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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. џ-carotene was the least polar molecule, and it traveled through the column faster than the chlorophyll.
Chlorophyll-A was next to travel through the column followed by chlorophyll-B. Because chlorophyll-A is more polar than џ-carotene and less polar than chlorophyll-B, this observation is reasonable.
Introduction џ-carotene is a yellow-orange pigment found primarily in fruits and vegetables. џ-carotene is a carotene 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 home in red blood cells) is an example of porphyry. In this experiment, chlorophyll and џ-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 arterial 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 carotene pigments. Reaction/Experimental Setup List of Reagents Reagent Molar Mass(g/mol) Boiling Point(Celsius) Melting Point(Celsius) Density Safety Data acetone 58. 08 56 _ 95 0. 791 g/CM Highly flammable, irritant hexane 86. 18 68. 5 -96 0. 6548 g/ml Flammable, corrosive methanol 32. 04 64. 7 -97. 6 0. 7918 g/CM Anhydrous sodium sulfate 142. 04 1429 2. 664 Exposure would cause minor irritation Pre-Lab Questions There were no pre-lab questions for the experiment.
Procedures Observations egg of spinach was placed in a 250-ml Erlenmeyer flask 0 150 ml of 100% ethanol was added D the mixture was stirred for 3 minutes C] the liquid was decanted into a 250-ml beaker D 50 ml of deceleration was added to the engaging spinach and stirred for 3 minutes O the mixture was filtered through a plug of glass wool into a 250-ml Erlenmeyer flask њ 50 ml of deceleration was added two more times and filtered 0 the deceleration extracts were combined O the deceleration extracts were poured into a separators funnel C] 50 ml of saturated Nasal was added and shaken C] the lower (deceleration) layer was collected in a flask C] Cacao was added and swirled D the solution was decanted into a beaker containing g of silica gel the solution was stored and allowed to dry 0 a glass column was obtained 0 a small plug of glass wool was shushed to the bottom 0 100 ml of hexane was added t] sand was poured in the top to form a 1 -CM layer on the glass wool a slurry of egg of silica and hexane was added to the column D the crude extract was added on top O the extract was eluted using hexane O the yellow џ-carotene was collected in a flask O the elution continued with a 1:1 mixture of hexane and ethyl acetate 0 chlorophyll-A and chlorophyll-B were collected in separate flasks 0 the pigments were covered, stored, and allowed to dry the total weight of the flasks were recorded 0 the dry pigment extracts were dissolved with deceleration 0 the extracts were oared into separate vials C] the flasks were re-weighed without the extracts C] the total weight of the pigment extracts were calculated and recorded Results The first band of pigment that was collected from the column was џ-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 Ref Value Chlorophyll a 0. 61 Chlorophyll b 0. 52 Cartooned 0. 93 Oceanography 0. 37 Weight of Pigment Collected Chlorophyll-A Chlorophyll-B џ-carotene 0. 04 g 0. 05 g Discussion 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 џ-carotene had weak interactions with the polar absorbent. Therefore, the џ-carotene band traveled very fast through the column, and this layer was collected first. The layer f chlorophyll-A was collected next. Chlorophyll-A is more polar than џ-carotene but less polar than chlorophyll-B. Therefore the chlorophyll-A band traveled slower than the џ-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 he 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 џ-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. Conclusion The two columns used in this experiment were successful in separating out the different pigments in the spinach that was provided. The TTL plate made the different pigments very clear to see under the UP light, and the iodine chamber re-enforced with the UP light was showing. From the TTL place was able to see carotenes, chlorophyll a, and chlorophyll b, oceanography, as well as some other pigments there were not listed in the lab book.