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Identifying Solution Components Through Paper Chromatography

Submitted By: Sam Goldstein Group Members: Cody Grace TA Name: Allison Konarske Chemistry 113 Section 102 Date: 02/24/10 Date of Lab: 02/3/11

Introduction: Paper chromatography is a widely used method of identifying compounds in complex mixtures on either small or large scales1. Paper chromatography, referred to as PC, is a convenient method due to its lack of high expenses and its lack of harmful side effects on the environment. Chromatography is one of the most important and widely used analytical practices due to its ease of use and ability to be very accurate. This technology is versatile and can be molded to fit many different experiments.2 High performance liquid chromatography (HPLC) is another method which is more controlled than PC due to being powered by computers and pumps3. When performing HPLC, the same results are desired as in PC, but HPCL is more costly and requires more manpower. A Russian scientist Mikhail Tswett was the first person to develop successful chromatographic techniques to study plant pigments.4 His initial use of chromatography showed that green plants contain more chlorophyll than different colored plants; Tswetts findings visually showed six different types of chlorophyll in green plants.4 Tswett, a student at the time, developed chromatography initially by crushing green leaves into a solution, then mixing the solution with a powder. He noticed that different colors that were initially in the mixed solution separated to different parts of the powder. He found that each different color had a unique polarity5. Paper chromatography is a very unintimidating process due to its clear visual findings and lack of complicated assembly. PC has a few main components: chromatography paper, a vessel, a mobile phase, a stationary phase, and a mixture to separate. The process of PC works by first blotting a small sample of each mixture on the special chromatography paper. The paper is then set in a liquid mobile phase which travels up the paper by capillary action bringing mixture
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components with it selectively due to each components specific polarity. In PC, the liquid mobile phase is non-polar, and the stationary phase is polar. PC separates components of mixtures by their polarities. The more non-polar components are carried by the non-polar mobile phase up a further distance on the paper whereas the more polar components stay closer to the more polar stationary phase, water. Contrary to initial belief, the stationary phase in this case is water, not the paper. Chromatography paper is made up of cellulose which contains a high quantity of hydroxyl groups.6 The hydroxyl groups act in hydrogen bonding with water. This layer covers the paper and acts as the polar stationary phase. Figure 1: Cellulose

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To create the best chromatogram, the goals are producing a chromatographic trial that has large component migration differences and small amounts component spreading. This is accomplished by finding the correct mobile phase that suits the complex mixture. In PC, when deciding on the correct mobile phase, different solutions with different polarities are tested and analyzed for the best results. The desired chromatogram will show clear migration distances with minimal spreading in order to pinpoint the center of each component.

Figure 2 shows how a chromatography trial is set up. Each dot on the starting line is a sample. The paper is set in the liquid mobile phase. Each sample then migrated up the chromatography paper according to their polarity. Paper Chromatography can be used as a forensic method to identify ink, as completed in this experiment, but another way of identifying different inks is done through spectroscopy.8 When this method is completed, each ink is looked at under a spectrophotometer and identified visually. The inks can also be examined under infrared cameras in order to provide more visual evidence of the components.

Figure 3 shows what a sample chromatogram could look like. There are varied distances traveled, component separations, and the final ink shows two components. The sample furthest to the right can clearly be identified due to minimal spreading and concentrated travel distances. This laboratory experiment consisted of many chromatographic trials in order to produce the best fit chromatogram. This chromatogram was then used to identify four unknown inks that were already analyzed. If the chromatogram was done correctly, the unknown ink would appear the same on the unknown chromatogram as it did on the preferred chromatogram. After each trial, the findings were recorded and the polarity of the mobile phase was changed in order to produce a better chromatogram. The best chromatogram shows large differences in component migration and minimal component spreading. The polarity of the mobile phase plays a large part in obtaining the desired results due to the intermolecular forces of the solvent. Spreading occurs when the component is attracted to the mobile phase more than it is to the stationary phase. When changing the polarity of the mobile phase, a balanced attraction between the mobile phase and the stationary phase is desired.

