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Using Paper Chromatography to Identify Unknown InksSamar Almarzooqi2/21/13Partners: Mariam Ahmed CHEM 113 Section 103TA: Steve Kennedy IntroductionChromatography is a way to separate mixtures of a solution into separate components. The technique is often used in a variety of different settings, like scientific research, medicine, and industrial processes, because it allows for the analysis of the components of many mixtures.1 Paper chromatography specifically is a type of chromatography that “separates dried liquid samples with a mobile and stationary phase.”2 Paper chromatography uses special cellulose paper that acts as a stationary phase. The different samples act as a mobile phase when dissolved in a solvent. The sample is originally at one side of the chromatogram paper, and it is pulled through the stationary phase through “capillary action.” The components of the sample are then separated through the movement of the samples through the stationary phase. The differing components of samples can be separated into their different pigments, and each pigment signifies another component of the sample.3Russian botanist, Mikhail Tsvet, first created chromatography in 1906 in an experiment that separated chlorophyll pigments. He did so by using organic solvents to extract green material from the plant, and he then allowed the material to filter through a solid powder. Using polar solvents, the extract was moved through the powder. He then observed different bands of color across the column, which he deduced showed the different types of chlorophyll within the extract. His approach signified a novel way to separate and “investigate the chemistry of complex natural mixtures.”2 The scientific community did not agree with chromatography as the best means of separating components of a sample, and it was not until the 1930s when the uses of chromatography were readdressed. In a type of chromatography termed partition chromatography, chemists Martin and Synge analyzed the structure of proteins through paper chromatography. Today, chromatography is a technique used in many different settings as a means for separating mixtures into their separate components. The different types of chromatography include: Liquid Chromatography, Gas Chromatography, Thin-Layer Chromatography, Ion-Exchange Chromatography, and Paper Chromatography.2 Chromatography separates mixtures into their components by utilizing the idea that different chemicals move at different rates through the liquid or solid stationary phases. Because the individual components of a mixture move at different rates, they will become separated from each other.4 In Paper Chromatography, the stationary phase is a liquid held within a solid, most often it is the cellulose paper and water complex. The mobile phase is the sample tested, and it travels through the stationary phase by placing the stationary phase in the mobile phase. The mobile phase is then pulled up through the stationary phase through capillary action, which allows for the separate components to be pulled up to different distances. In chromatography, the objective is to separate the individual components as much as possible, which is possible by maximizing the differences in the component migration and minimizing the spreading of the component process.3 Depending on the interactions between the stationary phase and mobile phase, the migration distances can be optimized. Using polar compounds as the mobile phase will not have as large of a migration distance because the polar compounds will be attracted to the polarity nature of the water. By using nonpolar compounds, the distance the molecule migrates will be farther because it will not react with the water in the stationary phase. Making sure both the solvent and the molecules have similar polarities will maximize the distance the components will travel.1 Paper Chromatography has a limited use in the effective application of the technique, mainly because it cannot separate complex mixtures and cannot lead to a quantitative analysis of the concentrations of individual components.5 The advantages of using Paper Chromatography, though, are that it is inexpensive and easy to reproduce. Also, because the chromatography paper is manufactured in a uniform structure, the chromatography for a particular sample can be compared to another without many variables affecting the difference in component migrations.3 In Forensic Science, chromatography can be used to separate unknown compounds and identify them based on the distance each component travels. In drug tests, the urine is tested through chromatography to identify drugs. In pathology, chromatography can be used to identify possible poisonous substances in dead bodies to determine if