Brian Gross Effects of the Polarity of the Mobile Phase in Paper Chromatography Penn State Chemistry 113 Section 103 Group Members: Molly Grove, Connor Donley, Katie Del Guercio, Shahkila Daniels TA: Ally Marianelli February 27, 2014
Introduction Chromatography is a process used to separate a mixture or solution into its components 1
using a mobile phase and a stationary phase. The name chromatography, translated from the Greek roots chroma and chroma and graphein graphein,, means “to write with colors”. The process was developed in 1903 by Mikhail Tswett, a Russian botanist who separated plant pigments using a column of 2
calcium carbonate. This specific method of separation became known as column chromatography. Since then, many other forms of chromatography have emerged. In 1941, British chemists Archer J.P. Martin and Richard L.M. Synge dev eloped a process called partition 3
chromatography in their study of the amino acid composition of wool. Soon after, Martin and his coworkers sought to develop a process with simpler, more replicable results. They revised their method, using a sheet of filter paper pap er as the stationary medium to separate the amino acids in wool. This became known as paper chromatography, and in 1952, Martin and Synge earned a 3
Nobel Prize for their work on the subject.
Paper chromatography, like all other forms of chromatograph y, involves the separation of a mixture or solution into its components using a mobile phase phase and a stationary phase. In paper chromatography, the stationary phase is a porous filter paper and the mobile phase is a solvent mixture. The mixture or solution that that is to be separated separated is applied to a small small spot near the edge of the paper. That edge is then placed in the solvent mixture, and the mobile solvent phase is 1
pulled through the paper by capillary action. The components of the test mixture mixture move with the the solvent up the paper. Separation of the test mixture occurs based on the relative solubilities solubilities of its components. The chromatography paper contains cellulose, making making it a very polar stationary stationary phase. Because of this, more polar components are more soluble in the paper and do not travel 4
as far as non-polar components, which do not bond as easily to the paper.
The polarity of the mobile phase plays pla ys a key role in how far the components travel as well. The more polar the solvent is, is, the shorter distance distance the components of the test sample will 5
travel because the solvent itself has an affinity for the chromatography paper.
Paper chromatography has a variety of uses that stem into a wide range of fields. fields. Its original use was to separate amino acids from a protein, but many other applications have since been discovered. One major use is in the forensic science field. Paper chromatography can be used for small samples of DNA found at crime sce nes to create a DNA fingerprint, which aids 6
forensic scientists in identifying potential criminals. In pathology, paper chromatography can 7
be used to detect alcohol, narcotics, hallucinogens, aspirin, and other chemicals in the blood.
Medical companies use paper chromatography to obtain pure compounds for use in developing 6
medicine.
One of the most common uses of paper p aper chromatography, the one used in this lab, is the separation of colored colored pigments. Specifically, it was was used to separate the inks of a pen. However, there are other processes for doing this. One such method is capillary electrophoresis. In this process, ink samples are placed into a porous gel, and a current is run through the gel. The current causes the inks to move through the gel, and components of the ink become separated based on their relative sizes. Larger components do not travel as far as the smaller components. This creates a “fingerprint” for the ink mixture, which can be used as a key for identifying other 8
ink mixtures. Another process, called thin layer layer chromatography, is similar to paper chromatography, except a very thin layer of material is placed on top of a rigid support, such as glass or plastic. Samples are applied as in in paper chromatography and the sample is placed in in a developing chamber. When the sample is finished, the component separations can be analyzed
1
and compared under an ultraviolet light. While these two methods can be used to separate 8
mixtures, paper chromatography is cheap, simple, and quick, and is often equally e qually effective. In our experiment, we were given a sheet of chromatography paper with 5 out of 15 mystery pens spotted on the paper. Our goal was to identify the mystery pens by creating
reference chromatograms suitable for distinguishing between each of the pe ns by varying the solvent mixture. In a previous experiment, we used a 2:1 propanol to water mixture mixture as the solvent, and the nonpolar components of the pen inks were not separated. Since this solvent did not separate the nonpolar components, we hypothesized that a higher polarity solvent would be needed. By using a more polar mixture, we anticipated that the the mobile phase would have a higher affinity for the stationary phase, so both the polar and nonpolar components compo nents of the ink would separate within the length of the chromatogram. Procedure The procedure for the experiment our group developed was partially adopted from PS U 1
Chemtrek and can be found in greater detail there. We were tasked with designing an experiment to identify unknown pens. This stemmed from a previous experiment, which I will refer to as the original experiment. In the original experiment, we had created a chromatogram by placing small dots from a variety of pens onto a piece of chromatography paper. Refer to Table 1 for the dotting order for the pens in the the original run. The chromatogram was placed in a solution of 2:1 1-propanol:water, covered with a pl astic cup, and allowed to run until the solvent front approached the top edge of the paper. However, the original run did did not effectively separate the inks in the pen, and we were tasked with developing a procedure to separate these inks in order to identify mystery pens.