Based off of the base trial where 2:1 propanol / water was the solvent, and each ink traveled the same distance and had a large amount of component spreading, a more polar mobile phase is needed to produce a better fit chromatogram for identifying unknown inks because a more polar mobile phase would show more concentrated areas of components and would show differences in travel distances between samples due to more balanced intermolecular attractions. Procedure: Initially, a base trial is performed to find out the extent of changes needed to be done to the mobile phase. A piece of chromatography paper is prepared by drawing a thin starting line approximately 1.5cm from the bottom of the paper according to given guidelines.6 Fifteen small marks are spaced evenly along the line where each ink is placed. A key is made so that each mark is specific to an ink for easy identification. Once each ink is marked on the paper, the paper is stapled in a ring with a small gap between both ends. The initial mobile phase of 2:1 propanol/water was then prepared and put in a petri dish.6 Next, the stapled chromatography paper is placed in the dish and a cup is placed over the paper promoting capillary action. (The setup of the apparatus and the chromatography paper are shown in Figure 2 and Figure 3 respectively.) After the mobile phase is allowed to travel up the medium for approximately fifteen minutes, or until it nears the top of the paper, it is removed from the liquid mobile phase and set to dry. Once dried, the center of each component on the paper is marked and analyzed for distance traveled. The distance the mobile phase traveled is also recorded. The distance the components and the mobile phase traveled are important to calculating the RF values7. The formula for calculating RF values is below: !"#$%&'( !"#$%&%' !" !! !"#$"%&%' !"#$%&'( !"#$% !" !! !"#$%& !!"#
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!! !"#$% =

After viewing the incoherence of the Base Trial, it is seen that a different mobile phase is needed to produce meaningful results. More trials were then completed until a suitable chromatogram was produced. The following trials were done with mobile phases of methanol, 2:1 water / methanol, 1:1 water / methanol, 2:1 water / propanol respectively. Finally, after finding a suitable chromatogram for unknown identification, trial 4, the same mobile phase of 2:1 water / propanol was used to test the given set of unknowns (set B). The unknown chromatogram was then visually and numerically compared to the chromatogram from trial 4 and to the RF values from trial 4 for conclusiveness. Table 1: Key for Identifying Each Given Pen9 Spot Number Ink Color Pen Name 1 Red 1 Pilot V-Ball 2 Red 2 Pilot EasyTouch 3 Red 3 Staples 4 Red 4 Papermate 5 Red 5 Bic 6 Blue 1 Pilot V-Ball 7 Blue 2 Pilot EasyTouch 8 Blue 3 Staples 9 Blue 4 Papermate 10 Blue 5 Bic 11 Black 1 Pilot V-Ball 12 Black 2 Pilot EasyTouch 13 Black 3 Staples 14 Black 4 Papermate 15 Black 5 Bic Table 3 shows which spot number coordinates to which pen color and specific pen name. Each spot number is marked on the chromatogram under the sample.

Results: The following are sequential images of each trials chromatogram and an explanation of what was noticed about each. All polarities were obtained from the Snyder Polarity Index10
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Figure 4: Base Trial Mobile Phase 2:1 Propanol / Water (Polarity Index: 4, 9)9

This Base Trial, using a mobile phase of 2:1 propanol / water shows identical travel distances for all fifteen samples. Also, for most of the samples, the components spread throughout the trial causing streakiness and making the evidence inconclusive. Figure 5: Trial 1 Mobile Phase Methanol (Polarity Index: 6.6)9

Trial 1, using a mobile phase of methanol, shows more component spreading which makes it harder to differentiate each sample than in the Base Trial. This trial shows no improvement in identifying each sample.

Figure 6: Trial 2 Mobile Phase 2:1 Water / Methanol (Polarity Index: 9, 6.6)

Trial 2, using a mobile phase of 2:1 water / methanol shows minimal component traveling and component spreading only for samples 1, 6, 11. This trial is still inconclusive because most of the samples did not travel far enough to be identifiable. This trial shows different results than the Base Trial, but is still inconclusive. Figure 7: Trial 3 Mobile Phase 1:1 Water / Methanol (Polarity Index: 9, 6.6)

Trial 3, using a mobile phase of 1:1 water / methanol shows a large amount of component traveling and also a large amount of component spreading. Trial 3 is inconclusive because each sample still cannot be differentiated easily.

Figure 8: Trial 4 Mobile Phase 2:1 Water / Propanol (Polarity Index: 9, 4)

Trial 4, using a mobile phase of 2:1 water / propanol proved to be the most conclusive trial. Each sample can be differentiated by either color intensity, combination of colors, streakiness and other purely visual characteristics. Component migrations were different enough to be conclusive and the samples had minimal separation. This is the trial that was used to identify the unknown samples because of the readability and distinguishability between samples. The final procedure adopted is simply to run a PC trial having a mobile phase of 2:1 Water / Propanol.

