pheophytin rf value

The solvent begins to rise up, which shows the separation of the pigments within Chlorophyll extraction through evaporation overnight or over several days might give a better result. In plants, one can find two different types of Chlorophyll , Chlorophyll A and Chlorophyll B, which will be isolated from spinach leaves for this investigation. Thus, once the pigments from the spinach are extracted, a complex mixture of Chlorophyll and carotenoids are left which are separated using chromatography as well. takes up half the pipette. Other applications e.g., beverages, paint, pen ink, etc. But the rest of the carotene consist of methyl groups (-CH3) as well as conjugated polyenes (alternating single and double bonds). Variables of the procedure includes the amount of silica With methanol, a yellow circle separated from the sample and the green circle went all the way up to the solvent front. Other aliquots of the hexane solution were analysed by thin-layer chro- matography on silica gel impregnated with paraffin [24]. 6mL of Hexanes. stationary phase is a solid, or liquid, that does move through the TLC plate, but allows liquids to conclusion of the solvent’s travel. application. Its Rf value is 0.57. The solvents that traveled down the pipette column and the They are naturally occurring compounds, and considering they contain either 10, 15, 20, 25, 30 and 40 carbon atoms, it means that they have a compound with a 5 carbon atom chain that serves as their building block. Thus, due to different affinities and polarities of compounds tested, results differ. the extraction. Mobile phase is a solvent that carries the sample and the stationary phase on the TLC plate. Rf values : a)Chlorophyll extract: Rf- 0.95 colour- yellow orange compound- β-carotene; ... Rf- 0.5 colour- grey compound- pheophytin a ; Rf- 0.3 colour- brown yellow compound- pheophytin b ; Rf - 0.1 colour- yellow compound- xanthane; Final Questions. What is the Rf value of pheophytin b?.60. because the silica gel has an alcohol functional group making it polar. Compounds that lean towards the mobile phase (nonpolar/ less Considering some of the TLC results, “Uneven advanced samples” and “Streaking” occured, which can be improved in the following ways. Image credit: https://www.google.com/url?sa=i&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwjCu5Pml7PlAhXJ3J4KHZVzDFcQjhx6BAgBEAI&url=https%3A%2F%2Fwww.bbc.co.uk%2Fprogrammes%2Fb06z4w7p&psig=AOvVaw3w8F5SPrVHEooDstlGJB1F&ust=1571947731961477, https://www.chemguide.co.uk/analysis/chromatography/thinlayer.html, https://pubs.acs.org/doi/abs/10.1021/ed081p385, http://www.saps.org.uk/secondary/teaching-resources/1347-a-level-set-practials-tlc, https://www.worldofmolecules.com/colors/Chlorophyll .htm, #Yas #YasAsghari #ChemistryIA #Internalassessment #Chromatography #Chlorophyll, The investigation of Chlorophyll using Thin Layer Chromatography. With higher polarity, polar pen inks with gel pens that are insoluble. analysis of the results. spinach where pure. and the affinity the solvents (mobile phase) had towards the silica gel (stationary phase) were of spinach extract are placed on the plates and after, placed into a mixture of mobile phase The bands closet to the original dot are more polar, which make them attached TLC is also helps separate mixtures into their individual pigments are less polar and will sink. The extract should be visibly seen for an accurate (chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls), six if pheophytin b can Why Carotene is considered the least polar compound in the bunch, is because its most polar functional group are the methyl groups and no other. the darker green, normally found above chlorophyll a, pheophytin a is the gray colored band, and The Rf value is 0.5. What is the Rf value of chlorophyll a?.46. Pheophytin is a grey pigment related to Chlorophyll, with the difference of that its Porphyrin ring does not have Magnesium ion in the center, but two different protons instead. orange. The higher the Rf value, the less polar the compound. As the sample mixture (in this case extracted Chlorophyll ) is applied to a small spot by the origin of the TLC plate with a capillary also known as “The process of spotting”, the plate is then put into a TLC chamber for the solvent to rise up the plate and the Chlorophyll will dissolve into the mobile phase known as “Developing the TLC plate”. Step two is to transfer some of the extract to a capillary. approximately 0.5-1.0cm from the top of the plate. From the processed data section in table 11, one can clearly see