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Addison Gasser Experiment 5

CHE 343-05

10/10/21

Spinach Pigment Extraction and Chromatography

The objective of this experiment was to perform a microscale extraction to isolate the organic
compounds from spinach leaves. The components were purified through thin layer and column
chromatography, with a solvent of hexane and ethyl acetate. After the components were separated and
purified, the pigments were identified by using the tlc data to calculate their Rf values.

Extraction Procedure
 Procedure
1. Approximately 0.5 g fresh spinach leaves
(without stems) were weighed out and
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the exact mass was recorded. The leaves
were torn into confetti-sized pieces and
subsequently placed into a mortar. About
1.0 mL hexanes, 1 mL acetone, and a
small bit of sand were added. The leaves
were ground up with a pestle until the
liquid became a bright and deep green.
2. With a 5” Pasteur pipet, transfer the
liquid to a centrifuge tube. Rinse your
mortar an pestle with another 1.0 mL of
acetone and transfer this to the
centrifuge tube also.
3. If more than 1.5 mL of acetone / hexanes
were still present on the centrifuge tube,
the volume was reduced using
evaporation in a warm sand bath.
4. Add another 1.0 mL of hexanes and 2.0
mL of distilled water to the extract in the
centrifuge tube. Using a Pasteur pipet,
gently draw the mixture in and out of the
centrifuge tube until the 2 layers have
been thoroughly mixed. To maximize
separation of the layers, allow the tube
to stand undisturbed for several minutes.
5. A 9” Pasteur pipet was used to draw off
the aqueous layer and transfer it into a
small beaker.
6. Dry the organic extract in the centrifuge
tube over anhydrous CaCl2 or Na2SO4.
7. A filter pipet was prepared to remove the
drying agent and filter the extract into a
small vial. To do this, a small cotton plug
was inserted into a 5” Pasteur pipet. The
pipet was secured on a ring stand using a
micro-clamp, and a clean collection vial
was placed below the pipet stem.
8. Obtain a tlc plate and spot it with the
extract. Develop the plate in a solution of
hexanes. Set a watch glass over the
beaker to prevent evaporation. Once the
solvent front is within 1 cm of the top of
the plate, remove the plate and mark the
solvent front.
9. Despite most spots being easily seen with
the naked eye, the UV lamp was used to
ensure that all possible spots have been
noted. Each spot was circled in pencil.
Observations
Exactly 0.5060 g of fresh spinach leaves were
weighed out using a scale. The leaves were
ripped into very small pieces and placed into
a mortar. 1.0 mL of acetone and 1.0 mL of
hexane were added to the mortar along with a
pinch of sand, and a pestle was used to grind
up the contents of the mortar thoroughly. The
color of the liquid was observed to be a light
green and it appeared to be mostly solid, so
approximately 2.5 more mL of acetonehexane
solution was added to the mortar.
After the contents of the mortar were
thoroughly ground, the color of the liquid was
observed to be a deep, vibrant green with a
more fluid consistency.
2 layers formed, one darker than the other
Slightly less than 1.5 mL of acetone / hexane liquid
were transferred to the centrifuge tube; therefore,
evaporation in a sand bath was not necessary.
Formed multiple layers.
The top layer was a dark clear green
The bottom layer was a cloudy lime green
An emulsion between the two layers formed that
was cloudy green
1.0 mL of water and hexanes was added, and
mixing was necessary to get rid of the emulsion
Using a 9” Pasteur pipet, the aqueous cloudy
layer was carefully drawn out from the bottom of
the centrifuge tube and transferred into a clean
beaker.
A small cotton plug was inserted into a 5” Pasteur
pipet to create a filter pipet. The filter pipet was
secured with a micro-clamp to a ring stand, and a
clean collection vial was placed underneath the
stem of the filter pipet.
Hexanes: ethyl: methanol (4:3:1 drop)
3 dots were added to the tlc plate
A UV lamp was used to observe all spots on the
TLC plate. Each spot—including both the spots on
the TLC plate visible to the naked eye and the
spots on the plate only visible to the UV lamp—
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Column Chromatography Procedure

