Chromatograph and Absorbance Analyses Show Vast Pigment Profile Differences in Capsicum Annum var. Grossum

By the Sabbath Scientists: Anthony Birkmeier, Rockford Coscia, Nicole Kwon, and Douglas Rasher

Abstract:

In a comparison of mature (red) and immature (green) bell peppers, it was our belief that there would be differences between mature and immature peppers. We thought that the mature variety would have more types and a higher concentration of sugars, less photosynthetic pigments, and both would have polyphenoloxidase (PPO). To test for carbohydrates, we used Barfoed’s, Selivanoff’s and the Iodine tests. Barfoed’s test showed a red/orange precipitate in both the green pepper solution and the red pepper solution, which suggests that monosaccharides are present in both the green pepper and red pepper. The green pepper produced a higher mass of precipitate indicating a higher concentration of monosaccharides in the green pepper. Both pepper solutions turned red before one minute in Selivanoff’s test, suggesting the presence of a monosaccharide ketose. In the Iodine Test, the addition of iodine produced no color change in either solution, indicating that starch was not present in either solution. These results contradicted our hypothesis in both quantitative and qualitative aspects.
During paper chromatography, the green bell pepper yielded chlorophyll a, chlorophyll b, and two unknown pigments on the chromatogram strips. The chromatogram strip from the red pepper yielded carotene and chlorophyll b with one unknown. This data was in agreement with our hypothesis.
The test for polyphenyloxidase was negative in both peppers, contradicting our hypothesis.
These results suggest that biochemical reactions must occur to produce the variations in pigments. Furthermore, there must be a chemical signal involved to trigger these reactions.

Discussion:
By: Rocky Coscia, Revised by: Nicole Kwon, Revised By: Doug Rasher

In our experiment, we investigated the differences between an immature (green) bell pepper and a mature (red) bell pepper. Our experiment was inspired by a previous experiment executed by the group SpaysKrafts from the LBS 145 course of fall 2002(Bonilla et. al., 2002). More importantly, it intrigued us that the green pepper ripens into a mature red pepper, though remained the exact same species (Luning et. al. 1995). In the previous research done by the SpaysKrafts, they compared differences between red, orange, and yellow peppers. From background research, we became aware that the green bell pepper matures into the red, orange, and yellow pepper! Do to this amazing feat of nature, we wanted to discover possible differences during its maturation. We hypothesized that the red pepper would contain more types of carbohydrates, that the green pepper would contain more pigments, and that both would contain polyphenyloxidase. It was surprising to see the results obtained in carbohydrate and photosynthetic pigment profiles throughout the life cycle of the pepper.
In the carbohydrate section of our experiment we employed various techniques to discover the carbohydrate makeup of the two peppers. Barfoed’s test showed a red/orange precipitate in both the green and red pepper solutions; this result suggests that monosaccharides are present in both the green pepper and the red pepper (Table 1, Figure 1). In our positive control of fructose, a red/orange precipitate also formed. From previous testing and knowledge, we knew that this result was correct in that fructose is a monosaccharide. Water, our negative control did not form a red/orange precipitate. Water is not a sugar thus would not have changed. As a quantitative test, we then employed a vacuum filtering technique to measure the amount of precipitate in Barfoed’s test and found that the green pepper contained more precipitate. This data suggests that the green pepper contained a higher quantity of reducing monosaccharides. 

In order to differentiate between ketones and aldoses, the red and green peppers were subject to Selivanoff’s test. The red pepper solutions turned red in approximately fifteen seconds in Selivanoff’s test, suggesting the presence of a monosaccharide ketose. The green pepper solutions turned red in approximately thirty seconds also suggesting the presence of a monosaccharide ketose (Table 1, Figure 3). Fructose was used as the positive control and the solution turned red, suggesting a monosaccharide ketose. This agreed with a previous experiment that was executed, running Selavinoff’s test with a 1% fructose solution. Our negative control of water did not produce a color change. With our knowledge that water is not a sugar, we knew that water would not have changed colors. In the Iodine test, the addition of iodine did not produce a blue-black color change in either the green and red pepper solutions (Table 1, Figure 4). This suggested that starch was not present in either the red and green pepper solutions. Starch was the positive control and there was a color change, indicating the presence of starch. There was not a color change in the negative control of fructose, indicating that there was not a presence of a polysaccharide, mainly starch. Also, starch is a polysaccharide whereas fructose is a monosaccharide, and thus would not contain starch. In summary, it can be inferred that both the green and red peppers contained monosaccharides, and further monosaccharide ketoses. The red and green peppers were absent of starches. A carbohydrate that may be present in the green and red bell peppers is fructose because this sugar yielded the same result in Barfoed’s and Selivanoff’s tests from our previous experiment (Maleszwski et. al. 2003). Fructose had similar test results; however, it cannot be assumed that fructose is the only sugar present in the peppers because a combination of a reducing monosaccharide and a monosaccharide ketose would also yield similar results. 

