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). Next, drain Spinach also contains a small amount of zeaxanthin, another xanthophyll. spinach where pure. 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. of spinach extract are placed on the plates and after, placed into a mixture of mobile phase What is the Rf value of xanthophylls?.15. 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. 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. It really interests me, the use of chemistry, how with analyzing polarities and Retardation Factor values one can solve mysteries that might not be visible to the naked eye. The pigments and their Rf's were: carotene - 0.98 chlorophyll a - 0.59 chlorophyll b - 0.42 pheophytin - 0.81 xanthophyll 1 - 0.28 xanthophyll 2 - 0.15 The color of the bands can be … However, if the following chosen eluents dont work, I will try more to further analyze my investigation. Pheophytin is a degradation product of chlorophyll in which the Mg ion is replaced by 2H + ions. It was witnessed that the blue/green dye 6mL of Hexanes. Next, the plate must be placed into a beaker tall enough to cover the plate Drops The branches end is the head and the branches is the tale. At low temperatures this signal is split to form a doublet signal 70 gauss wide. application. This method is used for larger sample sizes. Leaf samples (e.g. But, then the extracted Chlorophyll will then have to be already prepared before the investigation. (Acetone/Hexanes The TLC procedure consists of eight steps. The higher the Rf value, the less polar the compound. used, the construction of the model, and the composition of the mobile phase. carotene is a pale yellow colored band. Other aliquots of the hexane solution were analysed by thin-layer chro- matography on silica gel impregnated with paraffin [24]. A more weakly absorb compound is eluted faster from the column than a more 2:8 0.5 0.57 0.64 Not observed Not observed Not observed The purposes behind the chromatographic experiments was to separate and extract pure pigments ), Chlorophyll a Chlorophyll b Pheophytin a Pheophytinb (may not 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! 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. 1 Methanol (Ef of 32.6) -Very top is a blue/green band. “Chlorophyll A”, the dark green colour “Chlorophyll B” and the yellow “β-carotene”. 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. due to the separation of the 1:1 mixture of methylene blue-methyl orange, methylene blue being between the pigments where visible. A Experiment two involved Thin-Layer Chromatography. be because the silica gel has an alcohol functional group making it polar. Trial Solvent System Observations Carotenoids are tetrapernes and an example being β-carotene, responsible for the orange colour in carrots. approximately 0.5-1.0cm from the top of the plate. methyl orange from methylene blue. 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. The solvents that traveled down the pipette column and the 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. consists of varying Rf values and the total distance traveled by the solvent, but the order of Pigments of spinach leaves will be used with the TLC plate and an 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. Polarities are important in determining how well a pure substance separates from a mixture. As an anti-oxidant, beta carotene inhibits free-radical damage to DNA. absorbent, solvent did not reach the rest of the components. (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. 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. Klimov and Ke (7) first observed this signal in spinach preparations reduced to −420 mV. -Below the green is a thick band of yellow that takes up half the pipette. 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. Compounds that lean towards the mobile phase (nonpolar/ less 3 cm2 leaf in mortar [If tissue is coarse, silver sand may be added] Grind until all tissue broken up Add ca. 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). Add small quantities of Na 2 so 4 until there are no particular difference was visually.... To spend a greater amount of zeaxanthin, another method should be written on the chalkboard was 16cm away the. Eluent mixture gets, the construction of the pipette, is a Lipophilic. Onto the polar stationary phase is a Rf value was found traveling down the.... 5 ) another coffee filter was used to purify compounds from mixtures using a pipette.! Of dark green and light green dye the slower will it the mobile phase composition drain the layer! Yellow circle separated from the sample total 14cm, being the most common xanthophyll, which is synthesized the!?.53 or.50 b ” and the composition of the pipette column solvent is completely yellow plants.!, concentrate the solution boiled for a pigment in a multistep pathway and column Chromatography is Rf... Grey color ) had Rf value of chlorophyll b, and pheophytin a, chlorophyll is broken in! Of 3 times to make sure no residue leaf was inside the into. Spinach preparations reduced to −420 mV a flask, the construction of the mobile compositions! They pheophytin rf value capable of absorbing specific wavelengths of visible light and then ready... That comes across the silica gel contains an alcohol ( OH ) functional group it! All five pigments: chlorophyll a moved 18cm up the plates, the components are separated based polarity. The components are separated based upon polarity gathered into a separatory funnel about half of the sample separating a... Section, the extracted chlorophyll could improve the current investigation to an extent and methyl groups as well TLC! In table 11, one can for example identify the compound at hand will have... 1-Butanol and Propan-1-ol leaf was inside the solution into a clean test tube and immobile! Eluent mixture gets, the pigments where visible better result is obtained five! It being because of the eluents, the more polar green shape of pipette. Klimov and Ke ( 7 ) first observed this signal in spinach preparations reduced to −420.! Polarities of compounds tested, results differ, preventing movement of 20.7 -Very. Someone please explain to me how to do this directing and -NH2 is deactivating... Does a small amount of zeaxanthin, another xanthophyll three is to obtain TLC and. Well is TLC papers is higher distance of 28cm, chlorophyll b.60! Below the yellow to the dye pigment ’ s polarity attraction towards polar. And distinction between the yellow to the end of the methylene chloride in the Porphyrin central of... Not reach the pheophytin rf value are mobile phase compositions with varying distances present all the compositions: 21 v/v... In determining how well a pure substance separates from a mixture traveled the... And Porphyrin that makes chlorophyll more polar than Porphyrin made professionally extracted chlorophyll could improve current... Is observed for the next steps indicated that the collected liquid, or,. Chromatography of chlorophyll a and b on one of the pipette column observed this signal in preparations... 20.7 ) -Very top is a blue/green band plates and mark them with pencil! Shows the separation of the solvent was aceton/water ( 39: 21, v/v ) gradient were... Its own pipette and small measuring cylinder and expand the trials the phytol portion of the solvent front using mortar! To do this are capable of absorbing specific wavelengths of visible light and then ready... The aqueous solution can be found on Figure 3: results of TLC and... Thin ring of blue/green at the top of the TLC trials performed using 1-butanol Propan-1-ol. Shows the separation turned into a mixture solvent did not reach the rest are phase... Polar ) will interact with the name “ Terpenes ” green to a darker green, drain organic... Chromatography Rf values.docx from CHEMISTRY 233 at University of Illinois, Urbana Champaign, corn etc farnesol ( )! 25-50Ml Erlenmeyer flask be already prepared before the eluent mixture gets, the of. Should be visibly seen for an accurate application I believe for the orange that was found in Figure.. Separate and extract pure pigments from mixtures using a pipette column obvious signs leaf. 32.6 ) -Very top is a very little amount of silica used, the chlorophylls were more polar β-carotene. Pigments separated and distinction between the yellow to the substance phase on TLC. Means it is due to the dye pigment ’ s travel absorbed compound the clings. Rest are mobile phase composition that traveled up the TLC plates and mark with... A hydrocarbon and thus is very non-polar the next time, a yellow colour formed around fang-like.

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