AP Biology Lab Plant Pigment Chromatography PDF

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Biology lab on pigment chromatography...

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Alexis Labrada Ms. Wilson AP Biology February 13, 2018 Plant Pigment Chromatography with Spinach Bella Caracta, Trinity Hatchett, and Roshan Khan Data:

Table 1: Distance traveled of the pigments in Spinach Band

Trial 1 Distance (mm)

Trial 2 Distance (mm)

Trial 3 Distance (mm)

Trial 4 Distance (mm)

Orange

69.5

74

72

71

Yellow2

42.5

36

39

39

Yellow1

35

26

28

28

Blue green to bright green

22.5

14

18

20

Yellow green to olive green

11.5

7

11

13

Solvent line

70

80

76

72

Table 1: Demonstrates the distance traveled by each pigments. Each color traveled up the chromatography paper a different distance. According to the data the orange traveled the furthest and the green traveled the smallest.

Table 2: The effect of the type of pigment on the Rf value Color

Trial 1

Trial 2

Trial 3

Trial 4

Average Rf

Standard error

95% CI

Pigment ID

Orange

.99

.93

.95

.98

.962

.0137

+.9894/ -.9346

Carotene

Yellow1

.60

.45

.51

.54

.525

.0312

+.5874/ -.4625

Xanthophyll

Yellow2

.50

.33

.36

.38

.393

.0373

+.4675/ -.3184

Violaxanthin

Blue Green

.32

.18

.25

.27

.255

.0290

+.313/ -.1969

Chlorophyll a

Yellow Green

.16

.09

.14

.18

.142

.0193

+.1806/ -.1034

Chlorophyll b

Table 2: Demonstrates the Rf values which is the distance the pigment migrated divided by the distance solvent front migrated. The pigments were identified by comparing the Rf values to the known Rf values of these pigments. Carotene is the pigment that travelled the highest. The higher the Rf value, the further the pigment travelled.

Figure 1:

Figure 1: Demonstrates the distance each pigment travelled up on the chromatography paper. This is how the Rf values were determined. Each pigment has a distinct start and endpoint that allows the distance travelled to be determined.

Calculations:

Conclusion: ●

This procedure allows us to separate the pigments because of the relationship between the solvent, pigments, and chromatography paper. The solvent will dissolve the pigments of the Spinach when it begins to travel up the chromatography paper. The solvent moves upwards due to act of capillary action. Capillary action is when the hydrogen bonding in the solvent pulls the molecules upward and the hydrogen bonding between the chromatography paper helps defy gravity. The factors that contribute to the separation of the pigments is the solubility, size of particles, and their attractiveness to the paper.

Solubility of a pigment determines how far it will travel up the chromatography paper. The less soluble the quicker the pigment will appear on the chromatography paper. The solubility allows the pigments to be separated on the chromatography paper as they all have different levels of solubility. Polarity is what determines solubility because like dissolves like. Since the solvent is nonpolar the pigments that are more nonpolar will spend more time in the solvent, traveling a greater distance, rather than appearing on the chromatography paper as quickly as others. The size of the pigment particles affect the separation because the smaller the molecule, the further it will travel up the chromatography paper since it will be more dissolved in the solvent. Larger particles will travel a shorter distance because it is not dissolved in the solvent as much. The chromatography paper is a factor in the separation as it is made of cellulose which contains hydroxyl groups that will attract polar molecules. The polar molecules that are not dissolved in the solvent will be attracted to the paper and then separate.

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A mixture of two non-polar solvents were used because it attracts nonpolar pigments. Since the pigments are nonpolar they will be soluble in the solvent which would allow the pigments to travel up the chromatography paper and become visible. Some pigments moved further than others because the pigments have different levels of solubility which allows some to be more soluble in the solvent than others. This allows the more soluble ones to travel further up the chromatography paper with the solvent..

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The properties of the pigments found in spinach that affect their Rf value is their particle size, solubility, and polarity. These pigments contain polar and nonpolar properties. The hydrogen bonding properties will allow the pigment to attract to the chromatography paper and then travel up it through capillary action. A higher Rf value means that the

pigment is smaller and more nonpolar. The Rf value demonstrates how high the pigment went up the chromatography paper, the higher the value the higher it went up. Carotene was the most nonpolar pigment found in the spinach and the smallest. This pigment has a molar mass of 536.89 g/mol. The smaller the pigment the higher it traveled up the chromatography paper because it was more soluble in the solvent so it traveled up with it. Since Carotene traveled up the chromatography paper the highest it is the most nonpolar because the solvent was nonpolar and like dissolves like. The more nonpolar a pigment is the more soluble it is in the solvent and is therefore able to travel up the chromatography paper further. Violaxanthin was the pigment with the second highest Rf value. It had a molar mass of 600.85, which is bigger than Carotene. Since it did not travel up as far it is also less non polar than Carotene. Xanthophyll had a lower Rf value than the other two pigments and was found below them on the chromatography paper. Its molar mass is 568.87 g/mol yet did not travel as far as Violaxanthin. This is due to the fact that Violaxanthin is more nonpolar than Xanthophyll which is why it was able to travel up further and have a higher Rf value. Chlorophyll a had the second lowest Rf value and was find low on the chromatography paper. Its molar mass is 893.51 g/mol which is a lot more than the other pigments and did not allow it to travel up as far. It is also not as nonpolar as the rest as it did not attract to the solvent as much as the other pigments. Lastly, Chlorophyll b had the least Rf value and was at the bottom of the chromatography paper. Its molar mass is 907.47 g/mol which is the greatest out of all the pigments and is why it was not very soluble in the solvent, it was too big. This was the least nonpolar pigment in the spinach and the heaviest which is why it was found at the bottom.