According to our quantitiative tests, the green pepper actually contained a greater amount of carbohydrates, in the form of reducing monosaccharides, than the red pepper, contradicting our hypothesis that the red pepper has more types of sugars. (Table 2, Figure 2). The green pepper produced an average 0.016 grams of precipitate, while the red pepper produced an average 0.011 grams. This was determined through centrifuging and massing the precipitates formed in Barfoed’s test. The solution was centrifuged and vacuum filtered in order to isolate the precipitate. The precipitate was then dried on filter paper over night and massed to obtain the amount of reducing monosaccharides present in red and green peppers. The third trial from green peppers was significantly lower than all other masses, so it was dismissed so it would not disrupt the trend formed. During qualitative tests, coordinating results in Barfoed’s , Selavinoff’s, and Iodine tests suggest that both the green and red peppers possess the same types of carbohydrates. This negates our hypothesis that the red pepper contains more types of sugars. However, more tests may have to be performed in order to characterize all sugars found in both peppers. It is still unclear why a red pepper tastes sweeter than a green pepper. One explanation might be that even though the green pepper has more sugars, the red pepper could possess types of sugars that taste sweeter to the human tongue. These results also show that very little change in carbohydrate composition occurs during the lifetime of a bell pepper. However, any change is significant and the mechanisms involved with how a pepper loses or gains sugars are worth investigation. These results are also interesting in that they suggest the kinds of sugars present during a pepper’s life change.

The chromatogram tests showed the presence of various photosynthetic pigments present in the peppers. The green bell pepper solution yielded a pale yellow color pigment, a blue-green pigment, a more vibrant yellow pigment, and a pale green pigment on the chromatogram strips. The distances these pigments traveled, in relation to the distance of the solvent front (by the equation Rf = distance pigment traveled / distance solvent front traveled), yielded average Rf values of 0.44, 0.19, 0.30, and 0.12 respectively. In comparison to the Rf values calculated from our previous experiments (Maleszewski et. al. 2001), these observed Rf values suggest the presence of the pigments chlorophyll a, and chlorophyll b, with two unknowns. The attempt to determine the unknowns was unsuccessful after research in scientific journals and legitimate web sources. The Chi Square values for these two unknowns indicate that they derivate too far from known values for the 4 identifiable pigments. The chromatogram strip from the red pepper solution yielded a pink pigment, orange-yellow pigment, a pale yellow pigment, and a red pigment front. The distances these pigments traveled up the chromotagram strip, in relation to the distance the solvent front traveled (by the equation Rf = distance pigment traveled / distance solvent front traveled), yielded average Rf values of 0.450, 0.178, 0.276, , and 0.826 respectively. In comparison to the Rf values calculated in previous experiments (Maleszewski et. al. 2001), these Rf values indicate the presence of carotene, and chlorophyll b, with one unknown (Table 3, 4, and 5). The unknown Rf value has a Chi Square value too large to connect it with one of the four identifiable pigments. The presence of carotene is further supported by a correlating Rf value of 0.45, found in other research of carotenoids present red bell peppers (Gregory, 1987). The support for the identification of these pigments in green and red peppers agrees with our predictions that the green pepper would contain more pigments than the red pepper. The absorption spectrum test allowed us to produce a graph of absorbance vs. wavelength of the red and green pepper solutions. The absorbance of the green pepper solution illustrated a general trend of decrease as the wavelength increased (Figure 5). The absorbance reached a steady point from about 520 nm to 655 nm, and then proceeded to increase and then decrease. The solution had a higher absorbance closer to 400 nm because it absorbs blue light and a lower absorbance between approximately 470nm to 655 nm because it reflects green light. It increased around 700 nm because it absorbs red light. The absorbance of the red pepper solution illustrated a gradual decrease over the increase of wavelength. The absorbance was fairly level between 450 nm to 535 nm and then continued to decrease until 700 nm. The absorbance is higher closer to 400 nm because it absorbs green light. There is a general decrease until 700 nm because red light is reflected, not absorbed.

