CAU Chemistry Separation of Spinach Components Project

University of Washington – Tacoma
Organic Chemistry (TCHEM 261)
Separation of Spinach Components Lab
Student Version
Spinach leaves, like other leaves, contain a variety of compounds, called pigments,
which contribute to the color of the leaves. 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. They also contain
pheophytins (similar in structure to chlorophylls, but grey in color).
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.
H3 C
Mg N
H3 C
H3 C
H3 C
H3 C
R = CH3 or CHO
H3 C
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
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. TLC will be used to
evaluate the purity of your fractions. Using electronic absorption spectroscopy, the
wavelengths of absorbance peaks for the chlorophyll and carotenoid pigments will be
1. Read the Whole Lab, especially the post-lab write-up instructions.
2. Learn or Review any concepts or skills you are not confident about applying during
a. Column Chromatography, Nichols – Click on Chromatography tile, then on
“column chromatography” link and “2.4B: Microscale (Pipette) Columns or try this
3. Prepare your lab notebook as described below.
• Title – Use the one given for this lab.
• Clerical Information – Your name, the name of your assigned lab partner for that lab,
and the date on which the in-lab work was started. Be sure to also enter the lab in your
Table of Contents.
• Objective – After reading the entire lab, identify and write down the objective of the lab.
The objective is the question or questions your data will hopefully allow you to answer.
• Hypothesis – For this lab, please write a hypothesis about which compound, chlorophyll
or β-carotene, will absorb elute fastest based on its structure.

Physical & Chemical Properties Table – Use the format below.
Use/Purpose Relevant Physical Properties
in THIS lab
Response to

Pre-lab questions – Do these on a separate sheet of paper, NOT in notebook.
1. For each of the following pairs of compounds, circle the one that will absorb at
the longest wavelength.
2. You are given a sample composed of three compounds, A-C with different polarities.
The sample is loaded onto an alumina column and eluted with 30% methanol/water. After
eluting with 10 mL of solvent and collecting 1 mL fractions, compounds A and B are found to be
in the 3rd and 6th fractions, respectively. Compound C is still in the column.
(a) Which compound is more polar, A or B?
(b) Is compound C more or less polar than compounds A & B?
(c) After eluting with another 10 mL of 30% methanol/water, compound C has still not
eluted. Describe how you would obtain compound C.

