Extraction 3A of Spinach
Author: Holly Polk Instructor: Oleksandr Zhurakovskyi Organic Chemistry Lab 243A, Section 010 Date Work Performed: February 12, 2009 Date Submitted: February 19, 2009
Abstract The objective of the lab is to isolate different fractions of spinach components using column chromatography and to analyze the different fractions by TLC. Column chromatography and different eluents will be used to separate compounds based on differences in partitioning between mobile and stationary phases. When the extracts were used for TLC, the most distinctive spots were of carotene and chlorophyll. The spinach extract had a spot at 0.86, corresponding with carotene which had spots with Rf factors of 0.79 and 0.86. The spinach extract also had a spot at 0.23, corresponding with chlorophyll which had a spot at 0.16.
Introduction The objective of the lab is to isolate different fractions of spinach components using column chromatography and to analyze the different fractions by TLC. Column chromatography will be used to separate the different components in different fractions based on the polarity of the different compounds. Another objective of this experiment is to isolate and separate the spinach pigments using differences in polarity to effect the separation. A TLC plate will be used for analysis of the different pigments in spinach. In the crude extract, the following components may be seen (in order of decreasing Rf values): Carotenes (1 spot) (yellow-orange), Pheophytin a (gray, may be nearly as intense as chlorophyll b), Pheophytin b (gray, may not be visible), Chlorophyll a (blue-green, more intense than chlorophyll b), Chlorophyll b (green), Xanthophylls (possibly 3 spots: yellow). The different compounds should rise to different heights on the TLC plates. The basis of the experiment focuses on pigments. The leaves of plants contain a number of colored pigments generally falling into two categories, chlorophylls and carotenoids. Chlorophylls a and b are the pigments that make plants look green. There are also carotenoids which are part of a larger collection of plant derived compounds called terpenes. Spinach leaves contain chlorophyll a and b and b -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls which are oxidized versions of carotenes and pheophytins which look like chlorophyll except that the magnesium ion Mg2+ has been replaced by two hydrogen ions 2H+. Since the different components are colored differently, the separation can be observed visually.
Physical Constants
Chlorophyll a Blue-green, polar C55H72MgN4O5 M. W. 893.5026
Chlorophyll b Green, polar C55H70MgN4O6 M. W. 907.4862
beta-Carotene--yellow, nonpolar C40H56 M.W. 536.8824
Chlorophyll a C55H72O5N4Mg
a-carotene
xanthophyll
pheophytin Other Physical Constants: Compound name Structure
Mole cular weig ht (g/m ol)
Melting Point Celsius
Boiling Point Celsius
Safety considerations (if any)
Iodine
II
254 g/
mol
113.7 C
184.3 C
Iodine is very toxic if swallowed or inhaled. Iodine has a significant vapour pressure at room temperature which can lead to the build-up of dangerous levels of iodine vapour. Exposure to iodine may lead to reproductive damage. Iodine may be absorbed through the skin.
Hexane (C6H14)
8 6.1 8 g/ mol
95 C
69 C
Flammable. May cause impaired fertility. Harmful by inhalation. Irritant. May cause CNS depression. Flammable, irritant, toxic.
Acetone (C3H6O)
5 8.0 8 g 94.9 mol C
1
56.53 C
Anhydrous Sodium Sulfate (Na2SO4)
1 42. 04 884 C g/m ol
-
Eye irritant
Alumina (Al203)
1 01. 96 2054 g/ C mol
Not needed for this lab.
May be harmful if inhaled. May be irritating to mucous membranes and respiratory tract, causing cough and/or shortness of breath.
Safety Goggles and aprons to be worn Acetone is volatile and flammable Avoid breathing vapors of the reagents See safety of specific materials in physical constants. Procedure 1. Prewarm a water bath to 70C. 2. Preparation of spinach leaves: - Leaves can be prepared either for the whole class, or individually. The procedure is meant for the entire class. - Fill the blender about full with fresh spinach leaves, and add the following: ~10mL of water 2-3 tablespoons salt ~100 mL of a 75/25 hexanes/acetone mixture - Run the blender until almost pureed, and add more liquids if necessary - Pour the mixture into a beaker - The organic layer should be very dark green in color. If it isnt, add acetone a little bit at a time until the green color is mostly in the organic layer - Pour 3-4 mL of the mixture into each students centrifuge tube. Add some hexane if necessary - Centrifuge for 1 minute, and then transfer the upper organic layer to a test tube - Add anhydrous sodium sulfate to dry the solution - Filter the dried solution using a 25mL filter flask and vacuum suction. - Allow air to blow over the solution by leaving the vacuum on until all or most solvent has evaporated. - Add a minimal amount of hexanes to redissolve, and transfer to a test tube marked as E (extract) 3. Dry pack a microscale column in a 5 Pasteur pipet with alumina (~2cm) 4. Use hexanes as the solvent to moisten the column. Do not let the column run dry. 5. When the solvent has drained to the top of the packing, place about 0.5mL of the concentrated green solution on the column. 6. When the pigment has all absorbed on the column, keep using hexanes as the eluent. 7. Continue collecting the solvent eluting from the column (tube #1) until the orange band (carotene) is close to the bottom of the column. The orange band will be clearly visible. Collect the orange band in test tube #2. 8. After the orange band has eluted from the column, a more polar solvent mixture is added to the column, namely 75/25 hexanes/acetone solution. Use ~4mL of the mixture, and collect the eluent in test tube #3. This polar solvent will move the green chlorophyll band down the column. 9. Once the green band has eluted from the column, switch to test tube #4. Six (6) mL of acetone should be sufficient to elute organic all material off the column. The column can then be allowed to go dry.
