Plant Pigment Chromatography Lab By Katie Aman, Sarah Kohl, and Katrina Wheelan Results Experimental Error Random Errors Measuring Errors We hope you enjoyed our Prezi Conclusion Graphs Discussion Results/Analysis Introduction/Background Hypothesis/Prediction Materials Does Changing the ratio of one solvent to the other affect the Rf ratio? If so, how? What is the correlation and what does it look like on a graph? We predicted that varying the ratio of solvents would have an effect on the Rf values of each of the pigments. We also predicted that the correlation of the Rf values for each pigment graphed against the percentage of acetone would be a negative parabola with the maximum between the 1:2 and 0:3 ratios of acetone to naphtha .

*Formula: Percentage of Acetone as a solvent = (Acetone mL/3 mL) * 100 Looking at the results that we achieved and the graphs that could be made with the data, we can note a number of things. The graphs included both linear regression (a trend line) and quadratic regression. For all four pigments, the quadratic regression formed negative parabolas. Also true for all four pigments was that the r^2 value (the regression coefficient squared) was closer to 1 than the for the linear regression. This suggests that the quadratic model was a more accurate fit for our data, as we had predicted. Looking at our results, we can see a correlation between the solvent ratios and the distance that the pigments traveled, and therefore the Rf values. Based on our knowledge of the molecular composition of the two solvents, naphtha and acetone, this change makes sense. We experienced some error in distinguishing the barriers between pigment bands because colors sometimes tend to bleed together or are hard to tell apart. Many of the pigments were colored similarly and it was difficult to determine when one pigment ended and another began. This subjective element contributed some inaccuracy to the experiment.

Another factor in random error was the fact that all spinach leaves are not identical and some of the papers may have contained cells with different amounts of each pigment.

Though the spinach leaves were all pushed into the leaves with 10 rolls of the quarter, there may not have been an entirely consistent number of cells and therefore amount of pigments on each band of the papers. The number of pigment molecules may have affected the ability of the solvent to carry the molecules up the chromatography paper, and thus affecting the Rf values. As with any experiment, every measurement was associated with some error that may have contributed to the results. Measuring the amounts of each type of solvent may have been inaccurate by roughly ±0.05 mL. The amount of solvent could affect its ability to travel up the chromatography paper. We also allowed the chromatography paper to sit in the solvents for 10 minutes and staggered each paper and solvent pair by 1 and a half minutes, but our timing may have been slightly off. The error in the timing was likely ±0.2 seconds. We used a ruler to measure several marks on the chromatography paper: the place to rub the spinach leaf, the place to cut when trimming the strip, and the distances the pigments and solvents traveled. Each of these measurement may have been off by ±0.2 mm. Thank you for watching! 50 mL graduated cylinders (or test tubes)

