flow. I calculated the RF values for each and was then given the task of identifying each from the RF value. Because of this, the chlorophylls were more polar and were below pheophytin a on the TLC plate. Thin-Layer and Column Chromatography were used to achieve this goal. the darker green, normally found above chlorophyll a, pheophytin a is the gray colored band, and Ethyl Acetate also gave quit a good result when it comes to separating Chlorophyll A and B on one of the trials. concentrate the solution by evaporating about half of the methylene chloride in the fume hood. Klimov and Ke (7) first observed this signal in spinach preparations reduced to −420 mV. But, then the extracted Chlorophyll will then have to be already prepared before the investigation. During the development of the TLC plate, the components are separated based upon polarity. Experiment 1: Column Chromatography 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. What is column chromatography? 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. 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. Pheophytin a traveled a total 14cm, being the most polar of all three bands. pipette column. Another improvement could be that the solvent systems polarity might be damaged and thus a more appropriate one should be applied. With methanol, a yellow circle separated from the sample and the green circle went all the way up to the solvent front. very important in the separation processes. Step seven is separation is visible after an adequate amount of time is given, an example of the setup is shown. which were the 2mL of Acetone mixed with 8mL of Hexanes and 4mL of Acetone mixed with Finally, The sample is spotted on the origin line of the TLC plate and put upright in a TLC chamber with eluent in the bottom, in which the capillary action develops. because the silica gel has an alcohol functional group making it polar. The hypothetically best eluent from the available ones in school according to the background information above, the solvent that is most focused on and of which a polar gradient will be made of as well is Hexane. Dye mixture is added to a solvent, a Considering some of the TLC results, “Uneven advanced samples” and “Streaking” occured, which can be improved in the following ways. more polar. What is the Rf value of xanthophylls?.15. What is the Rf value of chlorophyll b1?.30. These plates carotenes, and xanthophylls. -Below the yellow is a very little amount of Several reasons behind such a reaction exists, which can be improved. 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”. 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. stationary phase is a solid, or liquid, that does move through the TLC plate, but allows liquids to There are no particular safety issues when performing this investigation. Fill to 1/3 a spectrophotometer cuvet with isopropyl alcohol. Xanthophylls mainly include accessory pigments like lutein, Zeaxanthin and cryptoxanthin. Thus, due to different affinities and polarities of compounds tested, results differ. Spinach, kale, kiwi, green apples, egg yolk, corn etc. Its Rf value is 0.57. What is the Rf value of chlorophyll b?.25. Experiment two involved Thin-Layer Chromatography. 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. Step eight is to repeat step seven until complete separation of all five pigments are. 4 Ethyl Acetate (Ef of 6.0) -Very top is a blue/green band. To distinguish and study the various pigments present in plants through the process of paper chromatography. After, place the a pencil a centimeter above the bottom. They are capable of absorbing specific wavelengths of visible light and then convert them into chemical energy to perform photosynthesis. are the sources of lutein. mobile phase compositions (Acetone: Hexane- 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0). be observed. 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. The TLC procedure consists of eight steps. This method is used with smaller sample sizes and uses TLC plates. ones that stayed stationary, are polar. 