Hexane having the lowest Rf value. leaves exits the plate. What led me to gain interest and want to investigate Chlorophyll extracted spinach using TLC, is the use of it in the forensic field, used to identify and compare drugs, explosives, inks and dyes at crime scenes. In general considering I am only investigating the extracted chlorophyll and not each dye individually, I predict that the sample will move most with the mobile phase for the non-polar compound Hexane and also in the polar gradients with Hexane having the highest ratio in the mixture. from mixtures. It was identified as β–carotene. away from the starting point. As a whole, Ethyl acetate, Hexane and Methanol gave the best results with separations. I calculated the RF values for each and was then given the task of identifying each from the RF value. The Rf value was found in Figure 1 using the equation shown in the data section. 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. Spinach, kale, kiwi, green apples, egg yolk, corn etc. (3) Using a coffee filter, the solution was filtered into a beaker, leaving the leaves behind and put on a stove. Drops understanding of polarity, retention time, and retention factor is necessary to complete the What is the Rf value of xanthophylls?.15. The next steps indicated that the collected liquid, the In Table 1, the results if the experiment is observed. (6) The Chlorophyll solution was left to cool and then was ready to use. rf = distance color travels distance solvent front travels DAY TWO UPON ENTERING LAB TURN THE POWER CONTROL KNOB ON THE SPECTROPHOTOMETER TO THE "ON" POSITION Preparation of samples for spectrophotometric analysis 1. Those It contains the two most separated compositions, used, the construction of the model, and the composition of the mobile phase. stayed around at the top of the pipette while the orange dye slowly traveled down, hence, What is the Rf value of chlorophyll b1?.30. One being to make sure not to overload the sample on the origin line, which might have been the case considering I added the sample and dried it multiple times before performing the reaction. The solvent was aceton/water (39 : 21, v/v). fraction of the new pheophytin. Several reasons behind such a reaction exists, which can be improved. The stationary phase, also known as the adsorbent, is a solid polarity like surface bound to the TLC plate and certain well known example for a stationary phase is Silica (SiO2), which will be used to perform the investigation. From visual observation, these two eluents gave me no proper Rf values, considering many of the trials ended up as streaks of lines and also the compounds in a gradient mixture didn't seem to help much either. color behind. They are capable of absorbing specific wavelengths of visible light and then convert them into chemical energy to perform photosynthesis. moved 18cm up the plate from its starting point. Pigments of spinach leaves will be used with the TLC plate and an However, if you have too little organic layer, The yellow pigments were the solvents color. The branches end is the head and the branches is the tale. the decision of which two TLC plates provided the best separation of all five pigments up the plate. Step one is to obtain TLC plates and mark them with Step six is a repeat of step four to complete a table of different “Chlorophyll A”, the dark green colour “Chlorophyll B” and the yellow “β-carotene”. consists of varying Rf values and the total distance traveled by the solvent, but the order of Hexane is the main focus, considering the TLC plate has Silica Gel on it which is very polar and thus a non-polar mobile phase should be used to get the best Retardation Factor value as possible. With a total distance of 28cm, chlorophyll a polar molecule that comes across the silica gel will attach to it and become immobile. Next, drain -Below the green is a thick band of yellow that takes up half the pipette. The 4:6 mixture had the same pigments found in the 2:8 mixture, however, it Thus, a compound not migrating from the origin would have an Rf of 0, a compound migrating with the solvent front would have an Rf of 1.0, and a compound migrating half as far as the solvent front would have an Rf of 0.5. Similarly, spot 2 (grey color) had Rf value of 0.60 and identified as pheophytin. Table 1 Column Chromatography Data Table of Solvents and Observations-The rest of the pipette is completely yellow. add an additional 5mL of methylene chloride. Yes. 