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

This technology is versatile and can be molded to fit many different experiments.Introduction: Paper chromatography is a widely used method of identifying compounds in complex mixtures on either small or large scales1. Paper chromatography. When performing HPLC. Paper chromatography is a very unintimidating process due to its clear visual findings and lack of complicated assembly. a mobile phase. A Russian scientist Mikhail Tswett was the first person to develop successful chromatographic techniques to study plant pigments. is a convenient method due to its lack of high expenses and its lack of harmful side effects on the environment.2 High performance liquid chromatography (HPLC) is another method which is more controlled than PC due to being powered by computers and pumps3.4 His initial use of chromatography showed that green plants contain more chlorophyll than different colored plants. Tswett’s findings visually showed six different types of chlorophyll in green plants. a stationary phase. a student at the time. He noticed that different colors that were initially in the mixed solution separated to different parts of the powder. but HPCL is more costly and requires more manpower. and a mixture to separate. the same results are desired as in PC. The paper is then set in a liquid mobile phase which travels up the paper by capillary action bringing mixture 1     . developed chromatography initially by crushing green leaves into a solution. PC has a few main components: chromatography paper. The process of PC works by first blotting a small sample of each mixture on the special chromatography paper. He found that each different color had a unique polarity5. Chromatography is one of the most important and widely used analytical practices due to its ease of use and ability to be very accurate. then mixing the solution with a powder. a vessel. referred to as PC.4 Tswett.

Figure 1: Cellulose Chemtrek6 To create the best chromatogram. In PC. PC separates components of mixtures by their polarities. The desired chromatogram will show clear migration distances with minimal spreading in order to pinpoint the center of each component. when deciding on the correct mobile phase. 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. not the paper. 2     . In PC.components with it selectively due to each component’s specific polarity. Contrary to initial belief.6 The hydroxyl groups act in hydrogen bonding with water. Chromatography paper is made up of cellulose which contains a high quantity of hydroxyl groups. water. the goals are producing a chromatographic trial that has large component migration differences and small amounts component spreading. the stationary phase in this case is water. different solutions with different polarities are tested and analyzed for the best results. the liquid mobile phase is non-polar. This is accomplished by finding the correct mobile phase that suits the complex mixture. and the stationary phase is polar. This layer covers the paper and acts as the polar stationary phase.

Each dot on the starting line is a sample. 3     . as completed in this experiment. Paper Chromatography can be used as a forensic method to identify ink. Each sample then migrated up the chromatography paper according to their polarity. The paper is set in the liquid mobile phase. The inks can also be examined under infrared cameras in order to provide more visual evidence of the components.8 When this method is completed. but another way of identifying different inks is done through spectroscopy.Figure 2 shows how a chromatography trial is set up. each ink is looked at under a spectrophotometer and identified visually.

The best chromatogram shows large differences in component migration and minimal component spreading. Spreading occurs when the component is attracted to the mobile phase more than it is to the stationary phase. There are varied distances traveled. This laboratory experiment consisted of many chromatographic trials in order to produce the best fit chromatogram. The polarity of the mobile phase plays a large part in obtaining the desired results due to the intermolecular forces of the solvent. component separations. This chromatogram was then used to identify four unknown inks that were already analyzed. the findings were recorded and the polarity of the mobile phase was changed in order to produce a better chromatogram. 4     .Figure 3 shows what a sample chromatogram could look like. a balanced attraction between the mobile phase and the stationary phase is desired. After each trial. The sample furthest to the right can clearly be identified due to minimal spreading and concentrated travel distances. When changing the polarity of the mobile phase. and the final ink shows two components. the unknown ink would appear the same on the unknown chromatogram as it did on the preferred chromatogram. If the chromatogram was done correctly.

(The setup of the apparatus and the chromatography paper are shown in Figure 2 and Figure 3 respectively. A piece of chromatography paper is prepared by drawing a thin starting line approximately 1. a base trial is performed to find out the extent of changes needed to be done to the mobile phase. A key is made so that each mark is specific to an ink for easy identification.6 Next. The distance the components and the mobile phase traveled are important to calculating the RF values7. or until it nears the top of the paper.5cm from the bottom of the paper according to given guidelines. the paper is stapled in a ring with a small gap between both ends. 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. The initial mobile phase of 2:1 propanol/water was then prepared and put in a petri dish. the stapled chromatography paper is placed in the dish and a cup is placed over the paper promoting capillary action. it is removed from the liquid mobile phase and set to dry. Once dried. and each ink traveled the same distance and had a large amount of component spreading. Once each ink is marked on the paper.Based off of the base trial where 2:1 propanol / water was the solvent.6 Fifteen small marks are spaced evenly along the line where each ink is placed.) After the mobile phase is allowed to travel up the medium for approximately fifteen minutes. 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 formula for calculating RF values is below: !"#$%&'(  !"#$%&%'  !"  !ℎ!  !"#$"%&%'   !"#$%&'(  !"#$%  !"  !ℎ!  !"#$%&  !ℎ!"# 5     !!  !"#$% = . Procedure: Initially.