Procedure Observations
1. Obtain a microcolumn and insert a cotton
plug. Clamp the column on a ring stand
and place a small beaker beneath it to
collect excess solvent during the column
preparation
2. About 2.0g of silica gel were weighed out Exactly 2.0009 g of silica gel were weighed out in
in a small beaker, and some hexanes a small beaker, and hexane solution was slowly
were stirred in to create a slurry that is stirred in until the solution had a slushy, but
not too thick, and not too thin — it mostly liquid, consistency, and it had become a
should not be so thick that it cannot be proper slurry that was not too thick or too thin.
drawn up into a Pasteur pipet.
3. Keep the solvent above the level of the of Sand was added to the top of the column
the solid absorbent. Pipet the slurry into The column was tapped to prevent air being
the column with a 5” Pasteur pipet. Tap trapped
the side of the column gently to prevent
any air from becoming trapped in the
packing and for better separation.
Continue adding slurry until the column
packing is about 1-1.5 inches from the
top of the glass column. Add a small
amount of sand to the top of the column.
4. To load the extract, use a 5” Pasteur Hexane was consistently added to keep column
pipet to put load sample onto the from drying out.
prepared column. Allow the extract to
travel into the sand layer then add
another 0.5 mL of hexanes to the
centrifuge tube with swirling and loading
that also.
5. Immediately start eluting with neat First fraction was collected when the yellow band
hexanes. When a tight yellow band is was ¾ of the way down the column.
approximately ¾ of the down the The band was a cloudy light yellow.
column, begin collecting fractions in Ethyl acetate was added to increase polarity.
numbered test tubes or vials. To increase An addition two separate fractions were
the polarity of the solvent system, collected.
gradually add more ethyl acetate solvent
mix to the eluent. Collect small fractions
for the pheophytins and chlorophylls.
Continue collecting fractions until the
final-colored band has been eluted.
6. Obtain a large tlc plate and number it The tlc was spotted several times but solutions
along the baseline with the number of were very diluted so there was barely any color
fractions collected. Spot each fraction on the tlc plate
and allow a few seconds for it to dry The capillary tube was cleaned in acetone
before spotting again in the same between spotting’s
position. Repeat 10 or 12 times for each
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fraction. After, clean the capillary tube by

dipping it in acetone and touching it to a
paper towel to draw out the acetone
wash. Do this 2- 3 times between
fractions.
7. Develop the tlc plate. Calculate Rf values The tlc was developed in a beaker that contain a
and make as many identifications as solution of hexanes
possible. Spots were circled but not many were seen
Rf values are calculated in results

Data/Results

Extraction TLC Plate Results

Spot Color Distance Distance Rf Value Compound

Traveled by Traveled by Identity
Spot (cm) Solvent (cm)
1 Light Yellow 0.2 4 0.05 Xanthophyll
2 Light Yellow 1 4 0.25 Xanthophyll
3 Yellow 1.2 4 0.30 Chlorophyll B
4 Yellow/Green 2 4 0.50 Chlorophyll B
5 Green 2.3 4 0.575 Chlorophyll A
6 Greyish 3.4 4 0.85 Pheophytin
7 Dark Yellow 3.7 4 0.925 Carotene

Column Chromatography TLC Results

Spot Color Distance Distance Rf Value Compound

Traveled by Traveled by Identity
Spot (cm) Solvent (cm)
A Yellow 1.3 4.5 0.29 Xanthophyll
B Green 1.8 4.5 0.40 Chlorophyll B
C Grey/Yellow 4 4.5 0.89 Carotene
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Example Calculation

Rf Value, Spot 1 = 0.2 cm / 4 cm = 0.05 (Xanthophyll)

Set-Up Sketches

Discussion

This experiment demonstrates a

principle most are
already familiar with and that is
that solids are more likely to
dissolve in a warm liquid than a
cold one. An impurity will not
dissolve when heat is applied,
and they can be removed from
the
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hot solution through filtration.

Once impurities have been
removed the purified compound
can
begin to cool and reform the
bonds that it typically exhibits.
The purpose of this experiment was to be introduced a variety of different techniques, including
microscale extraction, TLC plate development, preparation of a gravity filter pipet, elution, and
classification of eluted compounds. Spinach leaf pigments were utilized to practice these techniques by
extracting the pigments in both a traditional simple extraction procedure and using a chromatography
column. The data of this experiment was derived from TLC plates and their measurements to obtain Rf
values for each pigment compound.

For the extraction portion of this experiment, acetone/hexanes were used because they are
miscible with each other due to their similar structure. Both are non-polar due to hexanes carbon chains
and acetones ketone group. Hexanes are used in this extraction to selectively extract the desired non-
polar organic substances and leave the other components behind.

Chromatography is the process of separating a mixture by passing it into a mobile phase and
moving the mobile phase across the stationary phase until the compounds in the mixture separate at
different rates. In thin layer chromatography, the mobile phase is specifically a liquid solvent, and the
stationary phase is a solid absorbent also known as a ‘plate’. The mechanism behind a TLC plate is that
capillary action pulls the liquid solvent up the plate. Secondly, the solvent passes over a spot of the
mixture, and the mixture dissolves into that solvent. The solvent and mixture then continue to move up
the plate as the plate reabsorbs the solvent and mixture. As more solvent moves up, mixture is
continuously dissolved and reabsorbed as it moves up the plate. This overall process is called developing
the plate. Different compounds in the mixture will separate and move at different rates. The column
chromatography portion of the experiment was conducted to both filter and purify the spinach extract
pigments that were previously collected. A microcolumn was carefully set up, and a slurry concoction of
silica gel and hexanes was used to prepare it. Once the column was prepared with sand, slurry, and
cotton, the organic spinach extract was added to the column along with hexanes to ensure that nothing
it would not dry out.
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The data from my tlc was accurate. Spinach leaves mainly contain chlorophylls and carotenes,
which was shown, seven spots were visible. Xanthophyll, chlorophyll b, chlorophyll a, pheophytin, and
carotene were the pigments identified. The greatest Rf value was 0.925 which corresponds to the most
non-polar component, carotene. This was apparent because it traveled the farthest. The tlc data also
showed Rf values for xanthophyll which must have been the most polar component because it traveled
the least amount.