The first step was to create and test various solvent combinations to see if they could effectively separate the inks of the pens. The substances available for use were propanol, isopropanol, ethanol, methanol, and water. Each substance had a different polarity, polarity, as indicated by the Snyder Polarity Index, which ranks the polarity of compounds on a scale of 0-9. For the polarities of the compounds available to us, see Table 5. Substances were combined to create solvents of varying polarities. polarities. Each combination created was used as the solvent for a different different chromatogram, generating a key which for potential comparison on the mystery runs. The chromatograms were created by placing a sample dot from each of 15 pens near the bottom edge of a piece of chromatography paper and placing that edge in the solvent solution. solution. For a list of the pens used, see Table 3. The solvents were allowed to ascend the paper via capillary action and the pen inks were separated into their respective components. Solvents were tested until 2 were found that were thought to achieve good separation and thought to be good for distinguishing distinguishing between the pens. After finding these combinations, each group member was given two pieces of chromatography paper. Group members received different combinations of pens, but each individual was given 2 chromatograms with the same sequence of 5 pen spots. One chromatogram was run through each of the 2 selected solvents and compared to the key that had been created for for it. Based on the ink compositions determined from the mystery test and our comparisons, our task was to identify which mystery pen was used to create each of the 5 mystery spots.
Results
Table 1: Pen Layout for Brian’s Original Run Spot #
12
13
14
9
15
16
17
Papermate Papermate Papermate BIC Blue BIC Red BIC Black Blue Red Black *All numbering for the chromatogram spots increases from left to right (i.e. P en #1 is the farthest to the left, pen #15 is the farthest to the right) Pen
**Pen layouts for chromatograms that were made by group members that did not factor into the mystery runs are not listed.
Figure 1: Brian’s Original Run - 2:1 1-propanol:Water – 1-propanol:Water – Snyder Snyder Polarity of 5.87
Table 2: Mobile Phases Tested by Group Member Group Member
Test 1 Solvent
Test 2 Solvent
9
Test 3 Solvent
Brian Gross
2:1 Methanol:Water
4:1 Methanol:Water
3:2 Methanol:Water
Connor Donley
1:1 Methanol:Water
1:1 Water:Ethanol
Katie Del Guercio
2:1 Water:Methanol
2:1:1 Water:Propanol:Methanol
Molly Grove
1:1 Isopropanol:Water
3:1 Water:Isopropanol
N/A
Shahkila Daniels
9:1 Methanol:Ethanol
2:1 Water:Propanol
N/A
N/A N/A
Table 3: 3: Pen Layout for Brian’s First and Second Test Runs
9
1
Pilot Vball Black
6
Pilot Vball Blue
11
Pilot Vball Red
2
Papermate Black
7
Papermate Blue
12
Papermate Red
3
BIC Black
8
BIC Blue
13
BIC Red
4
Pilot Easytouch Black
9
Pilot Easytouch Blue
14
Pilot Easytouch Red
5
Staples Black
10
Staples Blue
15
Staples Red
Figure 2: 2: Brian’s First Test Run – 2:1 2:1 Methanol:Water – Methanol:Water – Snyder Snyder Polarity of 7.4
9
Figure 3: 3: Connor’s First First Test Run – 1:1 1:1 Methanol:Water – Methanol:Water – Snyder Snyder Polarity of 7.8
10
Figure 4: 4: Katie’s First First Test Run – 2:1 2:1 Water:Methanol – Water:Methanol – Snyder Snyder Polarity of 8.2
11
Figure 5: 5: Molly’s First First Test Run – 1:1 1:1 Isopropanol:Water – Isopropanol:Water – Snyder Snyder Polarity of 6.65
12
13
Pen layouts for Figure 6 can be found in Shahkila’s Laboratory Notebook
Figure 6: 6: Shahkila’s Shahkila’s First Test Run - 9:1 Methanol:Ethanol – Methanol:Ethanol – Snyder Snyder Polarity of 6.46
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Figure 7: 7: Brian’s Second Test Run - 4:1 Methanol:Water – Methanol:Water – Snyder Snyder Polarity of 7.08
Figure 8: 8: Connor’s Second Test Run – 1:1 Water:Ethanol – Water:Ethanol – Snyder Snyder Polarity of 7.1
9
10
Figure 9: Katie’s Second Test Run – 2:1:1 2:1:1 Water:Propanol:Methanol – Water:Propanol:Methanol – Snyder Snyder Polarity of 7.23
11
Figure 10: 10: Molly’s Second Test Run – 3:1 3:1 Water:Isopropanol – Water:Isopropanol – Snyder Snyder Polarity of 7.8
12
Figure 11: Shahkila’s Second Test Run – 2:1 Water:Propanol – Water:Propanol – Snyder Snyder Polarity of 7.43
