. H. A.; Ghafoor, K.; Norulaini, N. A. N.; Omar, A. K. M. Techniques for Extraction of Bioactive Compounds from Plant Materials: A Review. Journal of Food Engineering 2013 , 117 (4), 426–436. 2. Clark, J. Thin Layer Chromatography. https://www.chemguide.co.uk/analysis/chromatography/thinlayer.html (accessed Feb 6, 2020). 3. Clark, J. Column Chromatography. https://www.chemguide.co.uk/analysis/chromatography/column.html (accessed Feb 6, 2020). 4. Column chromatography. https://chemdictionary.org/column-chromatography/ (accessed Feb 6, 2020). 5. Bhanot, D. Choice of Solvents for Paper Chromatography. https://lab-training.com/2014/11/25/choice-solvents-paper- chromatography/ (accessed Feb 6, 2020).