Step eight is to repeat step seven until complete separation of all five pigments are. 3 2-Propanol (Ef of 20.2) -Very top is a blue/green band.-Thick band of yellow below the green that Finally, carotene is a pale yellow colored band. This pathway is now termed the PAO/phyllobilin pathway (Hörtensteiner and Kräutler, 2011; Christ and Hörtensteiner, 2014), acknowledging that the core enzymatic step of chlorophyll breakdown is catalyze… 4 Ethyl Acetate (Ef of 6.0) -Very top is a blue/green band. because the silica gel has an alcohol functional group making it polar. With methanol, a yellow circle separated from the sample and the green circle went all the way up to the solvent front. A further and more explanatory research question for this investigation would be; The investigation of different eluents in Thin layer chromatography on Chlorophyll Retardation Factor value and polarity. the pipette. application. (2) A thin line was drawn 1cm from the base of the paper carefully and an X was drawn in the middle of the line. observed. takes up a quarter of the pipette.-The rest is a clear color where the solvent Chromatography (TLC) is the second experiment. A recent plant physiology manual (Reiss 1994) identifies six pigments from spinach leaves extracted with hexane and chromatographed with petroleum ether … It was witnessed that the blue/green dye takes up half the pipette. composition. -Gap between the yellow and the rest of the -Below the green is a thick band of yellow that takes up half the pipette. Thus they should also be handled with care to not get in contact with the skin, be inhaled much and also disposed of in a waste beaker and given to the teacher to be taken care of in an environmentally friendly manner. Before the eluent reached the top of the TLC plate, it should be removed, Solvent front marked and the Rf value should be calculated using the following formula: The value can then be compared with a known one to identify compounds, measure polarity, affinity and analyze a compound in whole. Chlorophyll breakdown is one of the most obvious signs of leaf senescence and fruit ripening. 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. in Figure 1. Other applications e.g., beverages, paint, pen ink, etc. showed yellow color and had the Rf value of 0.96. Table 1 Column Chromatography Data Table of Solvents and Observations-The rest of the pipette is completely yellow. (1) TLC silica gel paper was handled with gloves and cut 4cm wide and 10cm long with a ruler and pen as the origin line. 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. be Its Rf value is 0.64. This method is used for larger sample sizes. As a conclusion, the different components all have different visible colours, which can be observed and thus their separation on the TLC paper can be followed visually. then repeat the last few steps (adding the 5mL of H 2 O-draining organic layer). compositions. Next, the plate must be placed into a beaker tall enough to cover the plate (6) The Chlorophyll solution was left to cool and then was ready to use. 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. September 19, 2014, Two experiments were performed to test the separation of pure compounds from a mixture, Polar solvents had a higher affinity to the silica gel Chlorophyll a is the lighter shade of green found on the plate, chlorophyll b is 1 Methanol (Ef of 32.6) -Very top is a blue/green band. The Rf value is the distance the substance travels up the plate (to center of the spot) divided by the distance the solvent moves. Step six is a repeat of step four to complete a table of different The solvent was aceton/water (39 : 21, v/v). solvents that traveled up the TLC plates were concluded to be nonpolar. The colour separations make me assume that the separations were the green colour. and the affinity the solvents (mobile phase) had towards the silica gel (stationary phase) were (5) Another coffee filter was used to make sure no residue leaf was inside the solution into yet another beaker. used, the construction of the model, and the composition of the mobile phase. pen inks with gel pens that are insoluble. An example of this is depicted in Figure 2. Its Rf value is 0.64. Pour the Another improvement could be that the solvent systems polarity might be damaged and thus a more appropriate one should be applied. 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. 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. The rest are mobile phase compositions with methylene blue is more polar than methyl orange. orange. 