POST-LAB QUESTIONS

1. Diethyl ether would be chosen as the most adequate solvent because KCl and H2SO4 are each
quite polar. The solvent chosen should be the least polar possible in order to ensure that a pure
organic product is collected. There should be a polarity difference between solvent and
byproducts to make certain that the byproducts are isolated rather than simply washed away. If
either isopropanol or acetone were used in this scenario, both of which are miscible in water
and highly polar, the byproducts would be washed away.
2. The darker top layer is the organic layer, and the bottom layer is the aqueous layer. The aqueous
layer is denser than the organic layer, so it separates to the bottom. In the separatory funnel,
the products dissolved were in the organic layer on the top. The byproduct of this experiment
was CO2 gas. This was released when the separatory funnel was vented after inverting and
shaking.
3. In this situation, it would be easiest to add a drop of water to the separatory funnel. The layer
that attracts the water droplet is the aqueous layer, and the layer that does not attract the
droplet is the organic layer. This method is simpler than the technique outlined in the Zubrick
textbook.
4. The pictures shown were not correct because a different solvent system should have been used.
For the first trial, too much ethyl acetate was used and not enough hexanes. The ethyl acetate is
polar, and hexane is non-polar. The 3:1 ethyl acetate: hexanes ratio of these would cause the
more non-polar components to move far because they are very polar. In the second, too much
hexanes were used and not enough ethyl acetate. This was seen when the components did not
travel far due to being polar and not moving in the more non-polar hexane solvent. I would
suggest trying a solution with an ethyl acetate : hexanes, 2 : 2 ratio because it would create a
nice balance between the too-polar and not-polar-enough solutions.
5. Darcy made the mistake of not putting a cotton ball in the bottom of the column and using the
ratio of 5:1 pentane: ethyl acetate solvent for the column. This solvent is used to develop the tlc
plate. Darcy should have instead used only pentane for the first fraction then he should have
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gradually increased the polarity by adding more ethyl acetate after each fraction until all the
tight bands were out of the column.
6. The more non-polar components would have moved farther on the plate. This would mean A
would be at the top, then D, then C, then B at the bottom.
7. First extraction:
Wt. of benzoic acid in ether: (10x g/ml) (40mL) = 400x g
Wt. of benzoic acid in water: (x g/mL) (300mL) = 300x g  400x g + 300x g = 700x g
Total weight of acid = 1.80 g
700 x g = 1.80 g x = 0.00257
Wt. of benzoic acid in ether: 400 (0.00257) g = 1.03 g
Wt. of benzoic acid in water: 300 (0.00257) g = 0.771 g
o Second extraction:
Wt. of benzoic acid in ether: (10x g/ml) (40mL) = 400x g
Wt. of benzoic acid in water: (x g/mL) (300mL) = 300x g
 400x g + 300x g = 700x g
Total weight of acid = 0.771 g
700 x g = 0.771 g x = 0.00110
Wt. of benzoic acid in ether: 400 (0.00110) g = 0.440 g
Wt. of benzoic acid in water: 300 (0.00110) g = 0.330 g
o Third Extraction:
Wt. of benzoic acid in ether: (10x g/ml) (40mL) = 400x g
Wt. of benzoic acid in water: (x g/mL) (300mL) = 300x g
 400x g + 300x g = 700x g
Total weight of acid = 0.330g
700 x g = 0.330 g x = 0.000471
Wt. of benzoic acid in ether: 400 (0.000471) g = 0.188 g
Wt. of benzoic acid in water: 300 (0.000471) g = 0.141 g
o Total benzoic acid collected: 1.66 g
- One extraction using 120 mL of ether each time:
o Wt. of benzoic acid in ether: (10x g/ml) (120mL) = 1200x g
o Wt. of benzoic acid in water: (x g/mL) (300mL) = 300x g
1200x g + 300x g = 1500x g o Total weight of acid = 1.80g
o 1500 x g = 1.80 g x = 0.00120
o Wt. of benzoic acid in ether: 1200 (0.00120) g = 1.44 g
o Wt. of benzoic acid in water: 300 (0.00120) g = 0.360 g
o Total benzoic acid collected: 1.44 g

8. If there are similar Rf values for the present compounds, then the TLC plate might show an
overlapping of spots, which would imply a similar polarity. Therefore, the solvent system should
be changed to a different polarity in order for the compounds not to overlap any longer on the
TLC plate.