Chromatography to separate plant pigments lab PDF

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Biology lab about chromatography to separate plant pigments...

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Laboratory report: Using Chromatography to Separate Plant Pigments

1. Observations: Table 1.1: Data collected for students analyzing spinach leaf pigments on a chromatography strip

Trial 1

Trial 2

Pigment Colour

Distance Travelled by Pigment (cm)

Distance Travelled by Solvent (cm)

Reference Flow Value (cm)

Name of Pigment

A = blue-green

0.70

10.0

0.0700

Chlorophyll a

B = olive green

1.70

10.0

0.170

Pheophytin

C = yellow/green

2.60

10.0

0.260

Chlorophyll b

D = yellow

9.90

10.0

0.990

Xanthophylls

A = olive green

1.00

12.3

0.0813

Pheophytin

B = blue-green

1.90

12.3

0.154

Chlorophyll a

C = yellow/green

5.00

12.3

0.407

Chlorophyll b

D = yellow

11.7

12.3

0.951

Xanthophylls

2. Sample Calculations Sample calculations for reference flow value: *Example of measurement calculations using Pigment A in trial 1* Step 1: Identify values for distance travelled by pigment and distance travelled by solvent Distance travelled by pigment = 0.70cm Distance travelled by solvent = 10.0cm Step 2: Identify equation used to solve reference flow value

Distance travelled by pigment Distance travelled by solvent = reference flow value Step 3: Insert values into equation

0.70 cm = 0.0700cm 10.0 cm

∴, the reference flow value for pigment colour A is 0.0700cm

3. Analysis 1. Insert picture of chromatography strip with digital labels Image 1.1: Chromatography paper with spinach pigments separated by a solvent - Trial one

Final distance travelled by solute Xanthophylls

Xanthophylls

Chlorophyllb

Pheophytin Chlorophyll a Where the spinach was cut (initial line)

Image 1.2: Chromatography paper with spinach pigments separated by a solvent - Trial two

Final distance travelled by solute Xanthophylls

Chlorophyllb

Xanthophylls

Chlorophyll a Pheophytin Where the spinach was cut (initial line)

1. Which pigment is (a) most soluble and (b) least soluble in the solvent you used? Explain how you decided For both trials, the most soluble pigment in the solvent was xanthophylls. This pigment took the longest amount of time to be fully visible on the chromatography strip, and travelled the longest distance of 9.90cm in trial 1, and 11.70cm in trial 2. This means that this pigment travelled close to the same distance that the solvent travelled, so therefore it was almost fully dissolved in the solvent. For trial 1 the least soluble pigment was chlorophyll a, which travelled 0.70cm. For trial 2, the least soluble pigment was pheophytin, which travelled 1.00cm. The short distance that these pigments travelled shows that they were barely dissolved into the solvent and could easily separate. The solubility of the pigments is also shown by the concentration of the color on the chromatography paper. The most soluble pigment, xanthophylls, is a very light colour spread across a longer distance, showing that it was fully dissolved in the solvent. The lesser soluble pigments, however, had very concentrated, darker colours, showing that majority of the pigment separated quickly due to the lack of solubility in the solvent 2. Identify sources of error in this investigation Some possible sources of error that may have affected the findings of this lab would be insufficient time allowed for pigment separation, variation in chromatographic behavior of different sheets, unevenness in the way that the paper was hung or movement of the chromatographic chamber and contamination of the solution. If the pigment was not given sufficient time to separate, the results would be inaccurate, because the distance travelled by each pigment would not accurately represent the full distance that the pigment could or would travel along the paper, thus altering the reference flow value. Variation in behavior of the paper could also greatly affect the findings. If the composition or surface of the paper was not uniform throughout the sheet, then the apparent distance that each pigment travelled would not be accurate which would affect the results of the lab. If the paper was hung unevenly or the chromatographic chamber was bumped or moved slightly, the way in which the solution and pigment moved up the paper would have been affected, altering the distance travelled by each pigment and changing the reference flow value. Another source of error that may have occurred is contamination of the solution. The presence of another, unknown substance in the solution due to unthorough cleaning of equipment could have affected the solubility of the pigments or the way in which the solution moved up the paper. Once again this could affect the distance travelled and the reference flow value. 4. Conclusion 5. B) Do you think additional pigments could still be present? Hypothesize how you would find out. Other pigments could still be present. In order to determine if more pigments are present, a longer strip of chromatography paper and more time given for the pigments to separate out would be required. This would allow the most soluble pigments to appear because they move up the paper the furthest and are the lightest in colour, so on a short piece of chromatography paper they may not be visible. Another possibility is putting more of, or a higher concentration of spinach on the paper. This could result in more pigments showing up because they may only be present in very small amounts or concentrations and therefore not visible in the lab because there may have not been enough spinach to begin with....