You are on page 1of 16

Adjusting and Analyzing the Mobile Phase of Paper Chromatography to Determine the Identity of Unknown Ink Stains

Corinne Renner February 21, 2013 Chemistry 113, Section 103 Teaching Assistant: Han Song

Renner 2 Introduction The Merriam Webster dictionary defines chromatography as a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the solutes as they flow around or over a stationary liquid or solid phase.1 Paper chromatography is a method that utilizes a very porous, absorbent paper for the stationary phase.2 The overall intention for this procedure is to separate chemical mixtures by suspending the paper in a shallow solvent that allows the components to diffuse the length of the stationary phase. With a proper solvent and conducive stationary phase, the chemical mixture should separate into independent components for careful analysis. The laboratory techniques and methods for chromatography arose in the early nineteen hundreds by Mikhail Tswett, a Russian botanist of the time. Tswett is credited with developing and publishing the original techniques for column chromatography due to his successful separation of chlorophyll, xanthophyll, and carotene.3 The concept and laboratory application of chromatography did not become popular until the 1930s when an Austrian scientist, Richard Kuhn, and his colleagues re-mastered the process by discovering an entirely new species of carotene. For this reason, the separation of alphaand beta-carotene by Richard Kuhn is considered the birth of modern chromatography.4 Paper chromatography is relatively simple to set up and perform, but the science behind the process is not as easy to explain. The structure of the actual paper is essential is this form of chromatography. The paper is made of loose cellulose fibers that are composed of polymers with hydroxyl (-OH) groups. These cellulose fibers attract water vapor from the atmosphere as well as and water that was present when the paper was

Renner 3 produced. This characteristic of the polymers creates a very thin layer of water molecules on the surface of the paper. It is the interaction between the solvent and the water that permits successful chromatography.2 A given chemical mixtures attraction to the polar phase, versus the polarity of the solvent, allows it to separate into its respective components. There must be some difference in the polarity of either phase for partition to occur. For instance, molecules in the mixture that are less polar than water will be less attracted to the water molecules on the paper and will dissolve within the more conducive, and less polar solvent up the paper. Thus, the lower the polarity of the component, the farther it travels from the base of the paper during the allotted time for the experiment and vice versa. This entire process is significant in laboratory work and research. The separation of chemical components is vital for chemical analysis. In order to properly identify a substance, it needs to be simplified as much as it can into its constituent components. These components are then compared to standards for identification.5 Chromatography is also useful for comparing chemical mixtures to one another. For instance, in paper chromatography, researchers measure each separate components degree of retention. The retention factor describes the average time that a molecule spends in the mobile phase, and is determined experimentally by dividing the distance moved by the component by the distance moved by the mobile phase front. This is useful in understanding the relative polarities of each component, and determining which components appear in other chemical mixtures due to equivalent retention factors.2 Additionally, chromatography has played pivotal roles in isolating new compounds, analyzing subtle differences between samples exposed to various environmental

Renner 4 conditions, and sequencing DNA.6 However useful, paper chromatography, and other chromatography methods are not the only process used in ink identification. Forensics scientists use a variety of methods to examine inks in order to detect fraudulent documentation. These scientists can compare inks through optical microscopy, infrared reflectance, luminescence, ultraviolet fluorescence, or solubility tests. Each individual method is designed to analyze inks without compromising the original document.7 With these experiments, forensic scientists can determine whether or not documents were subjected to alterations, as well as confirm the date of publication. In this laboratory, four unknown inks were placed on a chromatogram. Using paper chromatography, a proper mobile phase was created to analyze fifteen possible ink stains. The dry chromatogram of these fifteen different inks was then compared to the chromatogram of the four unknowns in order to identify them. A paper chromatography experiment was conducted with a 2:1 mobile phase of 1-proponol and water that was unable to separate the inks into its constituent components.8 The hypothesis is that a more polar mobile phase will allow the inks to separate on the paper and, thus, provide an easier key for identification of the unknown inks. Procedure The procedure for this laboratory was designed prior to the experiment and based off of the original instructions for paper chromatography in the PSU Chemtrek literature. First, a strip of chromatography paper was selected. Using a ruler, a line was drawn across the long edge of the paper about 0.5 centimeters from the bottom with additional tick marks for every centimeter along the lines length. There were fifteen different brands of pens under scrutiny. Five had blue ink, five had red, and five had black. Each