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The wavelength best absorbed by Carotene would be between 420nm-480nm. This wavelength range represents the color blue and violet and since Carotene is yellow it would be reflecting the colors of red and orange and absorbing the most of the blue wavelengths. The wavelength best absorbed by Violaxanthin would be very similar to Carotene because they are both yellow, it would be around the 420nm-480 nm range. This pigment also reflects the red and orange wavelengths and would absorb the blues and violets. The best wavelength absorbed by Xanthophyll would be between 440 nm-480 nm as well because it also appears yellow which means it reflects the pigments red and orange and absorbs the blues and violets. The best wavelength absorbed by chlorophyll a would be around 340 nm-420 nm and 640 nm - 680 nm because it appears a blue green color which means that it would reflect those wavelengths and absorb the wavelengths of the violets and the reds. The best wavelength absorbed by chlorophyll b would be around 440 nm- 470 nm and 630 nm-660 nm because it appears a yellow green color which means that it would reflect those wavelengths and absorb the wavelengths of blue, violet, and red which is in those ranges.

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Pigment 4 is chlorophyll a because it absorbs the most wavelength at the range 350-450 and 650-700 which are the violets and reds which is what the color green reflects. Pigment 5 is chlorophyll b because it absorbs the most of wavelength 400-500 and 650-700 which are the violets, blues, and reds. Pigment 1 is Xanthophyll because it absorbs the most of wavelength 400-500 which is the blue pigment. Pigment 2 is Carotene because it is very close to Xanthophyll since they are both yellow but it absorbs more of the wavelength than Xanthophyll. Pigment 3 is Violaxanthin because it absorbs the wavelength 400-470 which is the blue pigment but it absorbs less than the Xanthophyll and Carotene.

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The porphyrin ring is found at the head of a chlorophyll molecule which contains a magnesium atom in the center that absorbs light. Chlorophyll also contains a long hydrophobic tail that functions as an anchor. This is significant because the chlorophyll can anchor to other hydrophobic proteins in the thylakoid membrane. A plant with a magnesium deficiency will not be able to absorb light energy from the porphyrin ring because that is what the magnesium is used for.

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The role and adaptive purpose of having multiple pigments in a leaf is to absorb different wavelengths of light which allows for the plant to absorb more energy. This energy can be used to power photosynthesis. The different pigments also help protect the plant against the radiation from the sun so that the cells are not damaged. This is what the pigment in humans do as well.

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If Spinach contained Violaxanthin it would most likely be found closer to the bottom of the chromatography paper because it has a low Rf value out of all of the pigments in spinach. The Rf values determine how far up a pigment travels. Violaxanthin has an Rf value of .48 while the highest is .98. The higher the Rf value, the higher a pigment traveled. Since Violaxanthin is on the low end of the Rf values it would be found on the lower end of the paper.

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If a polar solvent were used in place of non-polar petroleum ether then the pigments would not travel far up the paper. This is due to the fact that the pigments are nonpolar and like dissolves like meaning it will dissolve in a nonpolar solution. If the solution is polar then the pigments will not really be dissolved and the pigment colors would not be seen travelling up the chromatography paper.

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If the experiment was done with chromatography paper that was twice as long for twice as long the time then the pigments with Rf values higher than the ones present would

most likely be seen because it would have finally separated from the solvent. These pigments were cut off on the original size chromatography paper so if it had more room and more time it would most likely be able to travel up further and eventually become visible. These pigments are smaller and more soluble than the others in the solvent which would allow it to hold in the solvent for a longer period of time. This could result in the presence of other colors. ●

Plants lose their green in the fall and start to show other colors because in the fall their chlorophyll begins to break down. When the green color of the chlorophyll is no longer visible the other pigments that are present in the leaf begin to show. If this procedure were to be done on a leaf that had already changed colors it would not work because the pigments are already broken down so they would not be present and therefore the color would not be seen on the chromatography paper.

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Error analysis: Some pigments do not match the Rf values in the table for a few reasons. If the chromatography paper was not fully dipped into the solvent then there may not have been enough solvent to pull the pigments further up. This would result in a shorter distance of the pigment being travelled which would affect the Rf value. Another error could be when the pigment of the spinach was transferred to the paper there was not enough. An insufficient amount of pigment would lead to a less visible line of how far the pigment travelled which would lead to a smaller Rf value than the one found in the table....