Experimental Plan – Use the Experimental directions below to write a plan for the work
you need to do in lab.
Data – Create spaces in your notebook to record all the data you will collect.
Have lab notebook checked and initialed
After preparing your lab notebook, please take the on-line pre-lab quiz to ensure
that you have a solid background for the investigation you will carry out in this lab.
Experimental Directions
Part I Extraction of the Pigments (Week 1)
1. Using a mortar and pestle, or a blender, grind up about 4 g of spinach in methanol. Use a
minimal amount, but it is important that all of the spinach comes in contact with
methanol (Lab staff does this.)
2. Add about 10 mL hexane to the spinach slop and stir thoroughly.
3. Pour off the liquid, squeezing as much juice out of the leaf residue as possible.
4. You now need to remove the methanol. To do this, place the mixture in a separatory
funnel. Add about 10 mL of water, mix thoroughly, and drain the water layer only out of
the separatory funnel. Repeat this step a second time. (Since methanol is more soluble in
water than in hexane, it will come out with the water. It’s important to get all of the
methanol out, so make sure you remove all of the water layer, even if this means you
remove a little bit of the hexane layer.)
5. No matter how well you do step 4, there will now be a little water in with the hexane.
This needs to be removed. To do this, transfer your hexane layer to an Erlenmeyer flask
and add a pinch of anhydrous sodium sulfate. If this coagulates, add a little more. Keep
adding until some of the sodium sulfate still looks powdery after swirling it around in
your hexane mixture.
6. Remove the hexane mixture from the drying agent and heat it gently, in the hood, on a
hot plate. Keep heating until the total remaining volume is about 0.5 mL. (Sometimes
it’s easier just to evaporate to dryness and then add back about 0.5 mL hexane.)
7. Save a small amount of your initial mixture to run a TLC tests after column
Part II Column Chromatography
1. Obtain 10 mL of acetone and 10 mL of hexane in separate 100 mL beakers.
2. Assemble the pipet column. CAUTION: Alumina and Silica powders are harmful if
inhaled. Use only in the fume hood.
a) Place a small piece of cotton into the bottom of the pipet followed by 0.5 cm of sand
and enough alumina to fill the pipet about 2/3 full. Note: Weighing paper may be
used to help fill the column with silica.
b) Gently tap the side of the pipet for 1 minute to pack the alumina.
c) Add another 0.5 cm of sand to the top of the pipet.
3. Place the pipet column in a clamp and secure it to the ring stand. It must be vertical.
4. Obtain six 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. Do not start after 3:30 pm. Read Step 8 carefully before continuing.
5. Prepare the column for chromatography:
a. 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 2.
b. Elute another 2−3 mL of hexane. Important: Do not let the column get dry during this
procedure. Replenish with solvent as needed.
c. Once the solvent has drained to just above the silica, pipet the green pigment solution
from Step I.7 evenly onto the column. Allow the solution to adsorb onto the silica.
d. A yellow band will appear and begin to separate from the green band. Continue adding
e. Collect the yellow fraction in a clean test tube.
f. Once you have collected the yellow fraction, change the solvent by adding acetone.
g. Continue adding the solvent and collect the clear portion in the waste beaker.
h. 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.
6. Save the fractions collected off the column.
Part III Thin-Layer Chromatography
1. Prepare a 2 development chambers: For each,
a. 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.
b. Add 5–10 mL of 30%:70% acetone/hexane mixture to one of the 400 mL beakers. Cover
with a watch glass.
c. Add 5-10 mL of either 100% hexane or 100% acetone or 100% methanol to the second
beaker. Try to use a different solution than the people around you, so that between
everyone you will have tested a variety of solvents. Cover the chamber with a watch
2. Obtain two TLC plates. Handle them carefully by the edges so that the adsorbent does not
flake off or pick up oils.
3. Prepare the TLC plates. Do the following on both plates as identically as possible.
d. 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).
e. Take a spotting (open-ended capillary) tube and dip one end into one of the solutions
containing the spinach extracts. Capillary action will draw the liquid into the tube.
f. 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. Let the spot dry. If it is not a
clearly visible green, again lightly tap the spotting tube on the TLC plate in the exact same
spot. Repeat until you see a distinctly green spot.
g. You should have a spot from your initial spinach mixture and from each of the fractions
eluted from the column which you think may contain material.
4. Place one TLC plate in each beaker and cover with the watch glasses. 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.
5. When the solvent has risen to within 1-5 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.
6. After the plate has dried, observe the TLC plate and lightly outline the spots with the pencil.
7. Draw a picture of your developed TLC plate. Colored pencils are available. Calculate the Rf
value for each spot and record the values in a data table.
University of Washington – Tacoma
Organic Chemistry I (TCHEM 261)
Spinach Lab Post Lab Write-Up
Part II. Column Chromatography
Number of fractions collected, volume of different fractions, time they were eluted, visual
appearance. A table might be good.
Part III Thin-Layer Chromatography
a) Drawing of TLC Plates
b) Show calculation of Rf values
c) Make a table reporting all Rf Values
Pigment color
Calculated Rf
Questions to Answer
1. Was column chromatography effective in separating different fractions from spinach? Be
sure to use your data to support your answer to this question.
2. Was our suggestion of a solvent system appropriate? Again, use data to support your
3. You wrote a hypothesis about which material, the chlorophyll or cartenoids, would elute
fastest. Was your hypothesis upheld by the data, contradicted by the data, or was the
experiment inconclusive?
4. Did Column Chromatography and TLC show similar relative movement of the different
compounds? Is this expected or surprising?
5. Can you suggest further experiments one might do to investigate the relationship between
conjugation and UV-Vis absorbance?
6. What applications might this have? You might consider the importance of sunlight
absorbance in plants, marine algae, or by solar cells for energy production. Don’t try to
cover everything; pick one of these or something else and just discuss the implications for
that one case. Use the introduction to provide background information about whatever
topic you chose to focus on, and come back to that same topic at the end of the

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