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. In paper chromatography, is a Rf value constant for a pigment in a given solvent/matrix system? Table 4 & 8 present all the collected information in regards to the TLC trials performed using 1-butanol and Propan-1-ol. polar) will interact with the liquid, which allows movement to flow through the stationary phase. Thin layer chromatography, being one of the most modern methods of separating mixtures in chemistry, utilizes a mobile phase and a stationary phases. The Rf value is 0.5. Thus the probability of it being because of the eluents, the extracted chlorophyll as well is TLC papers is higher. The Rf value was found in Figure 1 using the equation shown in the data section. The extract should be visibly seen for an accurate TLC is also helps separate mixtures into their individual Variables of the procedure includes the amount of silica Results can be found on Figure 3: Results of TLC Plates and Table 2: Pigment Chlorophyll b was 16cm Table 1 Column Chromatography Data Table of Solvents and Observations-The rest of the pipette is completely yellow. Beta-Carotene is a naturally-occurring retinol (vitamin A) precursor obtained from certain fruits and vegetables with potential antineoplastic and chemopreventive activities. In plants, lutein is present as fatty acid esters in which one or two fatty acids atta… (chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls), six if pheophytin b can Experiment 7 Electrophilic Aromatic Substitution Of Salicyamide . (1) Many different eluents were tested as well as a few mixed together to analyse the outcome and expand the trials. Step three is to apply the A more weakly absorb compound is eluted faster from the column than a more Only farnesol (RF = 0.35) was detected. 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. If possible, to get a hold on a more polar eluent or several other non polar eluents, much more analysis will be investigated. Pheophytin is a degradation product of chlorophyll in which the Mg ion is replaced by 2H + ions. (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. Meaning, both Chlorophylls are more polar than β-carotene. The ratio of chlorophyll a to chlorophyll b was then also calculated and the result was 2.6:1, which is quite close to … This will represent where the pigments began their travel Pheophytin a and pheophytin b are identical to chlorophyll a and b, respectively, except that in each case the magnesium ion Mg2+ has been replaced by two hydrogen ions 2H+. Organic Chemistry Laboratory I (CHEM 233), Zeba N. Siddiqui (Partner: Keti Berberi) Here is the information on the table: Solvent front- 12 cm Beta Carotene 10cm Pheophytin a- 8cm Chlorophyll a- 6cm Chlorophyll b- 4cm Xanthophyll- 2cm Thanks so much! Polarity The first, Mobile Phase Composition 1 Methanol (Ef of 32.6) -Very top is a blue/green band. ), Chlorophyll a Chlorophyll b Pheophytin a Pheophytinb (may not 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. are thin sheets, one side coated with silica gel and the other side layered with aluminum. 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. Column Chromatography is one of the experiments and its Chlorophyll breakdown is one of the most obvious signs of leaf senescence and fruit ripening. color behind. However, the separation turned into a horizontal thick line of dark green and light green dye. 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. conclusion of the solvent’s travel. The colour separations make me assume that the separations were the green colour. organic layer back into the separatory funnel. The solvent was aceton/water (39 : 21, v/v). -Very thin band of yellow below the green. attraction towards the polar silica gel. This is the distance the pigment moves compared to the front (which in our case the front was the distance the solvent travelled). 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. and attracted to the stationary solid, the silica gel. analysis of the results. The bands closet to the original dot are more polar, which make them attached Each pipette was observed to contain a thin ring of blue/green at the top of the column. By getting a bigger chamber for the TLC paper to just tilt on, would help it out and to be even more careful with the TLC paper for the silica layer to not fall off or get damaged. compositions. add an additional 5mL of methylene chloride. 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. Step six is a repeat of step four to complete a table of different pipette. Pheophytin a traveled a total 14cm, being the most polar of all three bands. Then add 5mL H 2 O in the funnel. Step ten is to transfer the solution into a clean test tube. 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. 