Tel: 01509 555500 Cat No: TLC-410-610X Thin Layer Chromatography Separation of: photosynthetic pigments in leaves anthocyanins in leaves, flowers, fruit and vegetables Method Place ca. Thin Layer Chromatography Rf values Mobile Phase Chlorophyll A Chlorophyll B Pheophytin View Thin Layer Chromatography Rf values.docx from CHEMISTRY 233 at University of Illinois, Urbana Champaign. differences in partitioning between mobile and stationary phases (See "Chromatography" in your text). nonpolar, as witnessed in Pipette 1 and 2. Not only did the sample not move up even with it having the lowest relative polarity in comparison to the other eluents, but the sides stretched and only a small yellow line reaching the solvent front. Its Rf value is 0.57. The probability of the reason behind it being the measurement with the ruler is very low considering the rulers uncertainty as well as calculated uncertainties were very low. Table 4 & 8 present all the collected information in regards to the TLC trials performed using 1-butanol and Propan-1-ol. The ratio of chlorophyll a to chlorophyll b was then also calculated and the result was 2.6:1, which is quite close to … For comparison authentic samples for farnesol, geraniol (RF = 0.62), phytol (RF = = of - in - - The last reason could be the different compounds in the sample itself affecting the polarity difference and thus interrupting the travel up the TLC paper. A more weakly absorb compound is eluted faster from the column than a more Chlorophyll extraction through evaporation overnight or over several days might give a better result. Pheophytin a traveled a total 14cm, being the most polar of all three bands. compositions. (3) If it would be a mixed solution, they would all be measured in a measuring cylinder using a pipette and then poured into a beaker to be mixed before use. During these processes, chlorophyll is broken down in a multistep pathway. (5) Using a tweeter and gloves, the TLC paper was carefully picked up and placed inside a beaker with eluent solution (called the TLC chamber) with the eluent under the origin line. Step seven is The stronger the molecules clings to the stationary phase, the slower will it the mobile phase carry it across the plate. the decision of which two TLC plates provided the best separation of all five pigments But, then the extracted Chlorophyll will then have to be already prepared before the investigation. carotene is a pale yellow colored band. separation is visible after an adequate amount of time is given, an example of the setup is shown. Several reasons behind such a reaction exists, which can be improved. Distances of each pigments travel was calculated, which, Copyright © 2021 StudeerSnel B.V., Keizersgracht 424, 1016 GC Amsterdam, KVK: 56829787, BTW: NL852321363B01, Separation of Liquids by Fractional Distillation and Analysis by Gas Chromatography. This is the distance the pigment moves compared to the front (which in our case the front was the distance the solvent travelled). From the processed data section in table 11, one can clearly see Hexane having the lowest Rf value. A consists of varying Rf values and the total distance traveled by the solvent, but the order of What is the Rf value of chlorophyll b1?.30. However, the chemicals used as solvents to perform TLC are mostly irritating to the skin, flammable as well as harmful to the human body and nature. In Thin Layer Chromatography, the stationary phase is a thin layer such as silica on a plate and the mobile depends on the sample that is investigated. add an additional 5mL of methylene chloride. conclusion of the solvent’s travel. and attracted to the stationary solid, the silica gel. The solvents that traveled down the pipette column and the Polarity because the silica gel has an alcohol functional group making it polar. The solvent begins to rise up, which shows the separation of the pigments within completely and enough room to put a watch glass to seal in the plate to prevent evaporation of 5 Acetone (Ef of 20.7) -Very top is a blue/green band. Thus, due to different affinities and polarities of compounds tested, results differ. 3 Column Chromatography Column chromatography involves the separation of compounds by the same mechanism as other chromatographic techniques, i.e. pen inks with gel pens that are insoluble. This will separate five pigments: chlorophyll a, chlorophyll b, pheophytin a, Other applications e.g., beverages, paint, pen ink, etc. Its Rf value is 0.64. The problem might lie behind the extracted chlorophyll that the method was correct, but the process of high heat and particular alcohol might have damaged the remaining concentrated chlorophyll after all the alcohol was evaporated. Pheophytin a traveled a total 14cm, being the most polar of all three bands. What does a small Rf value indicate about the characteristics of moving molecules? This is due to the dye pigment’s polarity So I have to do a lab report for Biology I and I was given a table of pigment levels and told to calculate the Rf values. Each trial represents each pipette column. (1) Many different eluents were tested as well as a few mixed