a possible murder took place.6 In cases where advanced technology cannot be used, chromatography can be used to analyze mixtures of unknown substances and is effective in doing so. Applicable in the use of chromatography is the use of paper chromatography to separate individual portions of candy coatings to determine the type of food dyes present.7 Paper chromatography is an inexpensive technique that identifies unknown solutions, so it is easy to use in practical applications where time and energy are limited.8 In the lab conducted, paper chromatography was used to identify both different color of inks (red, black, and blue), and brand of the ink (BIC Round Stic, Paper Mate, Staples, Pilot Easy Touch, and Pilot V Ball). The variability in the mobile solvent used was exploited in order to maximize the distance of the migration of the ink and be able to distinguish the different brands for the same color from each other. Previously in the first experiment, a 2:1 1-propanol/water solvent was used to separate the components of the six ink samples.3,10 Because all of the ink samples traveled the same distance at a very far distance along the chromatograph paper and 2:1 1-propanol/water is less polar than water, it could be concluded that the dyes were non-polar.3 Using the principle of “like dissolves like”, the assumption can be made that substances of more similar polarities would not travel as far as substances of extremely different polarities.11In order to maximize the distance the components of each ink traveled, using solvents that are more nonpolar will result in the ink traveling a farther distance. Therefore, less polar solvents were used in order to maximize the separation of the individual components of the ink. Because methanol is amphipathic, it was predicted that the individual components of the ink would spread more depending on the polarity of the individual components. Our hypothesis was that less polar solvents would be more effective in separating the individual components of the ink samples into different colors due the nonpolar nature of the inks. Therefore, spreading of the different components shown by different colors along the chromatogram would help distinguish different brands of ink from each other. In order to test the hypothesis and determine the identity of different ink samples, different samples of solvents with different degrees of polarities were used in order to find the best possible solvent mixture.Procedure Following the procedure from the PSU Chemtrek, students determined which solvent systems would be most effective in identifying unknown ink brands.3 Paper Chromatography is an easy and quick way to separate components of a solute, so in order to maximize the amount of samples of different solvent systems in separating the components of ink, a group of two people in the lab each conducted paper chromatography on two different solvent types for each color and brand of ink, 15 in total.9,10 The goal of the lab was to improve the separation of the individual components in each type of ink in order to determine the identity of the ink by comparing known ink Paper Chromatography samples to the unknown ones. Each student was given two similar chromatography papers, each with four different ink dots on it. In order to create a sample chromatogram that successfully separated the components of each ink to differentiate between the different brands, each student then made chromatograms with the fifteen different colored inks and brands (five of each color, each a different brand). A line was drawn across chromatograph paper 0.5 cm from the bottom, and each ink dot was placed at an equal distance away from each other. In order to avoid confusion for the order and identity of the fifteen known inks along the chromatogram, a number system was used where each dot was given a number from 1-15 and then in the lab notebook, their identity was accounted for. On the chromatograms, the order of the inks were:Blue Black Red 1 Pilot VBall BG05 6 112 Paper Mate 7 123 BIC Round Stic 8 134 Pilot Easy Touch 9 145 Staples 1.0 10 15 The chromatograms then needed to be rolled into a cylindrical shape and then stapled so that the end of the two sides were not touching. Different solvents were available to use, and using 1-propanol, ethanol, and 1:1 methanol-ethanol solutions to coat the bottom of each individual petri dish, the two different chromatograms were placed in the petri dish. A plastic cup was then placed over the chromatogram to limit the evaporation of the solvent used. A waiting time period of about 15 minutes was needed to provide ample time for the individual components to either travel or spread depending on their polar characteristics. Once the Paper Chromatography method was finished for each chromatogram, they