10. You should feel free to collect more fractions, as many as you feel are necessary. Be aware that not all fractions will be colored. 11. After the column chromatography, evaporate the solutions in the test tubes to almost dry. Remove the test tubes from the heat source as soon as only a few drops of solvent remain. Add 2-3 drops of hexanes to the residues. 12. To run TLC analysis of the different fractions, prepare a development chamber using the 75/25 hexanes/acetone mixture. Spot the four different solutions on the TLC plate using a capillary about 1.5-2cm from the bottom of the plate. Try to keep the spots as concentrated as possible. Run the TLC plate. -Remove the plate from the chamber when the solvent front is about 1-2cm from the top of the plate and mark the solvent front. 13. The spots on the TLC plate will be naturally colored. In the extract E you should be able to see all the different pigments listed here in order of decreasing Rf values: Carotenes yellow orange Pheophytin a grey Pheophytin b grey (may not be visible) Chlorophyll a blue green Chlorophyll b green Xanthrophyll yellow 14. The different pigments on the TLC plate can be viewed under UV light (both wavelengths) or after exposure to iodine vapors. 15. Calculate the Rf values for the different pigments. Draw the TLC plate in the lab notebook. Calculate Rf with the formula Rf = distance traveled by the spot/distance traveled by the solvent front. 16. Determine the components present in the different fractions collected from the column chromatography.
Results Column Chromatography Tube 1: colorless Tube 2: yellow color from orange band Tube 3: colorless- mostly from the hexane/acetone Tube 3a: green color from green band Tube 4: colorless- acetone wash to extract organic material After thin layer chromatography, the following results were observed:
Plate 1Standard A (Distance Traveled in cm) 1.8 (greenish color) 2.2 3.1 3.9 6.8 (yellowish color) Solvent Front Rf 0.23 0.28 0.39 0.49 0.86
Pure spinach extract
7.9cm
Plate 2 Tube A (Distance Traveled) Solvent Front Rf
Tube 1: clear solution from hexanes Tube 2: yellow color from orange band Tube 3: colorlessmostly from the hexane/acetone Tube 3a: green color from green band Tube 4: clear solution from acetone wash to extract organic material
Possible 6.2cm
7.3cm
0.85 0.79 0.86
5.8cm and 6.3cm
7.3cm
No observed spot
7.3cm
None observed
1.2cm
7.3cm
0.16
No observed spot
7.3cm
None observed
Calculations Rf = distance traveled by the spot distance traveled by the solvent front
Example: Tube 3a Rf = 1.2cm/7.3cm = 0.16
Discussion In the extraction of pigments from spinach leaves, salt and a hexane acetone mixture were initially blended with the spinach leaves to help separate organic material. Column chromatography was used to separate the different components in different fractions based on polarity. The less polar eluent was able to elute the carotene, and then the more polar eluent eluted the chlorophyll. A TLC plate is used for analysis of the different pigments in spinach. The behavior of the spinach components in the column and TLC was based on the components polarity. The compounds in the column interact with the stationary phase to different extents and separate as in TLC. The functionally groups on the compound effect its polarity. For example, since b -carotene is a hydrocarbon it is very nonpolar. Both chlorophylls contain C-O and C-N bonds which are polar and also contain magnesium bonded to nitrogen which is such a polar bond it is almost ionic. Both chlorophylls are much more polar than b -carotene. On close inspection of the molecules, it becomes obvious that the two chlorophylls differ only in one spot. Chlorophyll a has a methyl group (--CH3) in a position where chlorophyll b has an aldehyde (--CHO). This makes chlorophyll b slightly more polar than chlorophyll a. So, the pigments in order of decreasing Rf values are: carotenes, pheophytin a, pheophytin b, chlorophyll a, chlorophyll b, and then xanthophyll. The compounds were extracted form the column based on their polarities. In this experiment, column chromatography started with tube 1, which was colorless, then tube 2, which was a yellow color from orange carotene band, then tube 3 which was colorless- mostly from the hexane/acetone, then tube 3a which had a green color from green chlorophyll band, and lastly tube 4, a colorless- acetone wash to extract organic material.
When the extracts were used for TLC, the most distinctive spots were of carotene and chlorophyll. The spinach extract had a spot with an Rf factor of 0.86, corresponding with isolated carotene after column chromatography which had spots with Rf factors of 0.79 and 0.86. The spinach extract also had a spot with an Rf factor of 0.23, corresponding with isolated chlorophyll after column chromatography which had a spot with an Rf factors of 0.16. Another possible spot on the TLC plate was for tube 1, which was colorless. However, this was most likely just human error from smudging the plate after putting it in the iodine. Other errors may have resulted from not switching collecting tubes right when the band reached the bottom of the column. That would have resulted in carotene pigments in tube 1, which would explain the barely visible TLC spot. Choice of eluent is important for separation because it determines the rate that compounds can be eluted from the column. The more polar the substance being analyzed, the more polar the eluent should be. In the case of this lab, the spinach analyzed has two compounds significantly differing in polarities. Carotene is very non-polar, and there is barely any attraction between the carotene and polar stationary phase, so it easily elutes off the column using a nonpolar hexane eluent. However, more polar substances like xanthophyll are more polar and stick to the column, so a more polar solvent like the 75/25 hexane acetone mixture is needed to elute the column.