Organic Chemistry 12A
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.
List of Reagents
Highly flammable, irritant
Highly flammable, irritant
Anhydrous sodium sulfate
Exposure would cause minor irritation
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...
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