Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy

Extraction of Spinach Pigments and Analysis by Electronic Absorption Spectroscopy

Ultraviolet-visible spectroscopy is a useful tool for studying the electronic structure of unsaturated molecules and their conjugation. The electronic absorption spectra can generally reveal the degree of delocalization of the conjugated π system. The electronic transition between bonding and anti-bonding orbitals in organic molecules are large and normally require higher energy. As the number of π molecular orbitals increase in conjugated systems, the energy gaps between the filled and unfilled orbitals decrease. Lower energy is needed to promote electrons into an excited stated, resulting in molecules that can absorb in the visible region.

The pigments found in spinach are good examples of highly conjugated molecules that can absorb in the visible region. Spinach contains green pigments known as chlorophylls and yellow pigments known as carotenoids, both which are involved in the photosynthesis process. There are several types of chlorophyll, chlorophyll a and b being the most common. The difference between the two chlorophylls is that a methyl side-chain in chlorophyll a is substituted with a -CHO group in chlorophyll b. Carotenoids are a class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls). The yellow color due to the carotenoids is obscured by the chlorophyll pigments. Structures of chlorophyll and β-carotene are shown in Figure 1.


Chlorophyll


β-carotene

Figure 1 Structures of various visible light absorbing pigments found in spinach

In order to investigate the electronic absorption spectra of the pigments extracted from spinach, the chlorophylls and carotenoids need to be separated. Thin-layer chromatography (TLC) can be used to investigate the solvent system for separation of the compounds. In thin-layer chromatography, the stationary phase is the adsorbent (usually silica or alumina) coated on a sheet of glass, metal, or plastic. The sample is applied as a spot near the bottom of the plate. The TLC plate is then placed in a developing chamber containing a shallow layer of solvent, where the mobile phase (solvent) slowly rises by capillary action.


Under a given set of conditions, a specific compound will travel a unique fixed distance relative to the solvent front. Different compounds generally move at different rates. As a result, if the sample is a mixture of compounds, it will separate into a series of spots at varying distances up the plate (see Figure 2). TLC separation results are expressed in terms of Rf (retention factor) values. The Rf is a ratio calculated by dividing the distance traveled by the sample by the distance traveled by the solvent at the end of the experiment.

Figure 2 Example of a TLC plate at the end of the experiment

Column chromatography is a purification technique used to isolate compounds from a mixture. In column chromatography, the stationary phase is a solid adsorbent placed in a column and the mobile phase is a solvent that is added to the top and flows down through the column. Separation is achieved based on the polar and non-polar interactions among the compounds, the solvent, and the solid stationary phase.

In this experiment, you will extract the pigments from spinach leaves and isolate the chlorophylls and carotenoids using column chromatography. Using electronic absorption spectroscopy, the wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be identified.

OBJECTIVES

In this experiment, you will

·  Extract the pigments in spinach leaves.

·  Determine the Rf values of the pigments in spinach.

·  Isolate the green and yellow pigments using column chromatography.

·  Identify the wavelengths of absorbance peaks for the extracted spinach pigments.

MATERIALS

Part I Extraction

fresh spinach leaves / sodium sulfate, Na2SO4, anhydrous
mortar and pestle / acetone
three 12 ´ 75 mm test tubes / hexane
test tube rack / saturated aqueous sodium chloride
disposable Pasteur pipets and bulb / cotton plug
one No. 0 stopper / 10 mL graduated cylinder
hot plate / weighing paper
250 mL beaker / balance
Temperature Probe or thermometer / boiling stone

Part II Thin-Layer Chromatography

2.5 ´ 7.5 cm TLC plate / 3:7 acetone/hexane mixture
spotting tubes / filter paper
400 mL beaker / ruler
9 cm watch glass / pencil

Part III Column Chromatography

disposable Pasteur pipets and bulb / ring stand with utility clamp
three 12 ´ 75 mm test tubes / acetone
test tube rack / hexane
cotton plug / three 100 mL beakers
sand / 2.5 cm piece of rubber vacuum tubing
silica / weighing paper
spatula

Part IV Measure the Absorbance

LabQuest or computer / acetone
LabQuest App or Logger Pro / hexane
SpectroVis Plus spectrophotometer / samples from Part III
glass cuvettes with lids / tissues (preferably lint-free)
disposable Pasteur pipets and bulb

PROCEDURE

Part I Extraction

1. Obtain and wear goggles. Protect your arms and hands by wearing a long-sleeve lab coat and gloves. Conduct this reaction in a fume hood.