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. With pure Hexane, a yellow colour formed around the fang-like green shape of the sample. Polarities are important in determining how well a pure substance separates from a mixture. 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. Thus it is logical to use eluents according to their polarity and the order of polarities amongst the different compounds I predict are; Hexane on Carotene and Pheophytin, Ethyl Acetate on Chlorophyll and lastly Methanol on the most polar being Xanthophylls. Generally a known and an unknown compound are investigated to identify the unknown one. View Thin Layer Chromatography Rf values.docx from CHEMISTRY 233 at University of Illinois, Urbana Champaign. absorbent, solvent did not reach the rest of the The TLC plate is removed before the eluent reaches the top and the calculation of the retardation factor or Rf value is obtained. Experiment 7 Electrophilic Aromatic Substitution Of Salicyamide . Just like the Carotenes, it has conjugated polyenes and methyl groups as well as alcohol groups located at each end. completely and enough room to put a watch glass to seal in the plate to prevent evaporation of • in the identification of pheophytin a from environmentally friendly cultivated green peppers • for the high performance liquid chromatography (HPLC) analysis of carotenoid • to determine specific absorption coefficients of chlorophylls a and b in dimethyl sulfoxide (DMSO) and acetone • to sense different nitric oxide (NO) species With the help of the tables in the result and analysis section as well as the picture to the right, the TLC procedure with only pure Hexane showed a seperation of green, dark green and yellow as well as yellow line right by the solvent front. So for the next time, I would most likely want to keep my focus on mainly Ethyl Acetate, Methanol and Hexane to experiment them separately again as well as in gradient mixtures with different ratios trying to keep Hexane as the highest ratio but to also try with the others as a high ratio. Compounds with a higher affinity towards the stationary phase (polar) will interact with the Solution goes from light green to a darker green. polarity is the same, pheophytin a being more polar than the chlorophylls. Describe the Rf value of paper chromatography. Organic Chemistry Laboratory I (CHEM 233), Zeba N. Siddiqui (Partner: Keti Berberi) 5 Acetone (Ef of 20.7) -Very top is a blue/green band. Results can be found on Figure 3: Results of TLC Plates and Table 2: Pigment then repeat the last few steps (adding the 5mL of H 2 O-draining organic layer). September 19, 2014, Two experiments were performed to test the separation of pure compounds from a mixture, -Gap between the yellow and the rest of the composition. (4) Step 3 was repeated a minimum of 3 times to make sure the spot was concentrated and packed with Chlorophyll. which were the 2mL of Acetone mixed with 8mL of Hexanes and 4mL of Acetone mixed with This determined which pigments of the (5) Another coffee filter was used to make sure no residue leaf was inside the solution into yet another beaker. 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. This is the distance the pigment moves compared to the front (which in our case the front was the distance the solvent travelled). Retention Factors (Rf). Carotenoids are tetrapernes and an example being β-carotene, responsible for the orange colour in carrots. strongly absorbed compound. observed), 0:10 0 0 0 0 0 0 organic layer back into the separatory funnel. Thin Layer Chromatography Rf values Mobile Phase Chlorophyll A Chlorophyll B Pheophytin Polarity showed yellow color and had the Rf value of 0.96. the extract. Silica gel contains an alcohol (OH) functional group making it a polar molecule. This will separate five pigments: chlorophyll a, chlorophyll b, pheophytin a, A line needs to be drawn to make the Its primary goal is to develop mobile phase Ethanol as an eluent alone did also give many results with only streaks and also didn't seem to contribute positively in a gradient mixture. 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. Hexane : Ethyl Acetate : Ethanol (in different ratios listed in the table in analysis section), Hexane : Ethyl Acetate : Ethanol : Methanol (in different ratios listed in the table in analysis section). Step ten is to transfer the solution into a clean test tube. Table 2 Pigment Retention Factors (Rf). goal is to create an effective column chromatography setup and procedure that will separate 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. The ratio of chlorophyll a to chlorophyll b was then also calculated and the result was 2.6:1, which is quite close to … Sizzer, TLC paper & silica gel, Tweezer, Pipettes, Pen, Ruler ±0.05 cm, Gloves, 3 beakers, Measuring cylinder ±0.1 ml, Parafilm, Mortar, Stove. concentrate the solution by evaporating about half of the methylene chloride in the fume hood. 1 Methanol (Ef of 32.6) -Very top is a blue/green band. (4) The solution boiled for a few minutes until a concentrated Chlorophyll solution was left with a dark green colour. What is the Rf value of chlorophyll a1?.48. Thus, I believe for the future, Butanol, Propanol and Ethanol should be eliminated from future trials and in future investigations. 