You are on page 1of 5

The first part of the experiment is all about the separation of plant pigments using chromatography.

As stated, chromatography here is used as a separation technique. Table 4.1 summarizes the description of the materials and reagents used for this part. The leaves were pounded to extract the pigments. Acetone was the solvent used to extract these pigments from the leaves because acetone is polar and so is the pigments found in the leaves. After the pigments were extracted, spots were placed in the chromatographic paper and were observed. Leaves contain chlorophyll and this chlorophyll has different types. Chlorophyll A is green while chlorophyll B is yellow green or yellow. Other pigments found in plants are xanthophylls and beta-carotene. Below is the structure of these four types of chlorophyll. All of the types of chlorophyll are large molecules. Chlorophyll a and b has a magnesium central atom held in a polar porphyrin ring. The long hydrocarbon chain is attached to this ring and this chain is hydrophilic. Notice the difference in the structure of chlorophyll a and b. Chlorophyll a has a polar aldehyde group which makes it more polar than chlorophyll b. Three types of solvents were used as mobile phase to determine which solvent is the best eluting solvent. The spotted chromatographic paper is dipped in a solvent. The solvent then travels in the paper by capillary action. Difference in polarity of the solvent and that of the pigments of the plants gives way to the separation. Figure shows the appearance of the chromatogram after the procedure. It can be seen there that the spots travelled with different distances depending on the solvent used. It was also observed that the number of spots separated differ from each solvent. Generally, the yellow spot travelled a longer distance than that of the green spot. One of the solvents is pet ether only. is a nonpolar solvent. In the experiment, only the green pigment was observed in the chromatogram when this solvent was the one used. Because of the nature of the solvent and the pigment, it is expected that the one to be observed is chlorophyll b or color yellow because this is less polar relative to chlorophyll a. However, this result only means that pet ether is not a good eluting solvent because it was not able to separate clearly the pigments in the plant. This result also points out to some errors. The other solvent used is 9:1 pet ether and acetone. It is noticeable that one of the components of the solvent is polar and the other one is non polar. In this type of mixture, the components of the mixture doesnt act as a team. In fact, they act individually. Meaning, the polar pigment will migrate with acetone and the less polar pigment will migrate with pet ether. Since one of the components of the mixture of solvent is polar, the system (mobile phase and stationary phase) is comprised of two polar components. One is the stationary phase itself which is cellulose and the

other is the component of the solvent. The stationary phase and the acetone compete for the affinity of the polar pigments found in the leaves. Between the two, cellulose is more polar than acetone because of the ability of the cellulose to form H-bonds. Therefore, the more polar pigment will adhere more to the paper and hence, have a shorter distance of travel. The polar pigment will travel with the acetone and the least polar pigment will travel with the petroleum ether. Having these characteristics, this solvent is better than the first solvent because it is able to separate the more polar pigment from the polar pigment and from the less polar pigment unlike the first solvent, it can only separate the polar pigment from the non polar or less polar pigment. The last solvent used is 9:1:1 pet ether, acetone, and diethyl ether. Just like with the previously used mixed solvent, each of the components in this solvent acts as they are or individually. This solvent also has polar and non polar components. The system therefore contains two polar components and two nonpolar components. The polar components are acetone and the cellulose and the non polar components are diethyl ether and petroleum ether. Since the stationary phase, which is the water bound to the cellulose is more polar than the acetone in the solvent, it is expected that the more polar pigment will adhere more to the paper and will therefore have a shorter distance of travel. The polar component will travel along with acetone and will therefore have a longer distance of travel relative to the first one mentioned. On the other hand, the less polar pigment will travel with the less polar component of the solvent and will therefore have a longer distance of travel relative to the component that travelled with acetone. Lastly, the least polar component will travel with the least polar component of the solvent which is petroleum ether and will therefore have the longest distance of migration. Citing these observations it is concluded that the best eluting solvent is the last solvent mentioned because not only that it can separate non polar from polar components, but also it can separate the most polar, more polar, less polar, and least polar pigments from each other. Therefore, separation is more efficient and observable. In the experiment, only two pigments were separated from each other. However, compared to the distance travelled by the two pigments in the 9:1 pet ether acetone solvent, the distance between the spots of the former solvent is larger. Assuming that in a sample, the four types of chlorophyll are present and it is to be separated, the best solvent to use is the 9:1:1 petroleum ether-acetonediethyl ether. Each of the four types of chlorophyll varies in polarity. Chlorophyll a is the most polar, followed by chlorophyll b and then xanthophylls and the least polar is the beta-carotene. If the mentioned solvent is used and separation is efficient, there should be four spots in the chromatographic paper. The spot farthest from the origin is beta-carotene, running second would be the xanthophylls. This is followed by the polar chlorophyll b and the most polar among the chlorophylls which is