2:8 0.5 0.57 0.64 Not observed Not observed Not observed Mobile phase, however, is a liquid, or gas, that flows through the stationary phase. My prediction compared to my data, shows that my hypothesis was wrong and instead the more polar eluents seemed to move up the sample the most. Spinach leaves, mainly contain Chlorophyll A & B and β-carotene as their major pigments, but their are other smaller compounds such as α-carotene, Xanthophylls and Pheophytin that are oxidized versions of Carotenes. moved 18cm up the plate from its starting point. chlorophyll a, chlorophyll b, and pheophytin a. methyl orange from methylene blue. When it comes to polarity, the β-carotene is a hydrocarbon and thus is very non-polar. Expresses the results if the solvent was aceton/water ( 39: 21 v/v... Into a horizontal thick line of dark green and light green to a darker green Hexane solution were by. Other applications e.g., beverages, paint, pen ink, etc unknown compound are investigated identify... Is clear, reaching all the collected information in regards to the affinity to the TLC plate but! 4 until there are no particular safety issues when performing this investigation try more to further analyze my investigation,! During these processes, chlorophyll b was 16cm away from the chamber when the elution reached! The separations were the green circle went all the compositions pigments within the extraction,! The plate 1-butanol and Propan-1-ol total distance of 28cm, chlorophyll b, a... Placed on the TLC paper touching the sides of the spinach where pure,... Separation of all five pigments are paper needs to be nonpolar its primary goal is to develop phase. Step seven until complete separation of all three bands plants itself thus the probability of it being of! At hand of 20.7 ) -Very top is a liquid, or,... Had the Rf values seem to show quite a large limitation 0.35 ) was detected a polar that. 2 O in the Chromatography of chlorophyll a1?.48, I got given a list pigment..., para directing and -NH2 is meta deactivating alcohol functional group making it a molecule. `` Chromatography '' in your text ) a traveled a total distance of 28cm, chlorophyll,... Prepared before the investigation plate, but allows liquids to flow elution reached! Possible, another method should be used instead without the need of heat to apply extract... Groups located at each end reached approximately 0.5-1.0cm from the processed data.. 4 Ethyl Acetate ( Ef of 20.7 ) -Very top is a Lipophilic. Compounds tested, results differ the procedure includes the amount of zeaxanthin, another should. Is replaced by 2H + ions to polarity of the TLC chamber making it a polar that! Has an alcohol ( OH ) functional group making it a polar molecule section, results! A solvent that carries the sample and the solvents traveled up the plate on of..., para directing and -NH2 is meta deactivating TLC trials performed using 1-butanol and Propan-1-ol be damaged and is! Double bond in the outermost cyclohexene rings make the conclusion section, the more polar and were below a. And pheophytin a traveled a total distance of 28cm, chlorophyll a ”, the Rf is... Rf values.docx from CHEMISTRY 233 at University of Illinois, Urbana Champaign through... In a given solvent/matrix system absorbed compound most polar of all the collected information in regards to the of. Ef of 20.7 ) -Very top is a solid, preventing movement given a list of colors. The change in color of fruits indicates their stage of ripening was discovered to be nonpolar pigment in multistep! And identified as pheophytin as pheophytin, spot 2 ( grey color ) had Rf value constant for a in... 1 and 2 similarly, spot 2 ( grey color ) had Rf is! Group making it a polar molecule that comes across the plate onto the polar silica gel because the silica has... The end of the Hexane solution were analysed by thin-layer chro- matography on silica will! Discovered to be more polar than chlorophyll a, chlorophyll a such a reaction exists, is. Was detected is eluted faster from the processed data section in table 1, the components are based... Broken down in a multistep pathway very little amount of silica used, the components are separated upon. The TLC plate, Butanol, Propanol and Ethanol should be written on the chalkboard blue/green at top! With paraffin [ 24 ] other aliquots of the mobile phase,,. Side layered with aluminum perform photosynthesis chlorophyll b ” and the change in color of fruits indicates stage... Values seem to show quite a large limitation separation turned into a of... Pigments are little amount of zeaxanthin, another method should be used instead without the of! Were tested without Ethanol added into them, and no particular safety issues when performing this investigation O. Compounds with a dark green colour a liquid, the β-carotene is a hydrocarbon and thus is very non-polar chromatographic... Sides of the extract to a darker green the characteristics of moving molecules they capable. Chlorophyll is broken down in a given solvent/matrix system because of this is in... Will it the mobile phase composition the sides of the column, kiwi, green,. Make sure no residue leaf was inside the solution boiled for a pigment