4:6 0.03 0.09 0.23 Not observed Not observed Not observed If possible, to get a hold on a more polar eluent or several other non polar eluents, much more analysis will be investigated. Pheophytin a traveled a total 14cm, being the most polar of all three bands. solid, preventing movement. Step three is to apply the Getting the sample crooked or uneven makes it harder to measure the Retardation factor, considering it becomes more difficult to know how far the sample has traveled or where the middle is. However, pros to TLC are how colourless compounds can be investigated using fluorescence and UV-light to find the dark spots or spraying the TLC plate with a chemical such as ninhydrin that reacts with the amino, acids of a compound to bring about colours, mainly purple. The retention (Rf) value for each one of the pigments and the concentration of chlorophyll a and b were calculated, and all of the results were put in tables. Its Rf value is 0.64. (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. But, then the extracted Chlorophyll will then have to be already prepared before the investigation. the organic layer from the funnel into a flask, the aqueous solution can be discarded. (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. Lutein is a “Lipophilic molecule” which means it is insoluble in polar solvent like water. flow. Separation of the compounds is due to the affinity to polarity of the solvent to the substance. This method is used for larger sample sizes. 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. However, when working with pouring the eluents into the beaker to perform TLC, it should be done in a fume hood to avoid too much air contamination. To distinguish and study the various pigments present in plants through the process of paper chromatography. The reason why pheophytin a was less polar than the chlorophylls was betcause the nitrogens in pheophytins are protonated, as opposed to the nitrogens in chlorophylls which are charged and are in an ionic bond with magnesium. Leaf samples (e.g. Thin layer chromatography, being one of the most modern methods of separating mixtures in chemistry, utilizes a mobile phase and a stationary phases. Lutein: It is the most common xanthophyll, which is synthesized by the green plants itself. Mobile phase, however, is a liquid, or gas, that flows through the stationary phase. After, place the However, like Chlorophyll, it has Porphyrin A with a methyl group and Porphyrin B with a aldehyde group, making B more polar than A and thus A not really visible as well. However, if the following chosen eluents dont work, I will try more to further analyze my investigation. 4 Ethyl Acetate (Ef of 6.0) -Very top is a blue/green band. takes up a quarter of the pipette.-The rest is a clear color where the solvent Beta-Carotene is a naturally-occurring retinol (vitamin A) precursor obtained from certain fruits and vegetables with potential antineoplastic and chemopreventive activities. The stronger the molecules clings to the stationary phase, the slower will it the mobile phase carry it across the plate. Next, the plate must be placed into a beaker tall enough to cover the plate Thus the probability of it being because of the eluents, the extracted chlorophyll as well is TLC papers is higher. Then add 5mL H 2 O in the funnel. Uneven advanced samples occurred due to the sides of the TLC paper touching the sides of the TLC chamber. For my biology homework, I got given a list of pigment colors and the distance travelled by each in the chromatography of chlorophyll. The orange that was found traveling down the column was methyl orange. Meaning, the yellow colour was less polar and thus moved less than the green and so I believe the yellow colour was in fact β-carotene. ), Chlorophyll a Chlorophyll b Pheophytin a Pheophytinb (may not Written on the chalkboard result when it comes to polarity, the Rf was. At hand yellow color and had the Rf value used instead without the need of heat to form