Renner 5 individual pen was used to make a small dot on one of the tick marks, until all fifteen pens were organized on the chromatography paper. The following is a display of the organization of these pen brands that was then used as a key throughout the experiment. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

KEY: 1. Papermate Black 2. Pilot Vball Black 3. Max Pen Black 4. BIC Black 5. Pilot EasyTouch Black 6. Papermate Red 7. Pilot Vball Red 8. Staples Red 9. Pilot EasyTouch Red 10. Sharpie Highlighter Pink 11. Papermate Blue 12. Staples Blue 13. Pilot Easy Touch Blue 14. BIC Blue 15. Pilot Vball Blue Once the fifteen pen marks were drawn on the chromatography paper, the two short edges were stapled together, once at the top and once at the bottom of the two sides. It was important to ensure that these pieces were stapled together without touching to prohibit any spreading or diffusion between different inks. Ideally, the inks should spread directly upwards from the place where they are drawn, so that each ink undergoes an equivalent process. In a petri dish, a mobile phase was prepared. Because this part of the laboratory was purely experimental, several solvents were created and analyzed. During a chromatography experiment that used a 2:1 1-propanol to water solution, the pens that were sampled did not separate into their constituent components.8,9 Thus, in creating the mobile phase, solvents were designed from the available solutions 1-propanol, 2propanol, ethanol, methanol, and water. The mobile phases were prepared with alternating polarities. The polarities of these five substances was provided by the Snyder Polarity Index.10

Renner 6 Table 1: Snyder Polarity Index10 Substance Water Methanol Ethanol 1-Propanol 2-Propanol Polarity 9.0 6.6 5.2 4.3 4.3

In this experiment, a 15 mL solution of 1:1 methanol to water was prepared in a petri dish, as well as a 15 mL solution of pure methanol in a separate petri dish. To run the paper chromatography experiment, the chromatography paper with ink samples was placed in the center of the petri dish containing the mobile phase. The ink dots should be at the bottom of the paper so that the process can occur in an upwards fashion.11 The paper was then covered with a plastic cup to prohibit movement while submerged in the mobile phase. In order to test each mobile phase separately, the experiment had to be completely duplicated with an untouched chromatography paper with the same ink marks, because the process can only occur once per paper. When the mobile phase front was within 0.5 cm of the top of the paper, the experiment was complete, and the paper was removed. The mobile phase front was marked with a pencil for easy identification. The paper was unstapled and left to dry before careful analysis. Once a satisfactory chromatogram was obtained, the assigned chromatogram with the four unknown samples was ran using the exact same procedure. This ensures that the chromatograms are comparable for easy identification. The two chromatograms were then compared either by eye, or by placing the chromatograms under UV light. The UV light provided an alternative perspective of the different bands. With the successful

Renner 7 completion of these procedures, comparison amongst the chromatograms indicated the identity of the unknowns. Results The replicated chromatograms in this experiment were subjected to different mobile phases, each producing unique results. The following chromatogram, from a separate experiment, used a 2:1 1-propanol mobile phase. Chromatogram 1: 2:1 1-Propanol to Water Mobile Phase8,9

Only the last six marks, on the right, are from pens similar to the fifteen used in this experiment. It is evident that the majority of these pens did not separate into their constituent components. Other than the pens that produced the pink and yellow components, the other four pens remained concentrated at the mobile phase front. This chromatogram was used to examine the effect of the 2:1 1-propanol to water mobile phase. The 2:1 1-propanol to water mobile phase had a polarity of 5.87. This was determined by using the following calculation and the Snyder Polarity Index.10