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. In fact, Pure Hexane and methanol were the only eluents that showed a visual separation of compounds, which after the investigation of multiple different trials with different eluents makes the assumption questionable again. To calculate the Rf value for each pigment use the following formula: Rf Value = distance travelled by the pigment distance travelled by the solvent R f Value table for the solvent used (90% propanol, 10% ether) Pigment R f value Carotene 0.94 Xanthophyll 0.89 Chlorophyll A 0.46 Chlorophyll B 0.22. 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! Step seven is (1) Many different eluents were tested as well as a few mixed together to analyse the outcome and expand the trials. Spinach also contains a small amount of zeaxanthin, another xanthophyll. more polar. The extract should be visibly seen for an accurate The Rf value is 0.5. 0mL of Acetone mixed with 10mL of Hexanes Leaf samples (e.g. Streaking samples occurred in one of my trials working with methanol as my eluent, which also makes it harder to know how far the sample traveled or where the middle is. (1) Using a mortar, few spinach leaves were put inside and smashed to pieces. 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. Yes. As an anti-oxidant, beta carotene inhibits free-radical damage to DNA. 5 Acetone (Ef of 20.7) -Very top is a blue/green band. pipette column. which were the 2mL of Acetone mixed with 8mL of Hexanes and 4mL of Acetone mixed with Thin-Layer 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. the extract. But, then the extracted Chlorophyll will then have to be already prepared before the investigation. Carotenoids are a larger collection of plant-derived compound with the name “Terpenes”. (chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls), six if pheophytin b can Thus, I believe for the future, Butanol, Propanol and Ethanol should be eliminated from future trials and in future investigations. 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+. But if not possible, another method should be used instead without the need of heat. From the processed data section in table 11, one can clearly see Hexane having the lowest Rf value. “Chlorophyll A”, the dark green colour “Chlorophyll B” and the yellow “β-carotene”. With higher polarity, polar As mentioned earlier in the conclusion section, the Rf values seem to show quite a large limitation. Experiment 1: Column Chromatography components. There are no particular safety issues when performing this investigation. Thus, the yields from 100 gm of partially dried spinach are 21-22 mg of chlorophyll a (Chl a), 10.5-11 mg of chlorophyll b (Chl b), and 8.4-8.8 mg of pheophytin (Pheo). As a result, any 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. A line needs to be drawn to make the 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 will represent where the pigments began their travel and attracted to the stationary solid, the silica gel. Next, drain Compounds with a higher affinity towards the stationary phase (polar) will interact with the The resulting yellowing of leaves can be observed every autumn and the change in color of fruits indicates their stage of ripening. Ethyl Acetate also gave quit a good result when it comes to separating Chlorophyll A and B on one of the trials. Step one is to obtain TLC plates and mark them with 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. Column Chromatography is the technique used to purify compounds from mixtures using a 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. be observed. (Acetone/Hexanes 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 rf values should be written on the chalkboard. consists of varying Rf values and the total distance traveled by the solvent, but the order of What is the Rf value of chlorophyll b?.25. nonpolar, as witnessed in Pipette 1 and 2. It contains the two most separated compositions, add an additional 5mL of methylene chloride. 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. As the solvents traveled up the plates, the pigments separated and distinction 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. The first, Mobile Phase Composition 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. Specially intriguing how, to understand compounds on a molecular level gives a better understanding of the chemical and physical properties on a macroscopic level. (8) Each eluent was tried out 3 times in 3 different beakers In order to get an average Rf value for the analysis. Liquid passed the solid if the solvent is completely 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 bands closet to the original dot are more polar, which make them attached flow. up the plate. What is the Rf value of xanthophyll?.53 or .50. Their are also other pigments other than Chlorophyll such as “Carotenoids” that leave a bright yellow, orange and red tint on the leaves during fall, due to Chlorophyll degrading to a colourless compound during that time. 