Table 4: 4: Pen Layout for Brian’s Third Test Run
13
9
1
Staples Red
6
Pilot Easytouch Blue
11
Pilot Vball Black
2
Pilot Easytouch Red
7
Pilot Vball Blue
12
Pilot Easytouch Black
3
Papermate Red
8
Staples Blue
13
Papermate Black
4
Pilot Vball Red
9
BIC Blue
14
Staples Black
5
BIC Red
10
Papermate Blue
15
BIC Black
Figure 12: 12: Brian’s Third Test Run – 3:2 3:2 Methanol:Water – Methanol:Water – Snyder Snyder Polarity of 7.56
Figure 13: Mystery Run #1 – #1 – Using Using a 3:2 Methanol:Water Solvent
9
Figure 14: Mystery Run #2 – #2 – Using Using a 9:1 Methanol:Ethanol Solvent
9
9
Table 5: Snyder Polarity Index Values for Available Compounds Solvent
9
Polarity Index
1-propanol
4.3
Isopropanol
4.3
Ethanol
5.2
Methanol
6.6
Water
9 The polarity of a solvent can be considered as an average of the Snyder Polarity Index value of its components. Sample Solvent Polarity Calculations: Polarity2:1:1 Water:Propanol:Methanol = [2(9) + 1(4.3) + 1(6.6)]/4 = (28.9)/4 = 7.23 Polarity3:2 Methanol:Water = = [3(6.6) + 2(9)]/5 = (19.8 + 18)/5 = 37.8/5 = 7.56 Discussion Before we started the experiment, our group concluded that a more polar solvent would be needed to separate the inks in each of the pens. In the original experiment, 2:1 1 propanol:water was used as the solvent to create the chromatogram. However, this chromatogram, which had a Snyder Polarity Index value of 5.87, was ineffective and the inks from the pens ran all all the way up the chromatography paper. This meant that the polarity of the solvent was too low because it it was not binding to the polar chromatography paper. As a result, the nonpolar dyes ran all the way to the solvent front, and they were not separated. For our group’s solvent mixtures, we decided a solvent with a polarity higher than 5.8 on the Snyder Index would be necessary to separate the inks and allow us to distinguish between the pens. For my first solvent, I made a mixture that was 2 parts methanol methanol to one part water, resulting in a Snyder Polarity Index value of 7.4. However, the inks from the pens still ran all the way to the solvent front. There was some visible separation in the different pens, but the
results were not definitive enough (see Figure 2). There were not enough distinctive characteristics to tell tell apart the pens. While there was a small small amount of spread between components, there was not as much as we would have liked. One positive aspect of this this test was that it was somewhat effective at distinguishing between the bla ck and blue pens, a feature that the other first-run first-run chromatograms did not accomplish very well. We determined a polarity higher than 7.4 would be needed for the next runs. Connor, Katie, and Molly experienced similar results on their initial runs, and they also determined a new solvent combination would be necessary. Shahkila’s first chromatogram, on the other hand, had a favorable result. Despite having a relatively low low polarity of 6.46, her first first run produced a chromatogram that provided a pleasant amount of detail in telling apart the red pens, and a moderate amount of detail for the blues and blacks. Different shades of pink and tails of orange and yellow appeared from the red pens on the chromatogram, and our group decided the 6.46 Snyder Polarity Index solvent would be valuable for use in the unknown tests. Despite the majority of our group having a failure to produce a solvent for use in the mystery tests, we were able to successfully determine that our hypothesis was correct: correct: a higher polarity solvent than that of the original experiment would be needed to separate the components of the pens. For my second test, I tried to create a solvent with a Snyder Polarity Index value higher than 7.4. To accomplish this, I did a qualitative analysis (I did not make any formal formal calculations), and I determined a 4:1 methanol:water solvent would have a higher polarity than my previous run. However, I made made a mistake in in my analysis. Using more methanol for every part of water decreased the Snyder Polarity Index value from 7.4 to 7.08. A better test would have been decreasing the amount of methanol for every part of water, as this would have