Renner 8

In this previous experiment, the Rf values for each individual component were calculated and used for analysis. Table 2: Chromatography Data for Distance and Corresponding Rf Values Name of Sample Red #3 Red #40 Blue #1 Yellow #5 Yellow #6 Green #3 Cheddar Cheese Grape Kool-Aid Orange Kool-Aid Lemon-Lime Kool-Aid Prepared Black Dye Papermate Black Pen Papermate Blue Pen Papermate Red Pen BIC Black Pen BIC Blue Pen BIC Red Pen Distance Spot(s) Moved (cm) 4.0 2.6 2.8 1.4 2.4 2.8 1.5, 2.4 2.5, 2.9 1.3, 2.6 1.3, 2.8 2.8, 3.8 4.0 4.0 1.6, 4.0 4.0 4.0 1.3, 4.0 Rf Value 1.00 0.65 0.70 0.35 0.60 0.70 0.375, 0.60 0.625, 0.72 0.325, 0.65 0.325, 0.70 0.70, 0.95 1.00 1.00 0.40, 1.00 1.00 1.00 0.325, 1.00

These Rf values were calculated according to the following equation.

Example Calculation 1: Rf of the First Sample, Red #3 Dye

This calculation proves that the Red #3 Dye migrated 95% as far as the mobile front.

Renner 9 The ideal chromatogram should have a clear distinction between the different components of a given chemical mixture. For this reason, in composing a mobile front, the 2:1 1-propanol to water solvent was excluded. Chromatogram 2: 1:1 Methanol to Water Mobile Phase

The first mobile phase that was designed consisted of a 15mL 1:1 methanol to water solution. The majority of the inks separated into their basic components. Unfortunately, this chromatogram wasnt very clear or easy to read. Most of the color was concentrated in a single area, making it hard to differentiate between similar inks. Chromatogram 3: Pure Methanol Mobile Phase

The final chromatogram that was tested was placed in 15 mL of pure methanol. This produced the most defined results. Each ink separated enough to differentiate between its different components. And, each ink had some sort of tail or trail. Unlike the other two chromatograms, the different colors and clear definition of the various tails

Renner 10 was helpful in differentiating between inks that were exceedingly similar. Thus, the pure methanol mobile phase was used to generate the chromatogram with the unknowns. The adopted procedure specific to this experiment, was to create a mobile phase consisting of 15 mL of pure methanol. This solution, in a petri dish, was used to set the chromatography paper in. Chromatogram 4: Unknown Inks Using Pure Methanol Mobile Phase

This is the resulting chromatogram of unknowns using a pure methanol mobile phase. The unknowns from left to right were a BIC Black Pen, a Pilot Vball Black Pen, a Staples Red Pen, and a Papermate Black Pen. Discussion The first time a paper chromatography experiment was conducted, a 2:1 1propanol to water solution was utilized for the mobile phase. The results, apparent in Chromatogram 1, were not of fifteen ink samples, but of 17 different chemical compounds consisting of food dyes, colored drink mix, and various writing utensils. This information was valuable for the identification of unknown inks in the separate experiment because the chromatogram did not produce useful results with the 2:1 1propanol to water mobile phase. The retention factors for the black and blue pens were all at 1.00 which indicated that the components had traveled the entire way to the mobile

Renner 11 phase front. However, in order to compare inks to one another, more identifiable features were required. The solution to this problem was to search for alterations to this original experiment. The paper, or stationary phase, remained the same. Similarly, the amount of chemical mixture placed on each chromatogram, and the manner in which it was placed (0.5 cm from the bottom of the paper) would remain consistent. Altering the mobile phase was the only change that would affect the migration of the components. The major factor in designing the more productive mobile phase was polarity. The stationary phase is very polar due to the hydroxyl (OH-) groups in the cellulose fibers of the chromatography paper. The hydroxyl groups capture water molecules from the surrounding atmosphere, creating a thin layer of water. Water, according to the Snyder Polarity Index, has a polarity of 9.10 According to Chromatogram 1, the sample inks were substantially less polar than water and, thus, traveled the entire way to the mobile phase front because they were not attracted to the polar stationary phase. A more polar mobile phase would lower the retention factor of the ink and allow it to separate into its components because the inks affinity for the mobile phase would decrease as it became more polar. The design of the mobile phase was restricted to the use of methanol, ethanol, 1propanol, 2-propanol, and water. The 2:1 1-propanol to water solution had a polarity of 5.87. Relative to waters polarity of 9 on the stationary phase, this mobile phase was much less polar. The first mobile phase that was tested was a 1:1 methanol to water solution. 7.5 mL of each component were mixed and set in the petri dish. This solution had a polarity of 7.8. Chromatogram 2 validated this higher polarity because the inks separated into their constituent components, unlike in the experiment that subjected