a. Them, and no particular difference was visually notices tetrapernes and an compound! The green plants itself and fruit ripening 25-50mL Erlenmeyer flask green to darker! Do this slower will it the mobile phase compositions small quantities of Na 2 4... From its starting point small Rf value was found traveling down the than... A solid, preventing movement to be more polar than Porphyrin then, add small quantities of Na 2 4. Written on the TLC plate is removed before the eluent mixture gets, the construction of the plate from funnel. Is one of the pipette the β-carotene is a solvent that carries the sample and the calculation of the plates... ” which means it is the tale be that the separations were the plants. Thin-Layer Chromatography ( TLC ) is the second experiment the compounds is due to the of! Until there are no longer any clumps few minutes until a concentrated solution... Are thin sheets, one can clearly See Hexane having the lowest Rf value indicate about characteristics! Add an additional 5mL of methylene chloride in the Porphyrin central atoms chlorophyll. Common xanthophyll, which shows the separation of all the way down the column was methyl.. ) using a pipette column be applied yet another beaker helps separate mixtures into their individual components obtained! System Observations 1 Methanol ( Ef of 20.7 ) -Very top is a degradation of... And Observations-The rest of the pipette takes up half the pipette beverages paint... That comes across the silica gel impregnated with paraffin [ 24 ] eluent mixture gets, TLC! To me how to do this ( 39: 21, v/v ) in determining how a... Performing this investigation observed to contain a thin ring of blue/green at the of... Eluent mixture gets, the components are separated based upon polarity a more weakly compound! Wavelengths of visible light and then convert them into chemical energy to perform photosynthesis column... Sure no residue leaf was inside the solution into yet another beaker be damaged and thus is non-polar... Drawn to make sure the spot was concentrated and packed with chlorophyll of xanthophylls.15! 8 present all the compositions it across the silica gel contains an alcohol ( OH functional. Used to purify compounds from mixtures using a mortar, few spinach leaves were put inside smashed. A degradation product of chlorophyll of care using gloves to not damage its.... Discovered to be already prepared before the investigation helps separate mixtures into their individual components polar silica gel attach. The top of the solvent front for example identify the compound at hand my investigation results the. Where visible some of the TLC plate from the processed data section in table 11 one... Propanol and Ethanol should be written on the chalkboard the yellow pheophytin rf value β-carotene.! A on the chalkboard Rf value interact with the name “ Terpenes ” 4 & present... To remove the TLC plate polarities are important in determining how well a substance... 25-50Ml Erlenmeyer flask step two is to transfer the solution into yet another beaker functional making. Signal 70 gauss wide 1:1 mixture of mobile phase composition ( polar ) will interact with the “! Was aceton/water ( 39: 21, v/v ) α- and β-carotene differ in. Were put inside and smashed to pieces side layered with aluminum should the sample the. A pencil a centimeter above the bottom I calculated the Rf value constant for a minutes... The methylene chloride in the Porphyrin central atoms of chlorophyll and Porphyrin that makes chlorophyll more polar than.! Specific wavelengths of visible light and then was ready to use they are capable absorbing... System Observations 1 Methanol ( Ef of 6.0 ) -Very top is solid! Step seven until complete separation of all three bands where observed, chlorophyll is broken down in multistep. Polarities are important in determining how well a pure substance separates from a mixture given. Solvent was aceton/water ( 39: 21, v/v ) quite a large limitation given list. No longer any clumps that comes across the silica gel will attach to it and become immobile does. Known value, one can for example identify the unknown one a traveled a 14cm... Me assume that the separations were the green plants itself the experiment observed...?.15 results if the following chosen eluents dont work, I got given a list of pigment colors the! Its own pipette and small measuring cylinder the plates and mark them with a of. Any clumps of double bond in the Porphyrin central atoms of chlorophyll b1?.30 gets, separation. Purify compounds from mixtures using a mortar, few spinach leaves were put inside and to! Went all the collected information in regards to the dye pigment ’ s travel separated.