2:1 water / propanol respectively. the same mobile phase of 2:1 water / propanol was used to test the given set of unknowns (set B). 1:1 water / methanol. The following trials were done with mobile phases of methanol. it is seen that a different mobile phase is needed to produce meaningful results.After viewing the incoherence of the Base Trial. trial 4. All polarities were obtained from the Snyder Polarity Index10 6     . Results: The following are sequential images of each trial’s chromatogram and an explanation of what was noticed about each. 2:1 water / methanol. Each spot number is marked on the chromatogram under the sample. after finding a suitable chromatogram for unknown identification. Finally. More trials were then completed until a suitable chromatogram was produced. 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. 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.

Also. using a mobile phase of methanol. the components spread throughout the trial causing streakiness and making the evidence inconclusive. using a mobile phase of 2:1 propanol / water shows identical travel distances for all fifteen samples.Figure 4: Base Trial – Mobile Phase 2:1 Propanol / Water (Polarity Index: 4. This trial shows no improvement in identifying each sample. shows more component spreading which makes it harder to differentiate each sample than in the Base Trial. Figure 5: Trial 1 – Mobile Phase Methanol (Polarity Index: 6. for most of the samples. 7     .6)9 Trial 1. 9)9 This Base Trial.

6. 11. using a mobile phase of 2:1 water / methanol shows minimal component traveling and component spreading only for samples 1. 6.Figure 6: Trial 2 – Mobile Phase 2:1 Water / Methanol (Polarity Index: 9.6) Trial 3. but is still inconclusive.6) Trial 2. Trial 3 is inconclusive because each sample still cannot be differentiated easily. Figure 7: Trial 3 – Mobile Phase 1:1 Water / Methanol (Polarity Index: 9. This trial shows different results than the Base Trial. This trial is still inconclusive because most of the samples did not travel far enough to be identifiable. using a mobile phase of 1:1 water / methanol shows a large amount of component traveling and also a large amount of component spreading. 6. 8     .

9     .Figure 8: Trial 4 – Mobile Phase 2:1 Water / Propanol (Polarity Index: 9. This is the trial that was used to identify the unknown samples because of the readability and distinguishability between samples. Component migrations were different enough to be conclusive and the samples had minimal separation. streakiness and other purely visual characteristics. The final procedure adopted is simply to run a PC trial having a mobile phase of 2:1 Water / Propanol. using a mobile phase of 2:1 water / propanol proved to be the most conclusive trial. 4) Trial 4. combination of colors. Each sample can be differentiated by either color intensity.

8 0.4 3.5 / 2.89 13 3.5 3.50 2 3.Table 2: Trial 4 Information9 Distance Traveled Mobile Phase (cm) RF Value (cm) 1 3.92 / 0.68 5 3.89 11 3.82 Table 2 shows information about Trial 4’s chromatogram which was used to identify the Spot Number unknown samples.8 0.2 3.89 10 3.8 0. The distances traveled are measured from the line where the samples were placed to the most concentrated area of the sample.4 3.8 0. 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.89 / 0.8 0.8 3.68 12 3.68 6 3.6 3.5 / 3.6 3.8 0. 10     .8 0.8 0. Also notice that the RF values have no units because cm/cm cancel each other out.5 / 2.82 / 0.6 3.8 0.1 3.5 / 3.9 3.74 3 3.7 3. The mobile phase was a constant 3.89 7 3. Some samples show two distances traveled due to two components appearing after separation.92 / 0.4 3.8 0.8 cm from the starting line up the paper.92 / 0.92 / 0.5 / 2.92 4 3.92 / 0.58 8 3.5 / 2.8 0.5 / 2.82 15 3.8 0.1 3.8 0.92 / 0.4 / 1.1 / 1.92 / 0.55 9 3.8 0.8 0.45 14 3.5 / 2.92 / 0.1 3.4 3.

and spots 3 and 4.88 / 0. Also.00 / 0.70 Table 3 shows that the mobile phase varied by 0.9 / 2.0 / 2.2 cm between spots 1 and 2.67 0.0 Unknown Sample Red Pilot V-Ball Blue Pilot Easytouch Black Pilot V-Ball Blue Staples Spot Number 1 2 3 4 RF Value 0. This discrepancy is accounted for after calculating the RF value which is a ratio of two distances. All unknowns show two different components after separation.Figure 9: Unknown Trial – Mobile Phase 2:1 Water / Propanol (Polarity Index: 9.4 4.8 3.4 4.57 1.2 4. there is very minimal spreading of the components due to using a suitable mobile phase.8 Mobile Phase (cm) 4. This information was then compared to information from Trial 4 to identify the unknowns. Table 3: Unknown Trial Information9 Distance Traveled (cm) 3.00 / 0.7 / 2. 11     .0 4.85 1.2 4.93 / 0. Each component shows a distinct travel distance shown by the dots at the concentrated part of the color.0 / 3. 4) The given set of unknown ink samples was Set B.