Renner 12 Chromatogram 1 to the 2:1 1-propanol mobile phase. However, the observations were still not clear enough to differentiate between the different inks types, so an additional mobile phase was designed. The second mobile phase that was tested consisted of 15 mL of pure methanol. Methanol has a polarity of 6.6.10 The only alteration to this new mobile phase from the previous one was the removal of the water. The water in the 1:1 methanol and water mobile phase made the solvent too polar, which is evident on Chromatogram 2 because many of the components were concentrated towards the origin of the ink dots. While water was effective in increasing the polarity of the mobile phase, it made the solution too polar. Thus, the removal of water reduced the polarity of the mobile phase from 7.8 to 6.6. This value is almost in the middle of the original polarity of 5.87 and the polarity of 7.8. Chromatogram 3 showed substantial improvements from the previous two chromatograms. Almost every ink sample stretched the entire length of the chromatogram. There were obvious distinctions between the different colored components of each ink sample. These alterations supported the original hypothesis that a more polar mobile phase would provide a chromatogram that displayed the distinct features of each of individual sample so that comparison between those and unknowns chosen from the set of fifteen ink samples would be feasible. The bright colors and elongated tails provided the features necessary for comparison and identification of the unknowns. The unknown chromatogram, chromatogram 4, was subjected to the exact procedure as chromatogram 3 so that the results would be comparable. After undergoing

Renner 13 the chromatography process and allowed to dry, chromatogram 4 was compared to chromatogram 3. The first ink was from a BIC black pen. This ink sample was identified by visually comparing the stain to that of the other black inks on chromatogram 3. The black ink was clearly not from a Pilot Vball black pen, because that mark on the chromatogram was a thick black line that stretched from the bottom to the top of the chromatogram with a slight yellow appearance at the tip. Similarly, the Max pen and Pilot EasyTouch pen were eliminated from the possible choices because they had thick purple tails and the stain from the first unknown ink sample had a very faint tail. The true deciding factor between the Papermate pen and BIC pen was the hint of yellow in the center of the tail. The yellow portion of the Papermate was much brighter that that of the hue in the BIC pen. The unknown had a very faint yellow stain similar to the BIC pen and, thus, was identified as that sample. The second ink sample was from a Pilot Vball black pen. This ink was easy to identify because it had a thick black mark that stretched the length of the chromatogram. It looked unlike any of the other black inks, because of its dark, black stain. Near the mobile phase front the stain appeared light yellow, a further indicator that this unknown sample was from a Pilot Vball black pen. The third ink was from a Staples red pen. The unknown produced a vibrant hot pink mark that was concentrated more towards the mobile phase front than the marks produced by the Pilot Vball red pen and the pink Sharpie highlighter. Those two pens produced marks with long tails that stretched the entire length of the chromatogram. For this reason, these two choices were eliminated. The Papermate red pen and the red Pilot