chlorophyll a adheres the most to the paper and therefore will have the shortest distance of travel. This phenomenon wasnt observed in the experiment due to some possible sources of errors. One would be the chlorophyll contained in the leaf samples used. It is possible that the amount of pigments like xanthophylls and beta-carotene are not sufficient enough to make it visible in the chromatogram. Another error could be personal error where the observer wasnt able to see and mark the some spots in the chromatogram because it is very light. And because of this, after some time, the unrecorded spots disappear and therefore lead to wrong analysis of data. The errors discussed in the earlier part of this report can also apply here. Aside from the color of the spot, full characterization of the pigments observed and separated can be done by referring to the computed Rf values and comparing it to that of the standard. As discussed in the introduction, this is the ratio between the distance travelled by the spot and the distance travelled by the solvent. This value is a constant; however, this is affected by several factors such as humidity, temperature and others. The Rf value obtained for Other pigments found in plants as stated earlier are xanthophylls and betacarotene. Both of these are yellow and they only differ in intensity. The observation for both of these pigments is vague because of its color. The determination of how bright or how pale the intensity of the color yellow is differs from person to person. This makes the observation subjective, and in turn, leads also to a subjective result and not an objective one. This is why these two pigments were not given so much attention to this report relative to that of chlorophyll and b. In the second part of the experiment, chromatography was used to determine an unknown amino acid. This is a qualitative analysis. Amino acids are the building blocks of proteins and there are about 200 amino acids but only 20 amino acids comprise compounds in our body. This only shows how important the study of amino acids is. From the name itself, amino acid is an organic compound which has an acidic group and a basic group. The basic group is obviously the amine group and the acidic group is the carboxylic group. The amino acids only differ in their side chains (R). Below is the general structure of amino acids. Four types of amino acids were spotted on the chromatographic paper; 3 of which is known namely phenylalanine, tyrosine, and aspartic acid while one is an unknown. The identity of this amino acid was determined by comparing its Rf value with the known standards. The following are the structures of the amino acids. Notice their side chains. For phenylalanine, the side chain is a benzene ring CH2(C6H5). This comprises a bigger part of the total structure of phenylalanine so this nonpolar hydrocarbon chain makes the entire compound a non polar or a water hating one. Since the solvent used for this part of the experiment is ammonium hydroxide isopropyl alcohol which is nonpolar, this is expected to have a