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 reasons for choosing the makeup of the mobile phase makes sense. and non-polar compounds attract non-polar compounds. which was deemed the best for identification use. This 12     . it became more attracted to the already polar stationary phase. 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. Polar compounds attract other polar compounds. it was not polar enough. 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 results show that although the mobile phase was more polar than it was originally. The goal was to create a sample component movement up the paper so that the pigments in the samples did not smear and spread. there are clear differences between the chromatograms of the Base Trial and Trial 4. 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 samples traveled too far to be analyzed. 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. By increasing the polarity of the mobile phase. In Trial 2 the mobile phase’s polarity was increased again. In the Base Trial.Discussion: As seen even by the uninformed eye. Now knowing that the stationary phase is polar and the mobile phase is non-polar. 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.

After initial conclusions were made purely based off of visual evidence from each chromatogram. therefore will travel slowly up the page compared to a non-polar mobile phase. This factor clearly labeled unknown 1 as the Red Pilot V-Ball. the mobile phase of 2:1 water / propanol was selected as used in Trial 4. Unknown spot 1 had both a red and a yellow component as did four of the five red known inks. When compared with Trial 4. both spots 7 and 8 showed similar visual results. which was enough evidence to make a sound conclusion of what the correct unknown sample was. A more polar solvent is more attracted to the stationary phase. RF values were compared for similarities. and this unknown had the same characteristic. which ended up being the correct answers. Unknown spot 2 showed two clear components and a large amount of spreading.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 correct results for what the unknown samples ended up being are in Figure 11. Unknown spot numbers 2 and 4 had very similar RF values to the initial guesses. Unknown spot numbers 1 and 3 showed one very similar RF value to Trial 4. The speed at which the mobile phase traveled up the chromatography paper is directly related to the polarity of the solvent. In identifying the unknown samples. The balance of intermolecular forces was the main factor in finding the correct mobile phase. their visual qualities were comparable. Since both trials used the same mobile phase. After comparing the RF 13     . The unknowns were found by comparing visual evidence between the chromatograms from Trial 4 and the Unknown Trial. The differentiating factor was that only one sample did not travel as far as the mobile phase in Trial 4.

This unknown was easily visually observed because only one sample. Spots 7 and 8 on trial 4 were very similar to this unknown spot. one black and one yellow. There are blood tests that are used today as drug tests. For future study. spot seven was the best choice of being the same sample. After running a trial on unknown samples with a mobile phase previously used in a trial with known samples. spot 11 also showed these colors. resourceful alternative method of identifying components in a solution. the polarity of the mobile phase proved to be the deciding factor in producing a well-designed. 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. Unknown spot 3 showed two components. 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. PC techniques can be used to test urine samples for different drugs. The findings proved correct and unknown 2 was the Blue Pilot Easytouch. but Paper Chromatography is a very inexpensive. Spot 11 turned out to be the Black Pilot V-Ball which was the correct identification of Unknown 3. Instead of spending 14     . Spot 8 turned out to be the correct unknown which was the Blue Staples pen.values. the two chromatograms can be studied to reveal the unknowns. a more polar mobile phase must be used to make a better chromatogram for identifying unknown inks. Unknown spot 4 was similar to unknown spot 2 in that there was a large amount of spreading and two showing components. Conclusion: In this version of Paper Chromatography. but spot seven was already used. useful chromatogram for identification purposes.

accurate on machines that take up space and require maintenance. Following up on this experiment. Paper Chromatography could be used and would diminish money spent and space taken up by equipment used today. Both of those changes would produce more consistent. 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. 15     .

" US National Library of Medicine and National Insitute of Health. Cantu. 16     . 9) Goldstein.ncbi. Web. 7) Beaker with Stationary and Mobile Phases. 8) Kelley. 16 Feb.2 (1967): 123-34. 5) http://galileo. Stephen.smc. <http://www." The American Biology Teacher 29. A. <http://homepage. 2010. 16 Feb. Santa Monica College. J. The Chemistry of Natural Waters. "The Importance of Chromatography as an Analytical Technique from Quantitative Chromatographic Analysis. "Proposed Standard Methods for Ink Identification. 2011.virginia. Web. A Century of Separation Science.. Raymond. Nicholas W. Print. Jan. 16 "Chromatography Part 2. Print.buzzle.chromatography-online. Digital 17-21.Bibliography: 1) Eigsti." Chemistry 113 Notebook (2011): 12-15.htm>. Web.phys.html 4) Issaq. 2011. Haleem J. http://www. 2)>. 2002. New York: Marcel Dekker.nih. "Paper Chromatography for Student Research. and A.htm 6) Thompson.html 3) http://www. 2011. D. Sam. pgs 17-2." Chromatography as an Analytical Technique. In PSU Chemtrek. Hayden-McNeil Publishing: USA.

html   17     ." SanderKok. 16­‐Introduction/Importance.10) "HPLC . <http://www.Solvent Properties. 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.sanderkok.html>.                                                                                                                         1 http://www. Chemware.chromatography-­‐online.

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