In the enzyme portion of our experiment, we tested for the presence of a certain enzyme called polyphenyloxidase. This was done by adding catechol to a freshly sliced surface on both the green and red peppers. The result of no color change indicated the absence of PPO in both green and red peppers. This result contradicts our hypothesis that both peppers would contain this enzyme. The application of litmus paper to the freshly sliced surface of the green and red peppers produced an orange color on the separate pieces of litmus paper. This indicates a pH of about 5.5 in each type of pepper. Since there was no Polyphenyloxidase present in either the red or green pepper, testing for the effects of heat change or pH change on that enzyme became unnecessary. Since the absence of PPO concluded the experiment, further research was performed in order to determine other types of enzymes present in red and green bell peppers. The enzyme lipoxygenase, also know as LOX, is present in early green stages of the bell pepper’s life, as well as its mature stage. To perform a test for the presence and activity of this enzyme, a biological oxygen monitoring system is necessary. Since the LBS 145 lab does not have one, the data from this previous research was documented. This research indicated that the enzyme has 100% activity when at its optimum pH of 5.5-6.0. The research observed a 70% decrease in activity of the enzyme when it reached its mature stage (the ripening from green to red). In order to run pH and heat inhibiting tests, we would have needed 10mM n-propyl gallte and 340 mM of 5.8.11.14-eicosatetraynoic acid, which the LBS 145 lab does not possess. Their results indicate a 51% inhibition due to the altering of pH levels. (Luning et al 1995). Their data also supports the inference that heat would also cause inhibition of enzyme activity. The effects of pH and heat on this enzyme agree with our initial hypothesis of effect of heat and pH on the enzyme polyphenyloxidase.

Many possible errors presented themselves in these tests. We assumed that more precipitate in Barfoed’s test meant a higher quantity of sugar present in the green pepper. Although this concept is rather novel and makes sense, we have found no research in agreement with or against our approach. Also, while filtering the precipitate, it may have been possible that some of the precipitate was lost due to inexact equipment and variations in shaking time to lift the precipitate into the solution in order to decant. Furthermore, the rather small amount of precipitate made it very easy for simple weighing errors and other small changes to affect the results. Another possible source of error could have been from averaging the masses of the filter paper. Although the masses of the filter paper are all fairly similar they are not exactly the same and could have created differences in the outcome. Also, during the quantitative analysis portion of Barfoed’s test, our last value for the mass of the third precipitate (G3) for the green pepper was significantly lower than the other values (Table 2, Figure 2). This might due to the loss of precipitate during filtering. G3 was also the first sample filtered so the significant decrease is probably due to mishandling. When running the absorbance spectrum during our photosynthetic analysis, the main problem that arose was that our red and green pepper solutions were too dark for the absorbance spectrum. Diluting the solution ameliorated the problem. During the paper chromatography portion of our experiment, the chromatogram strips may have touched the sides of the test tube, altering the distance in which a pigment or the solvent traveled. 

In conclusion, our experiments indicated that red and green bell peppers contain monosaccharides, namely monosaccharide ketoses. They do not contain starches. There is also support that the green pepper contains a higher concentration of monosaccharides than the red pepper, which contradicts out hypothesis. Both the green and red peppers possessed equal qualities of carbohydrate composition, as far as our tests showed. This negates our hypothesis that the red pepper would have more types of sugars than the green pepper. The green pepper solution indicated the presence of chlorophyll a and chlorophyll b, with two unknowns. The red pepper solution suggested the presence of carotene and chlorophyll b, respectively. This data disagrees with our predictions that the green pepper would contain more identifiable pigments than the red pepper. The enzyme polyphenyloxidase was absent in both peppers, which is contradictory to our predictions.