Table 2: Trial 4 Information9 Distance Traveled Mobile Phase (cm) RF Value (cm) 1 3.1 / 1.9 3.8 0.82 / 0.50 2 3.5 / 2.8 3.8 0.92 / 0.74 3 3.5 3.8 0.92 4 3.5 / 2.6 3.8 0.92 / 0.68 5 3.5 / 2.6 3.8 0.92 / 0.68 6 3.4 3.8 0.89 7 3.5 / 2.2 3.8 0.92 / 0.58 8 3.5 / 2.1 3.8 0.92 / 0.55 9 3.4 3.8 0.89 10 3.4 3.8 0.89 11 3.5 / 2.6 3.8 0.92 / 0.68 12 3.4 3.8 0.89 13 3.4 / 1.7 3.8 0.89 / 0.45 14 3.5 / 3.1 3.8 0.92 / 0.82 15 3.5 / 3.1 3.8 0.92 / 0.82 Table 2 shows information about Trial 4s chromatogram which was used to identify the

Spot Number

unknown samples. The distances traveled are measured from the line where the samples were placed to the most concentrated area of the sample. Some samples show two distances traveled due to two components appearing after separation. The mobile phase was a constant 3.8 cm from the starting line up the paper. An example of calculating an RF value is below:

There are two RF values for multiple samples due to two components appearing after the movement of the mobile phase. Also notice that the RF values have no units because cm/cm cancel each other out.

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Figure 9: Unknown Trial Mobile Phase 2:1 Water / Propanol (Polarity Index: 9, 4)

The given set of unknown ink samples was Set B. All unknowns show two different components after separation. Each component shows a distinct travel distance shown by the dots at the concentrated part of the color. Also, there is very minimal spreading of the components due to using a suitable mobile phase. Table 3: Unknown Trial Information9 Distance Traveled (cm) 3.7 / 2.8 3.9 / 2.4 4.0 / 3.4 4.0 / 2.8 Mobile Phase (cm) 4.2 4.2 4.0 4.0 Unknown Sample Red Pilot V-Ball Blue Pilot Easytouch Black Pilot V-Ball Blue Staples

Spot Number 1 2 3 4

RF Value 0.88 / 0.67 0.93 / 0.57 1.00 / 0.85 1.00 / 0.70

Table 3 shows that the mobile phase varied by 0.2 cm between spots 1 and 2, and spots 3 and 4. This discrepancy is accounted for after calculating the RF value which is a ratio of two distances. This information was then compared to information from Trial 4 to identify the unknowns.

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Discussion: As seen even by the uninformed eye, there are clear differences between the chromatograms of the Base Trial and Trial 4, which was deemed the best for identification use. One notices small areas of concentrated pigment and variations in travel distances in Trial 4 which make each specific sample independent of the others. The one variation between the two trials is a different mobile phase holding all other variables constant. The mobile phase in Trial 4 of 2:1 water / propanol is distinctly more polar than the mobile phase of 2:1 Propanol / Water used in the Base Trial. Now knowing that the stationary phase is polar and the mobile phase is non-polar, the reasons for choosing the makeup of the mobile phase makes sense. Polar compounds attract other polar compounds, and non-polar compounds attract non-polar compounds. In the Base Trial, the samples traveled too far to be analyzed. That fact showed that the mobile phase ran up the paper very quickly causing the samples to also be pulled up the paper very quickly. The goal was to create a sample component movement up the paper so that the pigments in the samples did not smear and spread. By increasing the polarity of the mobile phase, it became more attracted to the already polar stationary phase. This caused the mobile phase to be pulled up the paper by capillary action in Trial 1 more slowly than it did in the base trial. The results show that although the mobile phase was more polar than it was originally, it was not polar enough. In Trial 2 the mobile phases polarity was increased again, but this time the mobile phase moved too slowly up the page causing a few samples to have too little or no movement. By Trial 4, the recipe of 2:1 water / propanol proved to be the right polarity so that the mobile phase traveled up the paper at a moderate speed showing clear visual differences between samples. This

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information supports the original hypothesis that a more polar mobile phase than originally used in the Base Trial was needed to create a useful chromatogram. The speed at which the mobile phase traveled up the chromatography paper is directly related to the polarity of the solvent. A more polar solvent is more attracted to the stationary phase, therefore will travel slowly up the page compared to a non-polar mobile phase. The balance of intermolecular forces was the main factor in finding the correct mobile phase. In identifying the unknown samples, the mobile phase of 2:1 water / propanol was selected as used in Trial 4. The unknowns were found by comparing visual evidence between the chromatograms from Trial 4 and the Unknown Trial. Since both trials used the same mobile phase, their visual qualities were comparable. After initial conclusions were made purely based off of visual evidence from each chromatogram, RF values were compared for similarities. Unknown spot numbers 2 and 4 had very similar RF values to the initial guesses, which ended up being the correct answers. Unknown spot numbers 1 and 3 showed one very similar RF value to Trial 4, which was enough evidence to make a sound conclusion of what the correct unknown sample was. The correct results for what the unknown samples ended up being are in Figure 11. Unknown spot 1 had both a red and a yellow component as did four of the five red known inks. The differentiating factor was that only one sample did not travel as far as the mobile phase in Trial 4, and this unknown had the same characteristic. This factor clearly labeled unknown 1 as the Red Pilot V-Ball. Unknown spot 2 showed two clear components and a large amount of spreading. When compared with Trial 4, both spots 7 and 8 showed similar visual results. After comparing the RF