increased the solvent’s polarity. polarity. I did not notice this mistake until after the second the second chromatogram was finished and I examined the results. My second test was not nearly as effective as the first one in distinguishing between the black and blue pens, although it was a little better at telling the reds apart (see Figure 7). Unfortunately, we already had a test that we figured would be sufficient to differentiate between red pens, so we did not use this 7.08 mixture in our final runs. After everyone completed 2 tests, our group decid ed definitively that Shahkila’s first solvent mixture would be used in our mystery runs due to its ability to discern the red pens. Despite having run 8 total chromatograms, we were still not confident in our previous tests’ ability to differentiate differentiate between the black and blue pens. We decided my first run, run, with a Snyder Polarity value of 7.4, did not separate the inks enough, so a more polar solvent would be necessary. Fortunately, Connor’s first run provided us with an upper boundary. He used a solvent mixture of 2 parts water to 1 part pa rt methanol, and we determined dete rmined the inks did not travel far enough up the chromatography paper, meaning the polarity of the solvent, 7.8, was too high (see Figure 3). We decided a solvent polarity of approximately approximately 7.6 would be sufficient to tell apart the blacks and blues. rd
I ran a 3 test using a solvent that was 3 parts methanol to 2 parts water to obtain ob tain a Snyder Index value of 7.56, which was very close to our target value of 7.6. This test yielded yielded favorable results. There were visible differences differences in the dye compositions of the black and blue pens, including yellow tails on the black streaks and noticeable shading variances among the blues (see Figure 12). Due to these advantages, we decided this solvent, combined with Shahkila’s first run for differentiating between differentiating between the red pens, would be our second combination for use in running the unknown pens.
After receiving my two mystery chromatograms, I ran one in the 3:2 methanol:water solvent (Figure 13) and the other in the 9:1 methanol:ethanol solvent (Figure (Figure 14). I called the 7.56 Snyder Polarity Index mixture “mystery “m ystery test 1” and the 6.46 unknown test “mystery test 2”. I compared these results to the keys created for each solvent combination (Figures 12 and 6 respectively). I noticed the first dot was clearly Pilot Vball black from from mystery test 1 due to its characteristic black color after comparison with the key. I was also able to determine the fourth dot shared a resemblance with the sixth dot on the key: key: they were the same color and they they were both characterized by a more dense coloration close to the original dot and a lighter blue coloring near the solvent front with with a gap between the two zones. From this analysis, I determined dot four was Pilot Easytouch Easytouch blue. My mystery test 1 was not as effective effective as I had hoped in nd
determining the identity of the 2 dot, which was either black or blue. I noticed a slight yellow yellow coloring at the top of the streak on the test chromatogram, but four different pens shared this quality. Not enough of the ink made it all the way to the solvent front, which is where I made many of my distinctions between blacks and blues, b lues, so I had to examine mystery test 2. Mystery test 2 did not yield the definitive results for the red pens that I had hoped for. It rd
was able to show me that the 3 mystery pen was Staples red due to the absence of a yellow tail. th
However, it was not very effective for the 5 mystery pen. There was a long yellow streak tailing a bright pink pigment pigment that ran all the way to the solvent front. Unfortunately, three pend nd
shared this property. Additionally, this test did not clear up my uncertainty about the 2 mystery nd
pen, so I was forced to make an educated guess. For the 2 pen, I guessed Papermate blue and th
for the 5 pen, I guessed Pilot Easytouch red. My guesses for mystery pens 2 and 5 were we re incorrect, but the three pens I confidently identified were correct. correct. Pen 2 turned out to be BIC black and pen 5 was Papermate red. In total,