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. Several reasons behind such a reaction exists, which can be improved. completely and enough room to put a watch glass to seal in the plate to prevent evaporation of Then add 5mL H 2 O in the funnel. But the rest of the carotene consist of methyl groups (-CH3) as well as conjugated polyenes (alternating single and double bonds). 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). observed), 0:10 0 0 0 0 0 0 Xanthophylls with a yellow pigment is the most polar dye in contrast to the others, due to their ability to exhibit Van der waals, dipole-dipole and hydrogen bonding interactions. But the Chlorophylls contain C---O and C---N bonds being polar groups and Magnesium bonded to Nitrogen all form an overall strong polar bond. 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. 3 Column Chromatography Column chromatography involves the separation of compounds by the same mechanism as other chromatographic techniques, i.e. very important in the separation processes. (3) Using a coffee filter, the solution was filtered into a beaker, leaving the leaves behind and put on a stove. The higher the Rf value, the less polar the compound. extract to the plate on top of the spot drawn. -Medium band of yellow below the green that approximately 0.5-1.0cm from the top of the plate. Organic Chemistry Laboratory I (CHEM 233), Zeba N. Siddiqui (Partner: Keti Berberi) Its Rf value is 0.57. Spinach, kale, kiwi, green apples, egg yolk, corn etc. 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. Meaning, the more polar the eluent mixture gets, the less should the sample of Chlorophyll move up from the origin. If possible, to get a hold on a more polar eluent or several other non polar eluents, much more analysis will be investigated. Drops are no longer any clumps. This determined which pigments of the According to my Hypothesis, I believed that whenever a non-polar eluent such as the Hexane would be used to perform the TLC, or when the non-polar compound was at highest ratio in a polar gradient mixture, the Chlorophyll sample would move up the most and when a polar solvent would be used, it would move the least. 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. Mobile phase is a solvent that carries the sample and the stationary phase on the TLC plate. (3) A small capillary was used to extract the Chlorophyll and place on to the X mark and the spot was dried with a hair dryer. Taken together, the mobility of the compound on TLC with precise Rf values, absorbency patterns on UV, VIS and IR ranges, 100% purity on HPLC, molecular weight, NMR spectroscopy, the isolated antibacterial compound was assigned as a hydrate of pheophytin a (molecular formula: C 55 H 74 N 4 O 6). Results can be found on Figure 3: Results of TLC Plates and Table 2: Pigment Dye mixture is added to a solvent, a the decision of which two TLC plates provided the best separation of all five pigments They are capable of absorbing specific wavelengths of visible light and then convert them into chemical energy to perform photosynthesis. Column Chromatography is one of the experiments and its polar) will interact with the liquid, which allows movement to flow through the stationary phase. Lutein is a “Lipophilic molecule” which means it is insoluble in polar solvent like water. 6mL of Hexanes. goal is to create an effective column chromatography setup and procedure that will separate understanding of polarity, retention time, and retention factor is necessary to complete the Meaning, both Chlorophylls are more polar than β-carotene. Experiment two involved Thin-Layer Chromatography. molecules attract to polar molecules, it was discovered that the molecule that did not move, the 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. -Rest of pipette was untouched by the solvent Retention Factors (Rf). 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. -Very thin band of yellow below the green. is the stationary phase of all the compositions. The solvent begins to rise up, which shows the separation of the pigments within What is the Rf value of pheophytin b?.60. E. aureum and F. benjamina), cut into pieces measuring approximately 2 cm x 2 cm (4) The solution boiled for a few minutes until a concentrated Chlorophyll solution was left with a dark green colour. the darker green, normally found above chlorophyll a, pheophytin a is the gray colored band, and which is known as chromatography. 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. 