This lab used techniques in column chromatography to separate β-Carotene and Chlorophyll pigments from fresh spinach. The spinach was dehydrated using 75 mL of 100% ethanol and stirred for at least three minutes in order to remove the water from the leaves. Dehydration of the spinach is required in order to make the DCM extractions more efficient since the water and wax present on the leaves may be insoluble in DCM.[1] After the spinach is dehydrated, the ethanol and water was removed from the flask to prevent the ethanol from dissolving the pigments needed. 25 mL of DCM was added to the dehydrated spinach and the mixture was stirred for another three minutes. The DCM is required to remove the pigments from the dehydrated spinach leaves.[1]
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A small amount of glass wool was placed into the bottom of a column followed by 25 mL of hexane and a small amount of sand. The sand is added before the silica to create an even base for the silica to lay on. [4] 15 grams of silica gel with a small amount of hexane was prepared into a slurry and added to the column. Even distribution is necessary for the stationary phase so the samples can travel evenly down the silica gel and allow the separation of the pigments to be accurate.[4] When the silica gel was evenly distributed, the silica gel with absorbed pigments was added, followed by a 9:1 mixture of hexane and ethyl acetate in order to elute the B-Carotene. B-Carotene is a non-polar pigment, so adding a non-polar solvent to the column will help it elute through the polar silica gel. [5] Having a 9:1 mixture of hexane and ethyl acetate helps the pigment elute faster through the silica gel. The column was allowed to run until the yellow band of B-Carotene eluted to the bottom of the column and was collected. To extract the chlorophyll, a 1:1 mixture of hexane and ethyl acetate was used. Chlorophyll is more polar than β-Carotene, so a more polar solvent is needed to allow the pigment to elute. [5] The green band that was formed was allowed to run through the silica gel and was collected in a separate flask. The two flasks were covered with aluminum …show more content…
Our yield for chlorophyll was 50.63%, which was around the expected yield for the experiment. Our 10.06% yield for β-carotene is low compared to the expected 25%. The low yield could be to the sensitivity of β-carotene. During evaporation, the mixture was overheated turning the liquid a brown color. β-carotene is extremely sensitive to heat, so the hair dryer could have burned the majority of
A small amount of glass wool was placed into the bottom of a column followed by 25 mL of hexane and a small amount of sand. The sand is added before the silica to create an even base for the silica to lay on. [4] 15 grams of silica gel with a small amount of hexane was prepared into a slurry and added to the column. Even distribution is necessary for the stationary phase so the samples can travel evenly down the silica gel and allow the separation of the pigments to be accurate.[4] When the silica gel was evenly distributed, the silica gel with absorbed pigments was added, followed by a 9:1 mixture of hexane and ethyl acetate in order to elute the B-Carotene. B-Carotene is a non-polar pigment, so adding a non-polar solvent to the column will help it elute through the polar silica gel. [5] Having a 9:1 mixture of hexane and ethyl acetate helps the pigment elute faster through the silica gel. The column was allowed to run until the yellow band of B-Carotene eluted to the bottom of the column and was collected. To extract the chlorophyll, a 1:1 mixture of hexane and ethyl acetate was used. Chlorophyll is more polar than β-Carotene, so a more polar solvent is needed to allow the pigment to elute. [5] The green band that was formed was allowed to run through the silica gel and was collected in a separate flask. The two flasks were covered with aluminum …show more content…
Our yield for chlorophyll was 50.63%, which was around the expected yield for the experiment. Our 10.06% yield for β-carotene is low compared to the expected 25%. The low yield could be to the sensitivity of β-carotene. During evaporation, the mixture was overheated turning the liquid a brown color. β-carotene is extremely sensitive to heat, so the hair dryer could have burned the majority of