were taken out of the petri dish and a line was drawn across the “solvent front line”.3 After being set down to dry, they were analyzed in order to determine if the separation of the components of the ink allowed for the distinction of the different types of ink. For the red ink, an ultraviolet light was provided in order to further analyze the type of red ink it was, but our lab group did not utilize it. From comparing the individual chromatograms, it was determine that the 1-propanol solvent better separated the components of the black and blue inks and allowed for the distinction of the different types, and that the 1:1 methanol/ethanol solvent allowed for better separation of the components.3 Therefore, for the two unknown chromatograms given, both solvents were used, one for each chromatogram. The same steps for preparing the chromatogram for the fifteen known inks and colors were then conducted again, this time for the unknown inks. The unknown inks were lettered A-D in order to avoid mistaking the numbers from the previous chromatograms for the unknown chromatogram in the Discussion section. Afterwards, a comparison between the chromatograms with the known identities of the ink and the chromatograms with the unknown was done in order to successfully determine the identity of the inks.3 Results Figure 1: Original Chromatogram with 2:1 1-propanol/water 10This figure is the original chromatogram using the solvent 2:1 1-propanol/water using six inks (#12-16). Figure 2: Chromatogram to Determine Unknown Inks-1:1 Methanol/Ethanol 9Figure 3: Chromatogram to Determine Unknown Inks- 1-propanol10Figure 4: Chromatogram to Determine Unknown Inks-ethanol10 Skewed color because the chromatogram was not placed in the petri dish so that the whole bottom was touching the same amount of solvent. Therefore, the mobile phase did not travel up the paper in a straight vertical line. Figure 5: Unknown Chromatogram-1:1 Methanol/Ethanol10Figure 6: Unknown Chromatogram – 1-propanol10 Skewed color because the chromatogram was not placed in the petri dish so that the whole bottom was touching the same amount of solvent. DiscussionThe goal of the lab was to distinguish between different brands of ink by maximizing the separation of each component of the ink through the experimentation with different solvents as the mobile phase. Because the chromatograms were distinguished depending on the color spread of the ink, finding a solvent that resulted in a variety of distinguishing colors for an ink was important. Using multiple solvents for the unknown chromatograms was done because the polarity of the components of each unknown ink was not known. Therefore, choosing solvents with different polarities was ideal in determining which solvent helped separate the colors within the ink. Therefore, the solvents 1:1 methanol/ethanol, 1-propanol, ethanol, were used to determine which was most effective. As shown in the Results section, the 1:1 methanol/ethanol and 1-propanol were used because they showed the best results.10 The chromatograms successfully showed the separation of each ink into distinct colors that could be characterized to one brand of ink. Both the chromatograms done using the ethanol (Figure 4) and 1-propanol (Figure 6) showed lines of ink migration that were not vertical, but skewed to a side. This probably occurred as a result of the way the chromatogram was stapled on either end, and due to the unleveled ends, not every surface of the bottom received the same amount of the solute. Though the Figure 4 was not beneficial in determining the distance each ink would have migrated, it was used in determining the ineffectiveness of the solvent ethanol in producing distinct colors for each ink. Figure 6 could also still be used in observing the color of the spreading due to the migration of the mobile phase.Before any Paper Chromatography was conducted, an observation of the ink stains on the chromatogram showed that each ink dot for a color looked the same except for the Pilot VBall brand, which was very dark. Therefore, an observation of the unknown dots revealed that A, the red ink, was most likely Pilot VBall because the ink dot was very dark and prominent.10 The chromatograms for the 1:1 methanol/ethanol showed a distinct color separation for the red inks, so the solvent was used to confirm the assumption of the red ink being Pilot VBall. The chromatogram for the 1-propanol showed a distinction for both the blue and black inks in the range and spread of the color, so it was used in order to confirm the identities of the two unknown blue colored and the one unknown black colored inks. From the Snyder Polarity Index, I did expect the 1-propanol solvent to be more effective than the 1:1 methanol/ethanol solvent in separating the individual components