Renner 14 EasyTouch pen produced marks that were similar to that of the unknown. However, these two pens displayed a faint orange color at the bottom of their tails. Because these three pens appeared to be so similar, chromatogram 3 and chromatogram 4 were placed under UV light together and compared. The UV light showed that the Papermate and Pilot EasyTouch pens had much more vibrant colors than that of the pure pink mark from the unknown pen. This solidified the fact that the unknown ink stain was from a Staples red pen, the only remaining choice. The fourth and final unknown ink sample was from a Papermate blue pen. This unknown sample manifested itself on the chromatography paper with a dark blue mark that was concentrated towards the mobile phase. There were only three blue inks that had a similar pattern. The remaining blue pen samples, those from the Staples blue pen and the blue Pilot V ball pen, had thicker blue tails that stretched the length of the paper. The blue Pilot Easy Touch pen was also eliminated as a choice because it had a light blue color rather than the dark blue color that the unknown ink exhibited. The final selection was between the Papermate blue pen and the BIC blue pen. The Papermate blue was selected because of its similar deep blue color and its more visible tail. The BIC blue pen produced a tail that was visible, but extremely faint. The unknown sample produced enough color to distinguish it from the BIC blue pen and identify it as an ink sample from a Papermate blue pen. The final chromatographic procedure, using a 15 mL pure methanol mobile phase was successful at identifying the unknowns within one attempt. The distinct features of each sample permitted the visual comparison of the two chromatograms. Discussion

Renner 15 This experiment explored the affect of mobile phase polarity on the migration and separation of ink sample components. It was discovered that the less polar a mobile phase, the less the ink samples were attracted to the paper stationary phase. In contrast, the higher the polarity of the mobile phase, and the closer that this polarity was to the polarity of water, the more attracted the molecules were to the polar stationary phase. The optimization of migration and spreading of all the ink components was achieved with a 6.6 polar mobile phase of pure methanol. This intermediate polarity value produced marks that each exhibited distinct features. These distinct features permitted the successful identification of four unknown ink samples from a possible set of fifteen samples. The hypothesis that a more polar mobile phase than 5.87 would produce a useful chromatogram for identification was proved by this experiment. The four unknown samples on chromatogram 4 were successfully identified in one attempt, from left to right, as a BIC black pen sample, a black Pilot Vball pen sample, a Staples red pen sample, and a blue Papermate pen sample. In future studies, it would be advantageous for the individual conducting the experiment to ensure that each mark on the chromatography paper was of equivalent proportions. If more ink is allotted for some samples, the results wont be as comparative because they would have measured different quantities of ink. Also, to save time while experimenting with different mobile phases, individuals should run several experiments at the same time. This experiment was designed purely for ink samples, thus polarity will always have to be adjusted to combat the polarity of the stationary phase and that of the samples of question. With many resources, paper chromatography is a useful and easily reproducible experiment to examine the separate components of chemical mixtures.

Renner 16 References 1. Merriam-Webster Dictionary. http://www.merriam-webster.com/dictionary/ chromatography (accessed February 9, 2013). 2. Clark, Jim. Paper Chromatography. http://www.chemguide.co.uk/analysis/ chromatography/paper.html (accessed February 9, 2013). 3. Column Chromatography. http://www.cyberlipid.org/fraction/frac0004.htm (accessed February 9, 2013). 4. Richard Kuhn and the Chemical Institute: Double Bonds and Biological Mechanisms. http://www.nobelprize.org/nobel_prizes/medicine/articles/states /richard-kuhn.html (accessed February 9, 2013). 5. History of Chromatography. http://www.umich.edu/~orgolab/Chroma/ chromahis.html (accessed February 9, 2013). 6. Chromatography and its Uses. http://www.pa.msu.edu/sciencet/ask_st/ 092293.html (accessed February 9, 2013). 7. Hu-Sheng Chen, Hsien-Hui Meng, and Kun-Chi Cheng. A Survey of Methods Used for the Identification and Characterization of Inks. Forensic Science Journal. [Online] 2002, 1:1-14. fsjournal.cpu.edu.tw/content/vol1.no.1/p1.pdf (accessed February 9, 2013). 8. Thompson, S. PSU Chemtrek Small-Scale Experiments for General Chemistry. Hayden McNeil. 2012; pp 10-15 10-22. 9. Renner, Corinne, Chem 113 Laboratory Notebook, pp. 7-14. 10. Snyder Polarity Index. http://www.sanderkok.com/techniques/hplc/elutropic_ series_extended.html (accessed February 7, 2013). 11. Burdge, J., Overby, J. Chemistry Atoms First; McGraw Hill Companies, Inc.: New York, 2012; pp 7.

You might also like