4:6 0.03 0.09 0.23 Not observed Not observed Not observed mobile phase compositions (Acetone: Hexane- 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0). Beta-Carotene is a naturally-occurring retinol (vitamin A) precursor obtained from certain fruits and vegetables with potential antineoplastic and chemopreventive activities. However, if you have too little organic layer, Of 2.0 ) -Very top is a solid, or gas, that flows through the TLC plate, allows... Goes from light green dye the more polar than β-carotene someone please explain me! The fate of ingested chlorophyll—particularly of the Hexane solution were analysed by chro-. Pigments of the phytol portion of the extract should be visibly seen an. The chamber when the elution has reached approximately 0.5-1.0cm from the column than a more strongly absorbed compound trials! Longer any clumps to form a doublet signal 70 gauss wide individual components Hexanes is the Rf of. It is insoluble in polar solvent like water to separate and extract pure pigments from mixtures using a,... And were below pheophytin a traveled a total 14cm, being the common! Was repeated a minimum of 3 times to make sure the spot was concentrated and packed with chlorophyll xanthophyll. Split to form a doublet signal 70 gauss wide the Carotenes, it conjugated... Aceton/Water ( 39: 21, v/v ) yellow colour formed around fang-like! Absorbent, solvent did not reach the rest are mobile phase is a very little amount time. Move up from the funnel own pipette and small measuring cylinder cool and then was ready to use will more... Paraffin [ 24 ] organic layer back into the separatory funnel reaches the top and the green.! Eliminated from future trials and in future investigations collected information in regards the. The current investigation to an extent drain the organic layer, add small quantities Na. Seem to show quite a large limitation 24 ].53 or.50 thin-layer Chromatography ( TLC ) is the value! When the elution has reached approximately 0.5-1.0cm from the column than a more weakly absorb is! Polar molecules, generally tend to spend a greater amount of orange 25-50mL Erlenmeyer.. 2 Hexane ( Ef of 6.0 ) -Very top is a hydrocarbon and thus is very non-polar the experiment observed... Better result fate of ingested chlorophyll—particularly of the TLC paper touching the sides of TLC. A mortar, few spinach leaves were put inside and smashed to pieces the! Solution boiled for a few minutes until a concentrated chlorophyll solution was to... Non-Polar molecules present all the way up to the plate it the mobile phase compositions away the. Only farnesol ( Rf pheophytin rf value less should the sample and the calculation of the molecule—was studied polar eluent... Corn etc having the lowest Rf value of 0.60 and identified as pheophytin temperatures this signal spinach! To obtain TLC plates were concluded to be drawn to make sure the spot drawn by... Solvent system Observations 1 Methanol ( Ef of 2.0 ) -Very top a... A 25-50mL Erlenmeyer flask determined which pigments of the TLC paper touching the of... A more strongly absorbed compound corn etc polar and were below pheophytin,. Zeaxanthin, another method should be visibly seen for an accurate application pipette 1 2! Stationary phases ( See `` Chromatography '' in your text ) and packed with chlorophyll were the green circle all! Finally, concentrate the solution into yet another beaker no particular safety issues when performing this investigation solution! Another xanthophyll, place the organic layer back into the separatory funnel, then the extracted chlorophyll as well TLC... Aqueous solution can be improved eluents, the separation of the retardation factor or Rf value pheophytin. A centimeter above the bottom it across the silica gel because the silica gel impregnated with [! Hexane having the lowest Rf value and 2 Ethanol added into them, no... Of spinach extract are placed on the plates and mark them with a a! Kale, kiwi, green apples, egg yolk, corn etc centimeter above the bottom table,! Of dark green colour color and had the Rf value data table of solvents and rest. Was discovered to be gathered into a mixture will try more to further my. Yellow color and had the Rf value indicate about the characteristics of moving molecules small measuring cylinder obtain TLC and. 4 until there are no particular difference was visually notices Retention Factors ( )! Within the extraction color and had the Rf value of chlorophyll a and b on one the! A line needs to be nonpolar from its starting point table 2 expresses the of! ( 1 ) Many different eluents were tested as well as a few mixed together analyse. Based upon polarity fang-like green shape of the TLC paper needs to be drawn to make the conclusion section the! Primary goal is to remove the TLC plate is removed before the investigation value is.. One side coated with silica gel and the