Elizabeth Ping | October 25, 2011| 1

Isolation of Chlorophyll and Carotenoid Pigments from Spinach

Introduction:

The purpose of this experiment was to extract chlorophyll and carotenoid pigments from spinach
leaves using a solvent and to use thin-layer chromatography.

B-Carotene

Procedure:
See attachment.
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Observations:

UV and Iodine Visualization of TLC Plates

TLC Plate 1 (solvent front 11.6 cm) TLC Plate 2 (solvent front 12.0 cm)
11.0 cm 11.5 cm
8.5 cm
7.5 cm 7.5 cm 7.5 cm 7.5 cm

3.0 cm
2.5 cm
Acetaminophen

Salicylamide
Ibuprofen

Reference
Pamprin

Pamprin
Caffeine
Aspirin

Calculations

TLC Plate 1
Drug Distance Solvent Front 𝑹𝒇 Calculation 𝑹𝒇
Traveled
Acetaminophen 7.5 cm 11.6 cm 7.5 𝑐𝑚 0.647
𝑅𝑓 =
11.6 𝑐𝑚
Aspirin 8.5 cm 11.6 cm 8.5 𝑐𝑚 0.733
𝑅𝑓 =
11.6𝑐𝑚
Caffeine 3.0 cm 11.6 cm 3.0 𝑐𝑚 0.2586
𝑅𝑓 =
11.6 𝑐𝑚
Pamprin 7.5 cm 11.6 cm 7.5 𝑐𝑚 0.647
𝑅𝑓 =
11.6 𝑐𝑚

TLC Plate 2
Drug Distance Solvent Front 𝑹𝒇 Calculation 𝑹𝒇
Traveled
Ibuprofen N/A N/A N/A N/A
Salicylamide 11.0 cm 12.0 cm 11.0 𝑐𝑚 0.917
𝑅𝑓 =
12.0 𝑐𝑚
Reference 7.5 cm 12.0 cm 7.5𝑐𝑚 0.625
𝑅𝑓 =
12.0 𝑐𝑚
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11.5 cm 12.0 cm 11.5 𝑐𝑚 0.958

𝑅𝑓 =
12.0 𝑐𝑚
Pamprin 2.5 cm 12.0 cm 2.5 𝑐𝑚 0.208
𝑅𝑓 =
12.0 𝑐𝑚
7.5 cm 12.0 cm 7.5 𝑐𝑚 0.625
𝑅𝑓 =
12.0 𝑐𝑚

Conclusions:

Thin layer chromatography was used to find the active ingredients that the over-the-counter
analgesic, Pamprin, contains. It was found that Pamprin contains acetaminophen and caffeine.
This is because the R f values for Pamprin were found to be 0.208 and 0.625 on plate 2 and 0.647
on plate 1. This correlated with the R f values of pure acetaminophen which was 0.647 and pure
caffeine which was 0.2586. The actual components of Pamprin according to the drug label are
acetaminophen, caffeine, and aspirin, and ibuprofen. Ibuprofen was not visualized with either
iodine or UV analysis in the pure form or in Pamprin. Furthermore, even aspirin did not appear
as component of Pamprin in either iodine or UV analysis. Also no spots were seen using the UV
analysis, and therefore R f values were calculated using the spots visualized during the iodine
analysis. All the spots were light to dark yellow-brown in color using iodine analysis.
Compounds of high polarity have R f values (caffeine with a R f value of 0.259) that are less than
polar compounds that have higher R f values (salicylamide, aspirin, and acetaminophen with R f
values all greater than 0.5) that develop on the same plate. This is because compounds that are more
polar interact strongly with the solvent and thus travel a shorter distance on the TLC plate, while
compounds that are less polar interact less strongly to the solvent and thus can travel a longer
distance on the TLC plate. Since only two out of the four possible analgesics were identified during
the TLC analysis, this experiment was a semi-success.

Possible sources of error include the cross-contamination between micropippets, not spotting
enough solution onto the TLC plates, not heating the Pamprin long enough to break down the
binding, and not visualizing the plates under the UV lamp in a dark enough area to see the
formation of spots.

Questions:

1) If the spots are too large, they might merge together.

2) If the spots are too small, then the spots may not be observed after development.
3) The spots must be above the level of the development because if the spots are below the
level of development, the solution in the spots would dissolve in the development
solvent.
4) Contamination might occur in the development of the analgesics because ink contains
organic molecules.
5) To observe minute differences in R f values, UV and iodine analysis such be used.
6) Aspirin may cause bleeding, while Tylenol does not. Aspirin can also be harsh on the
lining of the stomach, while Tylenol is not.

References:
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Pavia, Donald, Gary Lampman, George Kriz, and Randall Engel. Introduction to Organic
Laboratory Techniques: A Microscale Approach. Belmont: Thomson Brooks, 2007.