of the ink because the polarity of 1-propanol is 4.3 compared to 5.2 for ethanol and 6.6 for methanol12. As stated in the Introduction, the more nonpolar the solvent is, the greater the migration distance for the ink. Therefore, I expected the migration distance and the separation of the 1-propanol to be more prevalent than those of 1:1 methanol/ethanol because the nonpolar components of the ink would migrate a further distance while the polar components would not migrate as much of a distance. Once the unknown chromatograms were completed, it took quite a bit of time in determining the identities of unknown inks B (Blue) and C (Black) because for both inks, two different brands showed similar migration distances and spreading in the colors of the components of the ink. For both colors, both BIC and Paper Mate showed the similar patterns on the chromatograms. Each had minimal color spreading near the bottom, and a heavy dark color near the top.10 For Unknown A-Red, the identity was confirmed to be Pilot VBall because the spreading of the color of the ink started right at the original dot and continued to the top in a orange to pink color range, which was only unique to the Pilot VBall (Figures 2 and 3). The Unknown C- Black was determined to be Staples 1.0 because when comparing it to the chromatogram (Figure 2), #10 resembled the color range and spreading of the ink with minimal spreading near the bottom and a heavy purple-black color near the top. Therefore, the first guess made for the identities was: A Red-Pilot VBall, B Blue-Pilot Easy Touch, C Blue-Staples 1.0, and D Black-BIC. Unknowns B and D were wrong, so it was easy to determine that B was therefore the only other option, BIC, and D was also the only other option, Paper Mate. Conclusion As determined before in a previous experiment, the pen inks observed were nonpolar due their farther distance migration when using less polar solvents. Rather than just relying on their nonpolar qualities, our lab group determined that the individual components of the ink were at different ranges in polarity. Therefore, choosing solvent systems that were different in their polarities would yield different results for the different inks depending on their components. Therefore, the 1-propanol solvent with of 4.3 and a 1:1 methanol/ethanol, polarity index of 5.2 and 6.6 respectively, were used in order to maximize the possible identification of each ink.12 The hypothesis from the beginning of the experiment that the less polar solvents would results in a farther migration distance of the inks was accepted, but an addition to the hypothesis is the possibility of polar solvents allowing for better identification of the unknowns due to individual polarities of the components of the ink being at different polarities. For future experiments, an improvement for identifying the unknowns would be studied to further separate the components of the brands BIC and Paper Mate since the chromatograms showed similar spreading patterns for both brands for both blue and black inks. Also, a further study on natural dyes, as done in the first part of the experiment, could be done so that the polar-nonpolar interactions of Paper Chromatography could be applicable in the biochemical sense. This way, the results from the chromatograms of that experiment would be relevant because food dyes are prevalent in almost every food product. Finally, calculating Rf values for each unknown ink in further experiments could yield quantitative evidence to support whether an ink was most likely a specific brand.3Works Cited1Clark, Jim. "Paper Chromatography." Paper Chromatography. N.p., 2007. Web. 20 Feb. 2013.2"Paper Chromatography." Partnerships for Environmental Education and Rural Health (PEER). Partnership For Environmental Education and Rural Health, n.d. Web. 20 Feb. 2013.3Thompson, Stephen. PSU CHEMTREK: Paper, Thin Layer, and Liquid Chromatography. New Jersey: Hayden McNeil, 2012. Print4"Chromatography." HowStuffWorks. HowStuffWorks, Inc., n.d. Web. 20 Feb. 2013. <"Tank Chromatography.." . 20 Feb 2013 ? ?<, L. G. "Application of Paper Chromatography to Avian Pathology." Poultry Science 3rd ser. 38.668-676 (1959): n. pag. Poultry Science. Poultry Science Association Inc. Web. 13 Feb. 2013. <, Julia R. Chemistry: Atoms First. New York, NY: McGraw-Hill, 2012. Print.8"Uses of Paper Chromatography." TutorVista. , n.d. Web. 13 Feb. 2013. < chromatography>.9Ahmad, Mariam, Chem 113 Laboratory Notebook, pp. 14-15.10 Almarzooqi, Samar, Chem 113 Laboratory Notebook, pp. 14-15. 11"Like Dissolves Like and Molecule Ion Attractions." Vanguard Group, Inc., n.d. Web. 14 Feb. 2013. <"Properties of Solvents on Various Sorbents." HPLC. Kok ChemWare, Web. 21 Feb. 2013. <;. ................
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