longer distance travelled compared to the other amino acids. For aspartic acid, the side chain carries a carboxylic group which makes it more acidic and more polar than other amino acids due to the additional -OH part of the side chain. So this amino acid is known to be an acidic and polar or hydrophilic amino acid. Therefore, this amino acid is expected to travel a shorter distance compared to that of the phenylalanine. For the last known amino acid which is tyrosine, the side chain is also a benzene chain, however, this benzene ring has an OH attached to it which makes this side chain polar. Since the side chain and the rest of the molecule are polar, tyrosine is therefore a polar amino acid. Similar to aspartic acid, this is expected to travel a shorter distance than phenylalanine. At first, the spots were not visible. This is because amino acids make colorless spots. To visually observe the spots of the amino acids, a detecting agent is sprayed to it. This detecting agent is the ninhydrin. Ninhydrin is a polar In the last part of the experiment, chromatography was used to determine the number of component s of different types of ink. In this part, the stationary phase used is chalk or calcium carbonate and the mobile phase used was 70% isopropyl alcohol. Instead of using silica or alumina, chalk was used as substitute because it is also adsorbent making it feasible to use. One of the inks tested was Steadler and the other is the ink used in stamp pads. It was observed that different colors comprise the black ink of steadler. Some of the colors are pink, violet... In this experiment, it is assumed that the number of components is equal to the number of colors observed. As for the steadler ink, 4 colors were observed and it can be concluded that it is composed of four components. The same mechanism of chromatography applies in this part of the experiment. If an ink is has more than one component, the polarity of each component is differs from each other making separation using chromatography possible. In the case of the stamp pad ink, only one color was observed which is violet. From here, it can be concluded that this ink has only a single component and therefore, this is more pure than the first ink mentioned. This is ohw chromatography is used to determine the efficiency of purification process. If the ink or say any substance to be analyzed has only one color after subjecting to chromatography, the substance is said to be highly pure.

Aside from the uses of chromatography that was done in the experiment like separation of pigments, determination of unknown and test for purity of a substance, chromatography can also be used to monitor the rate of reaction. This can be done by spotting a TLC plate three times and is allowed to develop under uv light. The origin of the spot represents the authentic sample of the starting compounds. As it goes up, a second position is determined and this represents the reaction mixture. The last position observed is the authentic sample of the product. When the TLC plate shows no more starting material left, it is assumed that the reaction has gone to completion.

One limitation of chromatography is that it cannot separate and identify very volatile compounds. Since chromatography basically works through the concept of solubility, obviously, TLC and paper chromatography are not suitable for the identification of such compounds. Since the compounds to be analyzed are highly volatile, it will not adhere with either the mobile or the stationary phase. Therefore, no spots will be observed in the chromatogram and therefore, analysis will be impossible. SUMMARY AND CONCLUSION The basic concept and laboratory technique learned in this exercise is chromatography. Two specific types were studied namely paper chromatography and TLC. In the first part of the experiment, paper chromatography was used to separate plant pigments. Three types of solvents were used and the best eluting solvent among the three is the 9:1:1 petroleum ether:diethyl ether:acetone mixture because it is able to separate a more polar pigment from a polar one from a less polar and the least polar pigment. After assessing the best eluting solvent, the experimental Rf value were computed and since the Rf values of pigments in different solvents vary, it is difficult to rely only on the Rf value for the identification of the pigment. The most practical and easiest way to determine the identity of the pigment is by observing the color. Chlorophylls are either blue-green or yellow-green. Carotenes are generally yellow orange or orange and xanthophylls are usually yellow or bright yellow. On the second part of the experiment, paper chromatography was used to determine an unknown acid. 4 amino acids were tested, three of which are known and the other is an unknown. The known amino acids are phenylalanine, tyrosine and aspartic acid. among these amino acids, phenylalanine had the longest distance of travel, followed by tyrosine, and then aspartic acid. this behaviour is simply explained by their polarity. The nearest to the origin which is aspartic acid is the most polar among the three. The farthest from the origin which is phenylalanine is the least polar among the three and tyrosine lies between the two amino acids because it is less polar than aspartic acid and more polar than phenyalanine. Having computed the Rf values, the identity of the unknown amino acid of our group is tyrosine because the experimental Rf value of tyrosine is 0.70 adn the computed Rf value for our unknown is 0.69. In the last part of the experiment, Tlc was used to determine the component dyes of an ink. One of the inks tested is steadler ink and after subjecting it to chromatography, it displayed a variation of colors namely violet, pink, and yellow. Since more than one color was observed, it can be concluded that this ink has more than one component and therefore, has low purity. On the other hand, the stamp pad ink, after subjecting to chromatography only displayed a single color which is violet. This only means that this ink is comprised of a single component and is therefore more pure than the first mentioned ink. Chromatography is a very useful technique in many industries such as food, pharmaceuticals or even in forensics.