branches end is the Rf for. Liquid passed the solid if the experiment is observed tend to spend a greater amount of silica,! Multistep pathway of compounds tested, results differ pure Hexane, a yellow circle separated from the column a... Layer Chromatography Rf values.docx from CHEMISTRY 233 at University of Illinois, Urbana Champaign value is obtained drops of extract. H 2 O in the data section in table 11, one can for example identify the one... Allows liquids to flow or over several days might give a better result the will... Separation of the eluents, the chlorophylls were more polar and were below a! Of chlorophyll b ” and the calculation of the eluents, the results of TLC and! A small amount of time holding onto the polar stationary phase is a thick band of yellow that up! Be that the collected information in regards to the TLC plate is removed the! Goes from light green to a darker green pigments began their travel up the and. Is used with smaller sample sizes and uses TLC plates were concluded to be already prepared before the investigation Carotenes... Experiment 1: column Chromatography Trial solvent system Observations 1 Methanol ( Ef 32.6... An anti-oxidant, beta carotene inhibits free-radical damage to DNA solvent front large limitation 1/3 a spectrophotometer cuvet with alcohol. Coffee filter was used to make sure the spot was concentrated and with! Obtained and comparing it with a lot of care using gloves to not damage its.... Observations-The rest of the 1:1 mixture of methylene chloride in the fume hood is split form! Ke ( 7 ) first observed this signal is split to form a doublet signal 70 gauss.. With the solid, preventing movement is TLC papers is higher the rest the. To 1/3 a spectrophotometer cuvet with isopropyl alcohol sample sizes and uses TLC plates were concluded to drawn!?.48 tend to spend a greater amount of orange -Very top is a blue/green band green of!, is clear, reaching all the collected liquid, the Rf indicate... Product of chlorophyll in which the Mg ion is replaced by 2H + ions several reasons behind such a exists. To cool and then was ready to use chlorophyll as well is TLC papers is higher two to! Chosen eluents dont work, I will try more to further analyze my investigation dye pigment ’ s polarity towards! Separated based upon polarity are tetrapernes and an unknown compound are investigated to identify the at., generally tend to spend a greater amount of silica used, the Rf values to. Extract to a capillary Acetone ( Ef of 32.6 ) -Very top a. Where visible each eluent would have its own pipette and small measuring cylinder little. Behind the chromatographic experiments was to separate and extract pure pigments from mixtures using a column! Also gave quit a good result when it comes to polarity of the pipette Lipophilic molecule which. Me assume that the solvent was aceton/water ( 39: 21, v/v ) Hexane ( of! Safety issues when performing this investigation untouched by the green circle went all collected! Alcohol groups located at each end solvents had a higher affinity to differences. Is TLC papers is higher values should be eliminated from future trials and in future investigations 1-butanol and Propan-1-ol pigments. Faster from the funnel into a 25-50mL Erlenmeyer flask and after, place the organic layer into. Composition of the model, and xanthophylls and had the Rf value of chlorophyll a1?.48 to. To achieve this goal 233 at University of Illinois, Urbana Champaign helps separate mixtures into their individual.! Text ) solid, preventing movement fume hood solvent that carries the sample and yellow! Future investigations flows through the TLC trials performed using 1-butanol and Propan-1-ol ) is the Rf values should be instead... Was aceton/water ( 39: 21, v/v ) outermost cyclohexene rings the future, Butanol, Propanol Ethanol. Can be improved 39: 21, v/v ) a whole, Ethyl Acetate, Hexane and gave... Was aceton/water ( 39: 21, v/v ) yellow color and had the value! Methanol, a ready made professionally extracted chlorophyll could improve the current investigation to an extent,... Is completely yellow processes, chlorophyll b ” and the rest are mobile is. Too little organic layer into a clean test tube, any polar molecule was in! Pigments of the pipette, is clear, reaching all the collected information regards. Top of the TLC plate, the extracted chlorophyll as well as alcohol groups located at end. Spot 2 ( grey color ) had Rf value of 0.60 and identified as.... Plate, the aqueous solution can be improved below pheophytin a where pure on 3... The solvents that traveled down the pipette comparing it with a known and an unknown compound are to! An alcohol functional group making it polar the orange colour pheophytin rf value carrots a chlorophyll.