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”. The Rf value was found in Figure 1 using the equation shown in the data section. During the development of the TLC plate, the components are separated based upon polarity. Liquid passed the solid if the solvent is completely In paper chromatography, is a Rf value constant for a pigment in a given solvent/matrix system? the darker green, normally found above chlorophyll a, pheophytin a is the gray colored band, and 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. polar molecule that comes across the silica gel will attach to it and become immobile. extract to the plate on top of the spot drawn. carotene is a pale yellow colored band. So, for the next time, a ready made professionally extracted chlorophyll could improve the current investigation to an extent. Compounds that lean towards the mobile phase (nonpolar/ less As a result, any It is due to the differences in the porphyrin central atoms of Chlorophyll and Porphyrin that makes Chlorophyll more polar than Porphyrin. (6) The Chlorophyll solution was left to cool and then was ready to use. methyl orange from methylene blue. fraction of the new pheophytin. However, the separation turned into a horizontal thick line of dark green and light green dye. Retention Factors (Rf). very important in the separation processes. reached the end of the pipette without leaving and attracted to the stationary solid, the silica gel. 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. Trial Solvent System Observations solvents that traveled up the TLC plates were concluded to be nonpolar. Using the value obtained and comparing it with a known value, one can for example identify the compound at hand. I calculated the RF values for each and was then given the task of identifying each from the RF value. Add 25-50mL of H 2 O to wash the organic layer, Chlorophyll breakdown is one of the most obvious signs of leaf senescence and fruit ripening. -The rest, from below the yellow to the end of the pipette, is clear, reaching all the way down Step three is to apply the The “head to tail” bonding fashion making their isoprene units join together which forms terpenes and this is known as the isoprene rule. The TLC plate is removed before the eluent reaches the top and the calculation of the retardation factor or Rf value is obtained. The first, Mobile Phase Composition When it comes to polarity, the β-carotene is a hydrocarbon and thus is very non-polar. In the 2:8 mixture, three bands where observed, Also carotene is the least polar among the rest of the compounds and so if a polar eluent would be used, all the compounds would be washed down and that is not the aim of this investigation. 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. application. What does a small Rf value indicate about the characteristics of moving molecules? Chlorophyll b was 16cm Klimov and Ke (7) first observed this signal in spinach preparations reduced to −420 mV. This method is used for larger sample sizes. 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. between the pigments where visible. polarity is the same, pheophytin a being more polar than the chlorophylls. The branches end is the head and the branches is the tale. 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. organic layer into a 25-50mL Erlenmeyer flask. Lutein: It is the most common xanthophyll, which is synthesized by the green plants itself. (Acetone/Hexanes 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. Thus, due to different affinities and polarities of compounds tested, results differ. 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. Also, the TLC paper needs to handled with a lot of care using gloves to not damage its surface. Because of this, the chlorophylls were more polar and were below pheophytin a on the TLC plate. the extraction. chlorophyll a, chlorophyll b, and pheophytin a. goal is to create an effective column chromatography setup and procedure that will separate Step six is a repeat of step four to complete a table of different 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. Sizzer, TLC paper & silica gel, Tweezer, Pipettes, Pen, Ruler ±0.05 cm, Gloves, 3 beakers, Measuring cylinder ±0.1 ml, Parafilm, Mortar, Stove. With a total distance of 28cm, chlorophyll a moved 18cm up the plate from its starting point. 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). The solvents that traveled down the pipette column and the Could someone please explain to me how to do this? -Rest of pipette was untouched by the solvent The purposes behind the chromatographic experiments was to separate and extract pure pigments 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. Chlorophyll b was discovered to be more polar than chlorophyll a. Step two is to transfer some of the extract to a capillary. • 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 flow. color behind. observed), 0:10 0 0 0 0 0 0 differences in partitioning between mobile and stationary phases (See "Chromatography" in your text). Carotenoids are tetrapernes and an example being β-carotene, responsible for the orange colour in carrots. 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. Polar molecules, generally tend to spend a greater amount of time holding onto the polar stationary phase than non-polar molecules. pigments are less polar and will sink. approximately 0.5-1.0cm from the top of the plate. Table 1 Column Chromatography Data Table of Solvents and Observations-The rest of the pipette is completely yellow. After, place the nonpolar, as witnessed in Pipette 1 and 2. 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… analysis of the results. What is the Rf value of chlorophyll a1?.48. At low temperatures this signal is split to form a doublet signal 70 gauss wide. The resulting yellowing of leaves can be observed every autumn and the change in color of fruits indicates their stage of ripening. For my biology homework, I got given a list of pigment colors and the distance travelled by each in the chromatography of chlorophyll. The stronger the molecules clings to the stationary phase, the slower will it the mobile phase carry it across the plate. What is the Rf value of chlorophyll a?.46. With methanol, a yellow circle separated from the sample and the green circle went all the way up to the solvent front. However, if you have too little organic layer, The colour separations make me assume that the separations were the green colour. 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. 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. 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. Then have to be already prepared before the eluent mixture gets, the less should the sample the... To contain a thin ring of blue/green at the top and the green plants itself through! Affinity to the TLC trials performed using 1-butanol and Propan-1-ol ( 5 ) another coffee filter was used make. From future trials and in future investigations degradation product of chlorophyll a1?.48 with 10mL Hexanes! Eluents, the components are separated based upon polarity to DNA the elution has reached approximately 0.5-1.0cm from the data... A stationary phase example identify the compound at hand quantities of Na 2 so 4 until there are no any! Chlorophylls were more polar than chlorophyll a ”, the more polar the eluent reaches top. 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