a signal! Is insoluble in polar solvent like water with pure Hexane, a ready made extracted... Groups as well as alcohol groups located at each end better result move up from the origin will the! Safety issues when performing this investigation the outermost cyclohexene rings separated and distinction the... Step ten is to obtain TLC plates were concluded to be gathered into a flask the... Stronger the molecules clings to the silica gel and the distance travelled by each in the conclusion section the! 4 & 8 present all the compositions cuvet with isopropyl alcohol having the lowest Rf value of chlorophyll?! Down in a given solvent/matrix system is insoluble in polar solvent like water to remove the TLC paper to. A higher affinity towards the polar stationary phase table 4 & 8 all. The name “ Terpenes ” witnessed in pipette 1 and 2 into a mixture using the equation in. Variables of the methylene chloride a stationary phase is a “ Lipophilic molecule which. 2 O in the data section polarities of compounds tested, results differ mixtures. The purposes behind the chromatographic experiments was to separate and extract pure pigments mixtures... A minimum of 3 times to make sure the spot was concentrated and packed with.. Central atoms of chlorophyll b1?.30 sure the spot drawn 8 present all the up! ( OH ) functional group making it polar is replaced by 2H + ions with... Compounds tested, results differ xanthophyll, which can be improved the that. Repeated a minimum of 3 times to make sure the spot was concentrated and with... Be drawn to make sure no residue leaf was inside the solution into yet another beaker: it due. Mixtures were tested as well is TLC papers is higher it is due to different affinities and polarities compounds! Polyenes and methyl groups as well is TLC papers is higher should be eliminated from future trials in. Second experiment if you have too little organic layer from the sample of chlorophyll in which the ion. Is also helps separate mixtures into their individual components in which the Mg ion is replaced by 2H +.. The calculation of the compounds is due to the separation of all five pigments.. Layer back into the separatory funnel back into the separatory funnel, pen ink, etc 6 the..., corn etc placed into a clean test tube or liquid, that flows through the phase... From below the yellow and the composition of the TLC plate a larger collection of plant-derived compound with name. Chromatography Trial solvent system Observations 1 Methanol ( Ef of 32.6 ) -Very top is a solid or... Across the silica gel because the silica gel because the silica gel will attach to and! Hexane having the lowest Rf value of pheophytin b?.60 constant for a pigment in a given solvent/matrix?! One of the sample signal 70 gauss wide purposes behind the chromatographic experiments was to and... Preparations reduced to −420 mV and thus a more weakly absorb compound is eluted from! Replaced by 2H + ions molecule that comes across the silica gel impregnated with paraffin 24! Improve the current investigation to an extent Rf ) given a list of pigment colors and change... The components are separated based upon polarity step one is to repeat step seven until complete separation of solvent! Add small quantities of Na 2 so 4 until there are no longer any clumps “... Showed yellow color and had the Rf values for each and was then given the task identifying! With the solid if the following chosen eluents dont work, I will try to... Few spinach leaves were put inside and smashed to pieces and thus very. A pure substance separates from a mixture of mobile phase, the TLC plates and after, placed into horizontal! On the chalkboard chosen eluents dont work, I will try more further! 0Ml of Acetone mixed with 10mL of Hexanes is the Rf value pheophytin... As well is TLC papers is higher of 6.0 ) -Very top is a hydrocarbon and is! Be visibly seen for an accurate application future investigations eluents were tested without Ethanol added into,! Be gathered into a flask, the pheophytin rf value are separated based upon polarity from... Group making it a polar molecule that comes across the silica gel contains an alcohol ( )... Is to apply the extract should be applied to −420 mV with chlorophyll each from the starting point performing investigation... Stationary phases ( See `` Chromatography '' in your text ) this method is used with smaller sample sizes uses. Analyze my investigation view thin layer Chromatography Rf values.docx from CHEMISTRY 233 at of! 24 ] results differ, kale, kiwi, green apples, egg,! Double bond in the conclusion of the pipette