2. Weigh out 1.0 g of fresh spinach leaves. Record the mass to the nearest 0.01 g.

3. Tear the leaves into small pieces and place them in a mortar. Add 3 mL of acetone and 3 mL of hexane and grind the mixture for 3–5 minutes.

4. Create a filter pipet with a cotton plug and a disposable Pasteur pipet. Filter the green solution into a clean test tube. Try to avoid transferring the ground leaves.


5. Wash the solution in the test tube:

  1. Add 2 mL of saturated aqueous sodium chloride solution to the test tube.
  2. Place a stopper in the top and shake the test tube.
  3. Immediately vent the test tube by removing the stopper.
  4. Place the stopper back in the test tube, shake, and vent.

6. Place the test tube in the test tube rack and allow the mixture to separate.

7. Create a filter pipet using a disposable Pasteur pipet and cotton plug. Fill the pipet half-way with solid sodium sulfate. Note: Use the weighing paper to fill the pipet. The final traces of water are removed by treating the organic solution with a drying agent.

8. Filter the green organic layer into a clean test tube. Avoid removing the aqueous layer.

9. Prepare a 70°C hot water bath in a 250 mL beaker. Monitor the temperature of the water bath using a Temperature Probe or thermometer.

10. Evaporate the solvent to approximately 0.2–0.3 mL by placing the test tube in the hot water bath. Add a boiling stone to the test tube to prevent the solution from bumping.

Part II Thin-Layer Chromatography

11. Prepare a development chamber:

  1. Place a piece of filter paper against the side of a 400 mL beaker. The filter paper will help saturate the beaker with solvent vapors.
  1. Add 5–10 mL of 3:7 acetone/hexane mixture to the 400 mL beaker. CAUTION: Acetone and hexane are flammable. Keep away from open flames and hot plates.
  2. Cover with a watch glass.

12. Obtain one TLC plate. Handle it carefully and by the edges so that the adsorbent does not flake off.

13. Prepare the TLC plate.

  1. Using a pencil (NOT an ink pen), lightly draw a line across the plate, approximately 1 cm from the bottom. Across this line, mark the location indicating where the sample will be spotted, making sure it is not too close to the edge of the plate (see Figure 2).
  1. Take a spotting tube and dip one end into the solution containing the spinach extracts. Capillary action will draw the liquid into the tube.
  2. Lightly tap the tube on the mark on the TLC plate. Only a small amount of sample needs to be delivered. The spot should be 1-2 mm in diameter.

14. Place the TLC plate in the beaker and cover with the watch glass. Make sure the plate is not touching the filter paper. The solvent level must not be above the spots on the plate or your sample will dissolve into the solvent.

15. When the solvent has risen to within 1 cm from the top of the plate, remove the plate from the chamber and with a pencil, gently draw a line to mark the position of the solvent front.

16. After the plate has dried, observe the TLC plate and lightly outline the spots with the pencil.

17. Calculate the Rf value for each spot and record the values in your data table.

Part III Column Chromatography

18. Obtain 10 mL of acetone and 10 mL of hexane in separate 100 mL beakers.

19. Assemble the pipet column. CAUTION: Silica powder is harmful if inhaled. Use only in the fume hood.

  1. Place a small piece of cotton into the bottom of the pipet followed by 0.5 cm of sand and enough silica to fill the pipet about 2/3 full. Note: Weighing paper may be used to help fill the column with silica.
  1. Gently tap the side of the pipet for 1 minute to pack the silica.
  2. Add another 0.5 cm of sand to the top of the pipet.

20. Place the pipet column in the 2.5 cm (1 inch) piece of rubber vacuum tubing and secure it to the ring stand.

21. Obtain two test tubes to collect the column fractions and a 100 mL beaker for waste.
Important: Once you start the elution process, it cannot be stopped. You must go to completion. Read Step 22 carefully before continuing.