I identified 3 out of the 5 mystery pens correctly. The main problem I ran into during during the mystery runs was that the amount of ink used during our test runs was more than the amount of ink used in the mystery run. Because of this, our test test runs yielded darker and more more visible results, and there were more identifiable characteristics for each pen. In the mystery run, run, we were not able to dot the mystery pens ourselves, and the initial dots were lighter than in ou r tests. This made it more difficult to identify the unknown pens in cases where very subtle differences were the only distinguishing factors between pens. Conclusion Our hypothesis that a more polar solvent than the one used in the original experiment ex periment would be needed to separate the components of the pens was correct. We found that a more polar solvent had a higher affinity for the polar chromatography paper, and therefore did not carry the ink components all the way to the solvent front. There was a greater separation separation with increasing solvent polarity up to a certain point. At this point, which we determined to be roughly 7.8 on the Snyder Polarity Index, the solvent was too polar and the components of the pens did not travel far enough or they did not achieve ideal separation. From our group’s findings, I was able to identify 3 of the 5 mystery pens assigned to us. For future studies of this sort, even even more solvent combinations should be tested. tested. Due to our limited class time, our group was only able to test a limited number of solvent polarities. It is likely that a different solvent would be more e ffective in generating distinctions between the pens. However, due to lack of time, we were not able to find such a solvent. The fact that I could only identify 3 of the 5 pens reflected this. Despite my shortcomings in distinguishing
between the mystery pens, my overarching hypothesis held true: a more polar solvent was needed to improve the chromatographic separation of the components of the pens. References 1. Thompson, S. PSU Chemtrek: Small-Scale Experiments for General Chemistry 2013-2014; Hayden McNeil Publishing: Plymouth, MI, 2013; pp 17-1 – 17-1 – 17-22. 17-22. 2. "History of Chromatography." Chromatography." History of Chromatography. University University of Michigan, n.d. Web. 21 Feb. 2014. 3. “chromatography.” Encyclopaedia “chromatography.” Encyclopaedia Britannica. Britannica. Encyclopaedia Britannica Online. Online. Encyclopædia Britannica Inc., 2014. Web. 23 Feb. 2014. . >. 4. Chen, Hu-Sheng, Ph.D, Hsien-Hui Hsien-Hui Meng, Ph.D, and Kun-Chi Cheng, M.Sc. "A Survey of Methods Used for the Identification and Characterization of Inks." Forensic S cience Journal 1.1 (2002): 1-14. Forensic Science Journal. Web. 22 Feb. 2014. . 5. “paper chromatography.” Encyclopaedia chromatography.” Encyclopaedia Britannica. Britannica. Encyclopaedia Britannica Online. Online. Encyclopædia Britannica Inc., 2014. Web. 23 Feb. 2014. . >. 6. Mukherjee, Mukulika. "Paper Chromatography Uses." Buzzle.com. Buzzle.com, 06 July 2012. Web. 25 Feb. 2014. .
7. Allison, Keith. "Re: What Are the Different Different Uses for Paper Chromatography Chromatography in a Society?" MadSci Network: Chemistry. N.p., n.d. Web. 25 Feb. 2014. . 8. Egan, James M., Kristin Kristin A. Hagan, and Jason D. Brewer. "Forensic Analysis of Black Black Ballpoint Pen Inks Using Capillary Electrophoresis." FBI. FBI, 28 Feb. 201 1. Web. 25 Feb. 2014. . 9. Gross, Brian. CHEM 113 Laboratory Notebook, Spring 2014, pp. 6-13. 10. Donley, Connor. CHEM 113 Laboratory Notebook, Spring 2014, pp. 12-14. 11. Del Guercio, Katie. CHEM 113 Laboratory Notebook, Notebook, Spring 2014, pp. 6-12. 12. Grove, Molly. Molly. CHEM 113 Laboratory Notebook, Spring 2014, pp. 11-15. 13. Daniels, Shahkila. CHEM 113 Laboratory Notebook, Notebook, Spring 2014, pp. 16-21.
2 = http://www.umich.edu/~orgolab/Chroma/chromahis.html 3 = http://www.britannica.com/EBchecked/topic/115917/chromatography 4 = http://fsjournal.cpu.edu.tw/content/vol1.no.1/p1.pdf 5 = http://www.britannica.com/EBchecked/topic/441999/paper-chromatography 6 = http://www.buzzle.com/articles/paper-chromatography-uses.html 7 = http://www.madsci.org/posts/archives/2000-10/971129675.Ch.r.html 8 = http://www.fbi.gov/about-us/lab/forensic-sciencecommunications/fsc/july2005/research/2005_07_research01.htm 9 = My Notebook 10 = Connor’s Notebook 11 = Katie’s Notebook 12 = Molly’s Notebook 13 = Shahkila’s Notebook