chromatography paper

spinach leaves

coin

goggles

cork stopper

pencil

scissors

naphtha

acetone

ruler

paperclips

glass pipets Our experiment was to test the effect that different ratios of solvents would have on the Rf values of the pigments. The original experiment used a ratio of 9:1 (naptha to acetone) as the solvent. In our self-designed experiment we varied the ratio of solvents (naptha to acetone). A spinach leaf was ground into the chromatography paper. After 10 minutes, we measured the distance from the spinach leaf mark to the different color bands which each signified a different pigment. The four kinds of pigments we looked for were beta carotene (yellow-orange), xanthophyll (yellow), chlorophyll a (blue green), and chlorophyll b (olive green). In the original experiment, beta carotene moved the farthest because it was highly soluble in the solvent and made no hydrogen bonds with the chromatography paper. Xanthophyll moved the second farthest because it made some hydrogen bonds with the paper and was less soluble in the solvent. The chlorophyll a and b moved the shortest distances because they were bound more tightly to the paper. The Rf values from the original experiment in order from least to greatest were: beta carotene, xanthophyll, chlorophyll a, and chlorophyll b. In our self-designed experiment, we calculated the Rf values for each of the pigments with different solvent ratios. Methods In order to answer our question, we carried out a self-designed experiment, a modified version of the original lab experiment. First, we measured the acetone and naphtha in different ratios, keeping the constant total amount of solvent at 3 ml per test tube. Then we prepared the chromatography paper. We cut them into thin strips that were 1.3 cm wide. We then cut one end so that it was pointed. We drew a line with a pencil 2 cm from the tip of the paper and rubbed a leaf of spinach onto the line with a quarter. We then put the different papers into the varying solvents for ten minutes each. Stoppers and paper clips with tape on the end of them held the papers in the solution where we wanted them to be positioned. We took them out to observe and measure the separation of the pigments. The independent variable in our graphs was percentage of acetone as a solvent. The dependent variable in our graphs was the Rf values of the pigments. The control was 0% acetone as a solvent (0:3 ratio) and 100% acetone as a solvent (3:0 ratio). After concluding our experiment and analyzing our results, we found that our initial hypothesis was right about the shape of the graphs being a negative parabola. The most important thing that our experiment showed was that the Rf values of each kind of pigment do change when the ratio of solvents is changed. The Rf values will increase for awhile as the percentage of acetone as a solvent increases, but we also saw after a certain point that the Rf values will decrease when percentage of acetone as a solvent increases a lot. The specific reasons for these trends are stated in the discussion of our experiment. Tables Bibliography "Oil and Gas | Petrochemicals | Steel and Aluminum - Distributors and Suppliers." : July 2011. N.p., n.d. Web. 06 Feb. 2013. *Rf value = distance traveled by pigment (mm) /distance traveled by solvent (mm) Distance Pigments Traveled When Dissolved in Varying Solvent Ratios Molecular structure of acetone On the other hand, naphtha is a mixture of several hydrocarbons (consisting of hydrogen and carbon). Hydrocarbons (examples include fatty acids and other lipids) are non-polar. The non-polar molecules of naphtha can not form hydrogen bonds or be attracted to other molecules or pigments.

Since acetone is slightly polar while naphtha is non-polar, this means that varying the ratios and amounts of each will affect the pigments' ability to form hydrogen bonds with the solvent and travel up the paper. The ability of the pigments to form hydrogen bonds with the solvent molecules is important at first, but at some point it may slow down the rise of the pigments on the chromatography paper. This would explain our negative parabolas forming when graphing Rf values of each pigment against the percentages of acetone as a solvent (which is polar and can be a part of hydrogen bonds). Acetone is a slightly polar molecular containing two methyl groups (CH3), which are uncharged, and one carbonyl group (OH), which is what makes the molecule also slightly polar. This overall slight polarity of the molecule means that it can form weak hydrogen bonds and be attracted to other molecules such as the pigments. Chlorophyll A regression equations:

Quad: y=-0.000189x^2+0.02x+0.126 (r^2 = 0.400)

Linear: y=-.003x+0.336 (r^2=0.0670) Chlorophyll B regression equations:

Quad: y=-1.688x^2+0.0266x+0.017 (r^2 = 0.992)

Linear: y=.00967x+0.204 (r^2=0.781) Beta Carotene regression equations:

Quad: y=-0.000415x^2+0.0405x+0.108 (r^2 = 0.997)

Linear: y=-.000965x+0.569 (r^2=-0.00604) Xanthophyll regression equations:

Quad: y=-0.000183x^2-0.0256x+0.772 (r^2 = 0.933)

Linear: y=-0.00731x+0.569 (r^2=-0.6) Maximums of the Parabolas Formed by Quadratic Regression Chlorophyll A: 43.3% Acetone

Chlorophyll B: 78.6% Acetone

Beta Carotene: 48.8% Acetone

Xanthophyll: 32.1% Acetone

Average: 50.7% Acetone In our hypothesis, we predicted that the data would form negative parabolas with their maximums between the 2:1 and 0:3 ratios of acetone to naphtha. Every one of the pigments formed a negative parabola in quadratic regression, so that part of our hypothesis was correct. To see whether the second part was correct, we can look at where the maximums of the quadratic regression equations fell. We can see that all the pigments had maximums that fell between 30 and 80% acetone. The average maximum was 50.7%, which represents the "ideal" percentage of acetone to maximize the Rf value. Since 50.7% does not lie between our predicted range of 0-33.3% acetone, our hypothesis was only partly correct in that the graphs formed negative parabolas.

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# Copy of Plant Pigment Chromatography

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by Katie Aman
on 8 February 2013
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