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CHEM 282
Experiment #3: The Extraction of Green-Leaf Pigments and their Chromatographic Separation
Sophie Wolkoff (20107258) & Kaelen Partridge (20127197)
TA: Bily Deng
February 6, 2020
Experimental:
Step 1 of this experiment included a brief introduction to the classes of compounds that provide plants with their colour. These groups included chlorophylls, carotenoids and flavonoids. In step 2, the extraction of chlorophyll and carotenoid pigments was performed by grinding spinach leaves in a mortar with a 1:1 hexanes-
methanol solution. This mixture was filtered into a separatory funnel where it was rinsed
with hexanes and washed repeatedly with water. The resulting organic extract was collected to be dried with sodium sulfate and decanted in order to collect a dry liquid. This liquid was subsequently evaporated on a steam bath until about 1-2 mL remained.
Step 3 involved the thin-layer chromatographic (TLC) analysis of the plant extract. Three jars containing varying ratios of hexanes:ethyl acetate solution, 9:1, 7:3 and 5:5 by volume, were prepared about 10-15 minutes before their use. A TLC plate was then placed in each of the jars to develop, each plate containing a spot of the green
extract obtained from the lab and a spot of the provided β-carotene sample. The solvent
rose to within 0.5cm from the top of the plate before they were removed, dried, and measured for the final positions of their pigments. Rf values were calculated for each plate using their measurement values, and β-carotene was identified within the extract on the plates using a side-by-side comparison with the authentic β-carotene sample.
The column chromatographic analysis of the extract in step 4 was performed in groups of four, combining the organic extracts from both pairs. A slurry of silica gel in hexanes was prepared and added to the column in portions in order to form a chromatographic column. After the column was completely set up, being careful not to involve water, the green hexanes extract was added and drained to enter the silica. The
column was then eluted slowly with a 9:1 mixtures of hexanes:ethyl acetate as the
pigments separated within the column. The fastest moving pigment was collected into a pre-weighed flask to be analyzed again by TLC, using β-carotene as the reference, and evaporated to render a final mass of residue.
Results
Table 1: R
f
value for varying solvents Solvent Ratio of Hexanes:Ethyl-Acetate
Solvent height (cm)
Sample height (cm)
Reference β-carotene height (cm)
R
f
value
5:5
4.00
3.95
3.95
0.988
7:3
3.70
3.65
3.65
0.987
9:1
3.75
2.50
2.50
0.667
R
f
value calculations
R
f
= sample height (cm)/solvent height (cm)
5:5
R
f
= 3.95/4.00 = 0.988
7:3
R
f
= 3.65/3.70 = 0.987
9:1
R
f
= 2.50/3.75 = 0.667
Table 2: R
f
values for after column chromatography
Solvent ratio of Hexanes:Ethyl-Acetate
Solvent height (cm)
Sample height (cm)
Reference β-carotene R
f
value
height (cm)
9:1
3.35
2.50
2.35
0.746
R
f
value calculation for 9:1 solvent
R
f
= 2.50/3.35 = 0.746
Mass of β-carotene residue in sample
Mass = mass of flask and residue (g) - mass of flask (g)
Mass = 23.954 g - 23.887 g = 0.067 g
% of β-carotene in spinach
% = (mass of β-carotene residue/mass of spinach) x 100%
= (0.746 g / 5.022 g) x 100%
= 14.85%
β-carotene
TLC plates
5:5 7:3 9:1 9:1 after column chromatography
B-
carotene from extract
Referenc
e B-
carotene
B-
carotene from extract
B-
carotene from extract
B-
carotene from extract
Referenc
e B-
carotene
3.75
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