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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 mixture1

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


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: !"#$%&'( !"#$%&%' !" !! !"#$"%&%' !"#$%&'( !"#$% !" !! !"#$%& !!"#5

!! !"#$% =

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 Index106

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