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chlorophyll lab report

By alyssa-caparelli Nov 29, 2014 1231 Words
Alyssa Caparelli
Organic Chemistry 12A
Professor Alston
October 28, 2014

Isolation of Chlorophyll and Carotenoid Pigments from Spinach


The purpose of this experiment was to isolate ß-carotene, chlorophyll-A, and chlorophyll-B from spinach using column chromatography. Spinach was dehydrated using ethanol, and the pigments were extracted with dichloromethane. The spinach extracts were dried using CaCl2. Then, the solid pigments were run through a column using a non-polar solvent, hexane. The polar absorbent material in the column separated the different pigments by allowing the least polar molecules to travel through the column faster than the more polar molecules. The different pigment layers were collected, dried, and their weights were recorded. ß-carotene was the least polar molecule, and it traveled through the column faster than the chlorophylls. Chlorophyll-A was next to travel through the column followed by chlorophyll-B. Because chlorophyll-A is more polar than ß-carotene and less polar than chlorophyll-B, this observation is reasonable.


ß-carotene is a yellow-orange pigment found primarily in fruits and vegetables. ß-carotene is a carotenoid that is effective in preventing sunburn in sun-sensitive people, reducing the risk of breast cancer and other diseases, preventing asthma attacks caused by exercise, as well as many other uses. Chlorophyll is the green, light-capturing pigment found in plants. Chlorophyll (like heme in red blood cells) is an example of porphyrin. In this experiment, chlorophyll and ß-carotene were isolated using the technique of column chromatography.

Column chromatography is a method used to separate and purify components in a mixture. In gravity column chromatography (used in this experiment), a vertical glass column is packed with a polar absorbent and a solvent. Then the sample is allowed to pass through the column, which separates the different components.

In this experiment, pigment components were dehydrated and extracted from spinach, and the extracts were passed through a glass column using column chromatography. The absorbent allowed some molecules to travel faster through the column. The separate bands were collected and recorded. Hypothesis

This method will be successful in extracting chlorophyll and carotenoid pigments.

Reaction/Experimental Setup

List of Reagents

Molar Mass(g/mol)
Boiling Point(Celsius)
Melting Point(Celsius)
Safety Data
0.791 g/cm3
Highly flammable, irritant

0.6548 g/mL
Flammable, corrosive

Highly flammable, irritant
Anhydrous sodium sulfate
Exposure would cause minor irritation

Pre-Lab Questions

There were no pre-lab questions for the experiment.



100g of spinach was placed in a 250-mL Erlenmeyer flask  150 mL of 100% ethanol was added  the mixture was stirred for 3 minutes  the liquid was decanted into a 250-mL beaker  50 mL of dichloromethane was added to the remaining spinach and stirred for 3 minutes  the mixture was filtered through a plug of glass wool into a 250-mL Erlenmeyer flask  50 mL of dichloromethane was added two more times and filtered  the dichloromethane extracts were combined  the dichloromethane extracts were poured into a separatory funnel  50 mL of saturated NaCl was added and shaken  the lower (dichloromethane) layer was collected in a flask  CaCl2 was added and swirled  the solution was decanted into a beaker containing 5g of silica gel  the solution was stored and allowed to dry  a glass column was obtained  a small plug of glass wool was pushed to the bottom  100 mL of hexane was added  sand was poured in the top to form a 1-cm layer on the glass wool  a slurry of 50g of silica and hexane was added to the column  the crude extract was added on top  the extract was eluted using hexane  the yellow ß-carotene was collected in a flask  the elution continued with a 1:1 mixture of hexane and ethyl acetate  chlorophyll-A and chlorophyll-B were collected in separate flasks  the pigments were covered, stored, and allowed to dry  the total weight of the flasks were recorded  the dry pigment extracts were dissolved with dichloromethane  the extracts were poured into separate vials  the flasks were re-weighed without the extracts  the total weight of the pigment extracts were calculated and recorded


The first band of pigment that was collected from the column was ß-carotene. The second band collected from the column was chlorophyll-A. Chlorophyll-B was the last band collected from the column. After each band was collected in separate flasks, they were allowed to dry and then weighed. The total weight of each pigment layer was recorded in the table below. Pigment

Rf Value
Chlorophyll a
Chlorophyll b

Weight of Pigment Collected
0.04 g
0.04 g
0.05 g


As a result of the absorbent (the silica slurry) being polar, different molecules were allowed to pass through the column at different rates. The different rates of the molecules cause the different pigments to be separated in bands along the column. The polar absorbent is attracted to polar substances, and it binds to these molecules, which slow them down in the column. The non-polar ß-carotene had weak interactions with the polar absorbent. Therefore, the ß-carotene band traveled very fast through the column, and this layer was collected first. The layer of chlorophyll-A was collected next. Chlorophyll-A is more polar than ß-carotene but less polar than chlorophyll-B. Therefore the chlorophyll-A band traveled slower than the ß-carotene, but faster than chlorophyll-B. Finally, chlorophyll-B is the most polar substance. The interactions were very strong with the polar absorbent, which caused this layer to travel very slowly down the column.

The solvent (hexane) is used to affect the separation process. Hexane is a non-polar organic compound. Therefore, it does not interact at all with the polar absorbent. When the non-polar solvent is added to the top of the column, over the sample, the polar molecules are more attracted to the absorbent than the solvent. This causes the polar molecules to be left at the top of the column while the non-polar molecules travel down the column. This is what causes the molecules to separate in bands. In order to get the polar molecules to travel down the column, a more polar solvent (hexane and ethyl acetate mixture) would be used.

Due to the fact that both ß-carotene and chlorophyll are both extremely sensitive to photochemical air oxidation, the solutions must be protected from excess light and air. The solvents used in this experiment are very flammable. Therefore, no flames can be used to speed up this process.


The two columns used in this experiment were successful in separating out the different pigments in the spinach that was provided. The TLC plate made the different pigments very clear to see under the UV light, and the iodine chamber re-enforced with the UV light was showing. From the TLC place I was able to see carotenes, chlorophyll a, and chlorophyll b, xanthophyll, as well as some other pigments there were not listed in the lab book. These other pigments could very likely be a result of air oxidation, hydrolysis, or other chemical reactions involving the pigments previously discussed. Pheophytin a and pheophytin b were not seen on the TLC plate. Based on the UV spectra, the column separation was completed succesfully. Post-Lab Questions

Page 122 #1-4
1. Chlorophyll is more polar and has lower Rf values because they don’t travel as far on the solvent. 2. Pheophytins are less polar than chlorophylls.

3. Due to Acetone being polar, increasing its concentration would make the samples travel farther up the TLC plate, thus making the Rf values larger. 4. They did not consist of a single component and are not completely pure.

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