22. Prepare the column for chromatography:

  1. Place the waste beaker under the pipet column and elute 2-3 mL of hexane through the column. Note: If air bubbles or cracks appear in the column, discard and repeat Step 19.
  1. Elute another 2-3 mL of hexane. Important: Do not let the column get dry during this procedure. Replenish with solvent as needed.
  2. Once the solvent has drained to just above the silica, pipet the green pigment solution from Step 10 evenly onto the column. Allow the solution to adsorb onto the silica.
  3. A yellow band will appear and begin to separate from the green band. Continue adding hexane.
  4. Collect the yellow fraction in a clean test tube.
  5. Once you have collected the yellow fraction, change the solvent by adding acetone.
  6. Continue adding the solvent and collect the clear portion in the waste beaker.
  7. The green band should be moving down the column. Collect this fraction in a clean test tube. Note: Not all of the pigments will be removed from the column.

23. Save the fractions collected off the column.

Part IV Measure the Absorbance

24. Connect the SpectroVis Plus spectrophotometer to the USB port of LabQuest or a computer. Start the data-collection program, then choose New from the File menu.

25. Calibrate the Spectrophotometer.

  1. Prepare a blank by filling a glass cuvette with hexane and cover with a cap. Note: Always wipe the cuvette thoroughly to remove any trace amounts of solvent before placing in the Spectrophotometer. The exterior of the Spectrophotometer is not resistant to organic solvents.
  1. Place the blank cuvette in the Spectrophotometer.
  2. Choose Calibrate from the Sensors menu of LabQuest or the Experiment menu of LoggerPro.
  3. When the warmup period is complete, select Finish Calibration. Select OK.

26. Determine the wavelengths of maximum absorbance for the peaks in the yellow solution.
Note: If the sample is too concentrated, dilute with the appropriate solvent.

  1. Empty the blank cuvette and fill the cuvette about 3/4 full with the yellow solution and place it in the Spectrophotometer.
  1. Start data collection. A full spectrum graph of the solution will be displayed. Stop data collection. Identify the wavelengths of maximum absorbance for each peak and prominent shoulders. Record these values.

·  In LabQuest App, the displayed wavelength of maximum absorbance is automatically identified on your graph with a point protector. Tap on the screen to help identify the wavelengths of the smaller peaks and their shoulders.

·  In Logger Pro, choose Analyze and then choose Examine. Move the cursor to find the wavelengths of each peak and its shoulders.

c.  Store the run by tapping the File Cabinet icon in LabQuest, or choosing Store Latest Run from the Experiment menu in Logger Pro.

  1. Remove the cuvette from the Spectrophotometer and dispose of the solution as directed.

27. Determine the wavelengths of maximum absorbance for the peaks in the green solution.
Note: If the sample is too concentrated, dilute with the appropriate solvent.

  1. Recalibrate the Spectrophotometer with acetone by repeating Step 25.
  1. Repeat Step 26 using the green solution.

28. Dispose of waste as directed by your instructor.


DATA TABLE

Part I Extraction

Mass of spinach leaves (g)

Part II Thin-Layer Chromatography

Pigment color / Calculated Rf

Part IV Measure the Absorbance

Pigment color / Peak wavelengths (nm)

DATA ANALYSIS

1. Draw your TLC plate. Show the calculation of the Rf values.


Instructor Information

1. If a centrifuge is available, centrifuge the spinach extract and save the supernatant for chromatography.

2. Prepare a saturated aqueous sodium chloride solution by adding NaCl in distilled water until it no longer dissolves and a shallow layer of NaCl is visible at the bottom of the bottle.

3. Cut the filter paper in half. Place one piece in the beaker for TLC.

4. The TLC plate will show numerous spots, but this method of column chromatography will simply separate the yellow and green pigments. The data table will have several places to record the observations.

5. It is important to concentrate the spinach extract solution to 0.2–0.3 mL for good separation on the column.

6. Cut a 2.5 cm (1 inch) piece of rubber vacuum tubing. Cut the side open, place the pipet column inside and secure with the utility clamp.

7. The exterior casing of SpectroVis Plus is not resistant to organic solvents. Be careful when handling organic solvents around the Spectrophotometer. Avoid direct contact with organic solvents. Wipe the cuvette before placing it into the Spectrophotometer. Always use a lid with the cuvette to minimize spillage.

8. As an alternative to using SpectroVis Plus, you can also do this experiment with the original SpectroVis, the Vernier Spectrometer, as well as a variety of Ocean Optics Spectrometers.

9. If glass cuvettes are not available, 13 ´ 100 mm glass test tubes can be substituted.