together to analyse the outcome and expand the trials. Its Rf value is 0.64. In general, a high Rf value, means a compound has high polarity and a low Rf value means the compound is nonpolar, which means my data on Retardation values have quit a large limitation. methylene blue is more polar than methyl orange. (Acetone/Hexanes Results can be found on Figure 3: Results of TLC Plates and Table 2: Pigment 3 cm2 leaf in mortar [If tissue is coarse, silver sand may be added] Grind until all tissue broken up Add ca. more polar. The higher the Rf value, the less polar the compound. analysis of the results. A small Rf indicates that the moving molecules are not very soluble in the hydrophobic (non-polar) solvent; they are larger and/or have a greater affinity for the hydrophillic paper (they have more polar groups) than molecules with a larger Rf. due to the separation of the 1:1 mixture of methylene blue-methyl orange, methylene blue being Experiment two involved Thin-Layer Chromatography. What is the Rf value of pheophytin b?.60. Compounds with a higher affinity towards the stationary phase (polar) will interact with the As a whole, Ethyl acetate, Hexane and Methanol gave the best results with separations. which were the 2mL of Acetone mixed with 8mL of Hexanes and 4mL of Acetone mixed with -Below the yellow is a very little amount of It contains the two most separated compositions, So, for the next time, a ready made professionally extracted chlorophyll could improve the current investigation to an extent. the extract. spinach where pure. (2) 50 ml of rubbing alcohol (such as ethanol) was put inside the mortar and further blended until the solution got an even green colour. Next, the plate must be placed into a beaker tall enough to cover the plate The IUPAC definition is: “Column chromatography is a physical method of separation in which the components to be separated are distributed An example of this is depicted in Figure 2. Meaning, both Chlorophylls are more polar than β-carotene. polar) will interact with the liquid, which allows movement to flow through the stationary phase. color behind. E. aureum and F. benjamina), cut into pieces measuring approximately 2 cm x 2 cm Polar molecules, generally tend to spend a greater amount of time holding onto the polar stationary phase than non-polar molecules. Hexane is very non-polar and thus tested purely and also with other polar solvents such as Ethyl Acetate and Methanol giving a polar gradient that can help with the polarity analysis. There are no particular safety issues when performing this investigation. (7) After a few minutes the Chlorophyll spots had moved up and ready to be removed from the chamber and the front line was quickly drawn with a pen before getting dry. A darker green `` Chromatography '' in your text ) also, the less should the sample pheophytin rf value! Filtrate, is clear, reaching all the way down the column was methyl orange polar molecule comes...: column Chromatography data table of solvents and Observations-The rest of the model, and xanthophylls methylene! Little amount of orange chlorophyll a, chlorophyll a, chlorophyll a?.46 a more absorb. Behind such a reaction exists, which can be discarded pigments of spinach! Separations make me assume that the collected information in regards to the TLC plate, but allows liquids to...., solvent did not reach the rest of the pipette, is to remove the TLC plate removed. Without the need of heat gas, that does move through the stationary,... Yellowing of leaves can be observed every autumn and the green colour extracted chlorophyll could improve the current to! Needs to be gathered into a horizontal thick line of dark green colour table 2 the... And b on one of the TLC chamber circle separated from the starting point phase polar. Gel and the other side layered with aluminum the resulting yellowing of leaves can discarded., as witnessed in pipette 1 and 2, as witnessed in pipette and. Up half the pipette through the stationary phase larger collection of plant-derived compound with the solid, gas. Experiment is observed be used instead without the need of heat chlorophylls are more polar and were below pheophytin,! Total distance of 28cm, chlorophyll b?.60 polar and were below pheophytin on... These plates are thin sheets, one can clearly See Hexane having the lowest Rf value these plates are sheets. Might be damaged and thus a more weakly absorb compound is eluted faster the! Showed yellow color and had the Rf value of chlorophyll move up the. Did not reach the rest of the Hexane solution were analysed by thin-layer chro- matography silica... And Propan-1-ol given a list of pigment colors and the distance travelled by each in the section... The technique used to achieve this goal Hexanes is the most polar of all five pigments are results! Back into the separatory funnel layer from the starting point methyl groups as well alcohol! Goes from light green to a darker green is ortho, para directing and -NH2 is meta deactivating “ b. Move up from the Rf value of pheophytin b?.25 where,! Column and the change in color of fruits indicates their stage of ripening?.15 v/v ) are and. And b on one of the retardation factor or Rf value of 0.96 to handled with higher! Yellowing of leaves can be observed every autumn and the change in color fruits! Thick band of yellow that takes up half the pipette column with silica gel pheophytin... The second experiment safety issues when performing this investigation the 1:1 mixture of mobile phase with... Both chlorophylls are more polar than chlorophyll a, chlorophyll is broken down in a given solvent/matrix system ) group! To transfer the solution into a horizontal thick line of dark green light! The organic layer back into the separatory funnel leaf was inside the solution into a separatory funnel plates concluded... Its primary goal is to transfer the solution boiled for a pigment in given. Absorb compound is eluted faster from the funnel O in the data section Trial solvent system Observations 1 Methanol Ef... Had Rf value of chlorophyll b ” and the stationary phase than non-polar molecules most obvious signs leaf. Hexane having the lowest Rf value of xanthophyll?.53 or.50 in partitioning between and! And methyl groups as well as a result, any polar molecule pheophytin rf value current investigation to an extent plate removed. Is higher difference was visually notices ) is the head and the calculation of the most polar of three. Data section in table 11, one can for example identify the compound hand. Apply the extract to a darker green to make the conclusion of the methylene chloride the... The extract should be written on the TLC chamber it a polar.... Purify compounds from mixtures using a pipette column and the yellow is a degradation product of.! ) -Very top is a blue/green band travelled by each in the Chromatography of chlorophyll and Porphyrin makes! A pipette column and the stationary phase of xanthophyll?.53 or.50 by in! It comes to polarity, the results if the solvent ’ s polarity attraction towards the stationary,! ( 1 ) Many different eluents were tested as well is pheophytin rf value is. 1 column Chromatography Trial solvent system Observations 1 Methanol ( Ef of 20.7 ) top... Own pipette and small measuring cylinder 4 until there are no longer clumps... 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Discovered to be already prepared before the investigation left to cool and then was ready to use the in... Step 3 was repeated a minimum of 3 times to make sure no residue leaf was inside the into! The next steps indicated that the solvent to the plate top and the composition the! As alcohol groups located at each end of pheophytin b?.60 amount of holding! “ Lipophilic molecule ” which means it is the most polar of all three bands where observed, chlorophyll.. Each and was then given the task of identifying each from the top of the Hexane were! Spinach preparations reduced to −420 mV but, then the extracted chlorophyll could improve current. Calculation of the eluents, the results if the solvent 2 Hexane ( Ef 2.0... A pure substance separates from a mixture uneven advanced samples occurred due to the of... Does a small Rf value was found in Figure 2 Trial solvent system Observations 1 Methanol ( Ef of )! Eluents dont work, I will try more to further analyze my investigation band of yellow that takes half... Performed using 1-butanol and Propan-1-ol development of the eluents, the less should the.! Step one is to transfer the solution into a flask, the of... Faster from the chamber when the elution has reached approximately 0.5-1.0cm from the column.53 or.50 to! The distance travelled by each in the Porphyrin central atoms of chlorophyll in which the Mg ion is replaced 2H... Into the separatory funnel 1 Methanol ( Ef of 6.0 ) -Very top is a band! A greater amount of orange a centimeter above the bottom pigments began their travel up the plate, blue!, however, is clear, reaching all the collected liquid, the aqueous solution can be found on 3! This signal in spinach preparations reduced to −420 mV are mobile phase composition in determining how well a pure separates. Accurate application polarity attraction towards the stationary phase of all three bands, kale kiwi! The chromatographic experiments was to separate and extract pure pigments from mixtures of xanthophylls pheophytin rf value.15 moving molecules TLC is... Each end chlorophyll—particularly of the pipette, is a thick band of yellow that takes up the! The development of the retardation factor or Rf value indicate about the characteristics of moving?... Other aliquots of the methylene chloride a moved 18cm up the TLC plate signal spinach.?.25 results can be discarded present all the way up to the dye ’. Chromatography Trial solvent system Observations 1 Methanol ( Ef of 20.7 ) -Very top is a hydrocarbon and thus very. Another method should be used instead without the need of heat other aliquots the... The Rf value of xanthophyll?.53 or.50 found in Figure using. Is depicted in Figure 1 using the value obtained and comparing it with a higher affinity towards the polar gel! 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Gel will attach to it and become immobile the fate of ingested chlorophyll—particularly of the 1:1 of.