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values, spot seven was the best choice of being the same sample. The findings proved correct and unknown 2 was the Blue Pilot Easytouch. Unknown spot 3 showed two components, one black and one yellow. This unknown was easily visually observed because only one sample, spot 11 also showed these colors. Spot 11 turned out to be the Black Pilot V-Ball which was the correct identification of Unknown 3. Unknown spot 4 was similar to unknown spot 2 in that there was a large amount of spreading and two showing components. Spots 7 and 8 on trial 4 were very similar to this unknown spot, but spot seven was already used. Spot 8 turned out to be the correct unknown which was the Blue Staples pen. Conclusion: In this version of Paper Chromatography, the polarity of the mobile phase proved to be the deciding factor in producing a well-designed, useful chromatogram for identification purposes. Numerical data such as RF values are also conclusive in providing answers once a chromatogram is produced that has minimal component spreading and variations in component travel distances. After running a trial on unknown samples with a mobile phase previously used in a trial with known samples, the two chromatograms can be studied to reveal the unknowns. This procedure proved that based off of a base trial where each ink sample traveled the same distance and where there was a large amount of separation inside each component, a more polar mobile phase must be used to make a better chromatogram for identifying unknown inks. For future study, PC techniques can be used to test urine samples for different drugs. There are blood tests that are used today as drug tests, but Paper Chromatography is a very inexpensive, resourceful alternative method of identifying components in a solution. Instead of spending
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money on machines that take up space and require maintenance, Paper Chromatography could be used and would diminish money spent and space taken up by equipment used today. Following up on this experiment, the only way to improve results could be to have more accurate solvent combinations and a more consistent way of blotting the samples on the chromatography paper. Both of those changes would produce more consistent, accurate results.

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Bibliography: 1) Eigsti, Nicholas W. "Paper Chromatography for Student Research." The American Biology Teacher 29.2 (1967): 123-34. Print. 2) Scott, Raymond. "The Importance of Chromatography as an Analytical Technique from Quantitative Chromatographic Analysis." Chromatography as an Analytical Technique. Web. 16 Feb. 2011. http://www.chromatography-online.org/quant/HistoricalIntroduction/Importance.html 3) http://www.buzzle.com/articles/types-of-chromatography.html 4) Issaq, Haleem J. A Century of Separation Science. New York: Marcel Dekker, 2002. Print. 5) http://galileo.phys.virginia.edu/outreach/8thGradeSOL/Chromatography.htm 6) Thompson, Stephen. The Chemistry of Natural Waters. In PSU Chemtrek; Hayden-McNeil Publishing: USA, 2010; pgs 17-2, 17-21.

7) Beaker with Stationary and Mobile Phases. Digital image. Santa Monica College. Web. 16 Feb. 2011. <http://homepage.smc.edu/walker_muriel/chromatography_of_gel_ink_procedure.htm>. 8) Kelley, J. D., and A. A. Cantu. "Proposed Standard Methods for Ink Identification." US National Library of Medicine and National Insitute of Health. Jan. 1975. Web. 16 Feb. 2011. <http://www.ncbi.nlm.nih.gov/pubmed/1141146>. 9) Goldstein, Sam. "Chromatography Part 2." Chemistry 113 Notebook (2011): 12-15. Print.
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10) "HPLC - Solvent Properties." SanderKok. Chemware. Web. 16 Feb. 2011. <http://www.sanderkok.com/techniques/hplc/eluotropic_series_extended.html>.

http://www.jstor.org/stable/4441625?&Search=yes&searchText=chromatography&searchText=paper&list=hide& searchUri=%2Faction%2FdoAdvancedSearch%3Fq0%3Dpaper%2Bchromatography%26f0%3Dall%26c1%3DAND%2 6q1%3D%26f1%3Dall%26acc%3Don%26wc%3Don%26Search%3DSearch%26sd%3D%26ed%3D%26la%3D%26jo%3 D&prevSearch=&item=9&ttl=37048&returnArticleService=showFullText 2 http://www.chromatography-online.org/quant/Historical-Introduction/Importance.html

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