Paper Chromatography Lab Prepared for: Mrs. Freeman September 6, 2013 1
Introduction The separation of mixtures is an important part of chemistry. One such method of separation is called chromatography, which utilizes the solubility differences in substances (of a mixture) in a given solvent. In its most basic form, chromatography is used to separate a mixture into its components. There are many different kinds of chromatography, including column chromatography, high performance liquid chromatography, gas chromatography, thin layer chromatography and paper chromatography. However, they all use the same principle: they all have a stationary phase and a mobile phase. The mobile phase, which is a liquid or gas, flows through the stationary phase (solid, or a liquid on a solid), and carries the components of the mixture with it. What separates the components are the rate at which they move across the stationary phase and the distance they travel. This travel (or lack thereof) is caused by an affinity to either the stationary phase, the mobile phase, or both (General Chemistry Online, 2011) This lab uses paper chromatography as opposed to other forms of chromatography. In paper chromatography, the stationary phase is the chromatography paper, and the mobile phase is a liquid solvent. A line is drawn almost at the end of the paper, and a drop of the mixture is put on the line. The paper below the line is put into the solvent, as to allow the solvent to diffuse up the paper. If the solvent is polar, the components of the mixture that are polar to some extent will dissolve in the solvent and travel up the paper, as opposed to any other nonpolar components. This is reversed when using a nonpolar solvent. In most cases, the components of the mixture will have slightly different polarities, and will travel different differences up the paper (Clark, 2012) 2
After this point, an Rf value (distance travelled relative to the solvent) can be calculated for each component. To obtain this, the distance travelled by the component is divided by the distance travelled by the solvent. These can be used to compare the components of a known mixture vs. an unknown mixture. Purpose In this lab, you will use 5 known acid base indicators and their Rf values to analyze two unknown substances. Acid base indicators are usually colorful, which change colors according to the acidity of the substances they are present with. Methods 1. Place all strips of chromatography paper on flat, dry surface. 2. Mark a line with pencil 2 centimeters from the end of each strip. 3. Dip pipette into one of the acid base indicators and touch the tip to the filter paper (not chromatography paper). The spot of liquid should be no more than 5 mm in diameter. Repeat until application procedure has been mastered. 4. Dip pipette back into the indicator solution and place a spot in the middle of the line on one of the strips of chromatography paper. Immediately label the paper as to identify which indicator was placed on this strip. 5. Repeat step 4 for each of the remaining indicators and the two unknown samples. Use a clean pipette for each substance. 6. Fill 4 flasks with 25 ml of Ethanol saturated with NH3 (solvent). Stopper each flask 3
7. After the strips are dried, remove the stopper from one of the flasks and position two strips on either side of the stopper. 8. Place the stopper and the strips so that the solvent touches the strip below the pencil line, and so that the sample spots do not come in contact with the solvent. 9. Repeat steps 7 and 8 for the remaining strips. 10. Allow the solvent to diffuse up the strips until the solvent front (the highest point of solvent saturation) is at least 9 cm above the pencil lines. 11. Remove the strips and place them on a dry sheet of paper. Allow them to dry for a few seconds 12. Draw a pencil line on the solvent front of each strip. 13. Draw a point in the middle of the sample spot. If this is not possible because of the paper still being wet, poke a small hole in the middle of the sample spot. Note: phenolphthalein spot will become colorless quickly. 14. When the strips have completely dried, measure the distance the sample spot(s) is from the pencil line for each strip. In addition, measure the distance from the solvent front to the pencil line. Record this in data table. 15. Clean the station and wash hands. 16. Calculate the Rf values for each sample spot and analyze the unknown substances. 4
Data Table 1- Paper Chromatography of ph Indicators Sample Distance solvent moved (cm) Distance sample moved (cm) R f Values Bromothymol blue 5.11 cm 4.62 cm.904 Congo Red 5.55 cm 0.00 cm 0.00 Methyl orange 6.00 cm 4.69 cm.782 Phenol red 5.09 cm 4.38 cm.861 Phenolphthalein 5.03 cm 4.28 cm.851 Unknown A 4.71 cm 3.61 cm.766 4.71 cm 0.00 cm 0.00 Unknown B 5.19 cm 4.08.786 5.19 cm 4.98 cm.960 NOTE: R f = migration distance of substance migration distance of solvent front Analysis/Discussion As the results indicate, Unknown A was composed of both Congo red (Rf = 0.00) and Methyl orange (Rf =.782), and Unknown B was composed of Methyl orange (Rf =.782) and 5
Bromothymol blue (Rf =.904). This is clear because Unknown A had two sample spots, separated from the original sample spot, and the Rf values of both (Rf =.766, Rf = 0.00) corresponded closely enough to only two of the indicators, Methyl orange (Rf =.782) and Congo red (Rf =0.00), respectively. Similarly, the two Rf values of Unknown B (Rf =.786, Rf =.960 corresponded with the Rf values of both Methyl orange (Rf =.782) and Bromothymol blue (Rf =.904), respectively. In addition to the quantitative observation, a qualitative observation can be made as to the colors of the sample spots, which corresponded accordingly (matching the Rf value result). While the Rf values were not exactly the same, they were close enough to create a solid conclusion. Because of a time constraint, my lab partners and I were not able to keep the strips of chromatography paper in the solvent until the specified 9 cm in the directions. Perhaps if this had been completed, the Rf values might have been even closer. Looking at the Rf values of all the different indicators, we can see that they all have very different values. There are several reasons for this. Our solvent (ethanol saturated with NH3) was extremely polar, and consequently, the more polar indicators moved farther up the strips. For example, we can conclude that Congo red was completely non-polar, as it did not dissolve and travel up the strip at all, while Bromothymol blue was the most polar, as it had the highest Rf of.904. In addition, while the solvent front of all the strips were not uniform, this is insignificant. Because an Rf value is essentially a ratio, and each indicator s travel is rationally proportional to the solvent travel, the Rf values are sound. Conclusions At the conclusion of the experiment, we can conclude that Unknown A was made up of Congo red and Methyl orange, and Unknown B was composed of Methyl orange and Bromothymol blue 6
References Chromatography. (n.d.). General Chemistry Online. Retrieved September 5, 2013, from antoine.frostburg.edu/chem/senese/101/matter/chromatography.shtml Clark, J. (n.d.). paper chromatography. chemguide: helping you to understand Chemistry - Main Menu. Retrieved September 5, 2013, from http://www.chemguide.co.uk/analysis/chromatography/paper.html#top 7