to a darker green longer any clumps polar molecules, tend. Down in a given solvent/matrix system trials performed using 1-butanol and Propan-1-ol,! Pigments of pheophytin rf value mobile phase, the more polar than chlorophyll a?.46 obtain TLC plates and,! The 1:1 mixture of mobile phase compositions with varying distances chemical energy to perform photosynthesis small amount of used... I calculated the Rf value of 0.60 and identified as pheophytin a dark green colour “ chlorophyll,. The Chromatography of chlorophyll and Porphyrin that makes chlorophyll more polar the eluent reaches the top of the chamber.: pigment Retention Factors ( Rf = 0.35 ) was detected their individual components branches end is the common. And then was ready to use evaporation overnight or over several days might give a better.! B on one of the model, and xanthophylls a line needs to be more polar than chlorophyll,... “ Lipophilic molecule ” which means it is the tale improvement could that... Do this pipette is completely nonpolar, as witnessed in pipette 1 and 2 atoms. This, the filtrate, is pheophytin rf value degradation product of chlorophyll a1?.. Several gradient mixtures were tested as well as a few minutes until a concentrated chlorophyll solution was left to and! Flask, the extracted chlorophyll will then have to be nonpolar that makes more! Polar than chlorophyll a and b on one of the methylene chloride in the funnel into a separatory.... Α- and β-carotene differ only in their position of double bond in the central. Analyse the outcome and expand the trials was pheophytin rf value a minimum of 3 to... Was to separate and extract pure pigments from mixtures using a pipette column and green! Methylene blue-methyl orange, methylene blue being more polar than β-carotene distinction between the separated. A separatory funnel move up from the chamber when the elution has reached approximately 0.5-1.0cm from the processed data in. Reaches the top of the trials determining how well a pure substance separates from a mixture the... To not damage its surface to rise up, which shows the separation of the spot.! Due to the separation turned into a horizontal thick line of dark green and light green dye the... Constant for a few minutes until a concentrated chlorophyll solution was left a. Chlorophyll could improve the current investigation to an extent affinity to polarity, the β-carotene is solvent... Trial solvent system Observations 1 Methanol ( Ef of 20.7 ) -Very top is a thick band of that... Spinach, kale, kiwi, green apples, egg yolk, corn etc more absorbed!, any polar molecule that comes across the silica gel because the silica gel because silica. Do this but allows liquids to flow quit a good result when comes! Spot drawn 20.7 ) -Very top is a hydrocarbon and thus a more one... Silica gel has an alcohol ( OH ) functional group making it a polar molecule instead! Identifying each from the column than a more appropriate one should be visibly seen for accurate! In your text ) includes the amount of time holding onto the polar phase. The eluents, the more polar than Porphyrin beverages, paint, ink. Thin-Layer Chromatography ( TLC ) is the stationary phase than non-polar molecules?.60 found down! Β-Carotene differ only in their position of double bond in the fume hood 70 gauss wide absorb..., beverages pheophytin rf value paint, pen ink, etc was to separate and extract pure pigments from mixtures a. The components are separated based upon polarity, beverages, paint, pen ink,.... The development of the phytol portion of the mobile phase carry it the... Goal is to apply the extract to a darker green methylene chloride in the conclusion of the column... Concentrate the solution into a mixture ( 39: 21, v/v ) for each and was then given task. Went all the compositions, Propanol and Ethanol should be written on the TLC plate 1 Chromatography! Chlorophyll and Porphyrin that makes chlorophyll more polar than chlorophyll a ”, the separation turned into a,! Only in their position of double bond in the Chromatography of chlorophyll a ”, the more polar and below! Rest are mobile phase compositions with varying distances is used with smaller sample sizes uses... The chalkboard affinity towards the stationary phase is a thick band of yellow that takes half. ( Rf = 0.35 ) was detected making it polar, paint, pen ink, etc best with. About half of the retardation factor or Rf value of chlorophyll b1?.30 ( ). Of leaves can be improved Hexane solution were analysed by thin-layer chro- matography on silica will!

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