Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05

Date: 9/13/2021

Pre-Lab Submission Lab Section: AX05

Project 1: Separations, Purification of Plant Pigments
Amanda Haner, Kaitlyn Bergeron, Kemal Rifky

Goal
The goal of the project is to extract and isolate plant pigments using solvent extraction and column
chromatography. The relationship between absorbance of light and concentration will be explored using
the UV/VIS spectrophotometer.

Background
Chromatography is a technique that dates to the mid-1800s that is used for chemical separation
(2). There are different types of chromatography, including gas chromatography, thin-layer
chromatography, column chromatography, etc. Specifically, column chromatography is a widely used
technique that depends on differential interactions of molecules between a stationary phase and a
mobile phase (4). The stationary phase of column chromatography is typically a solid or liquid that stays
fixed within the column while the mobile phase is the solvent moving through the column and helping
the material being separated throughout the stationary phase. The mobile and stationary phases of
chromatography have different polarities (6).
Electronegativity is the ability of an atom to attract electrons to itself in a chemical bond, which
results in both polar and ionic bonds (7). The trend of electronegativity can be seen on the periodic
table, generally increasing across a row from left to right and decreasing down a column.
Electronegativity is inversely related the size of an atom; the larger an atom is, the less able it is to
attract electrons in a chemical bond (7). The electronegativity difference between two bonding atoms
affects the degree of polarity of a chemical bond. The greater the electronegative difference there is
between two bonding atoms results in greater polarity of a bond. On the other end, if two atoms have
identical electronegativities, it results in a nonpolar bond since they share the electrons equally (7). The
polarity of a bond is quantified by the size of its dipole moment, which occurs anytime there is a
separation of positive and negative charge (7). A dipole moment can be calculated using the equation:
µ=qr

where q is the magnitude created by separating two particles of equal but opposite charges and r is the
distance (7).
Molecules can also be polar depending on their shape and the nature of the bonds present (7).
For instance, if a diatomic molecule, a molecule composed of two atoms, has a polar bond, the entire
molecule is polar. In a polyatomic molecule, a molecule composed of three or more atoms, the presence
of polar bonds does not determine if the molecule is polar, the molecular geometry does (7). The
molecule is polar if the molecular geometry has dipole moments from individual polar bonds that add
up to a net dipole moment. The molecule is nonpolar if that sum is zero, meaning that the dipole
moments of individual polar bonds cancel each other out (7).
Separation of mixtures into its different pure components results from the components having
different solubilities throughout the phases (6). If the stationary phase used in chromatography is polar,
then the nonpolar components of the mixture being separated will easily pass through the stationary
phase while the more polar components will spend more time in the stationary phase (6). This
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

difference in time is what separates the mixture since the least polar components will elute first and the
more polar components elute last (6).
Plant pigments are among the many mixtures that can be separated using chromatography.
Plant leaves have two major classes of pigments: chlorophylls and carotenoids. Chlorophyll is
responsible for the green color of most plants as it reflects predominantly green light and absorbs other
colors (5,7). It is the primary pigment that helps plants undergo the process of photosynthesis, when
light energy absorbed from the sun is converted into chemical energy (7); chlorophyll is the main site for
this light absorption (5). In addition to chlorophyll a and chlorophyll b, plants also contain other
pigments that become noticeable in the fall since the leaves die and the chlorophyll decomposes (2).
The most common among these pigments are carotenoids, which include xanthophylls and beta-
carotene. These carotenoids can be found in plant leaves but are most commonly found in specific
fruits. They are responsible for the red, orange, and yellow colors found in these fruits (2).

Figure from (2)

Methods and Justifications

Part 1: Column Chromatography
Column chromatography will be used to separate the pigments from one another (1). Chromatography
is a technique used for separating mixtures of liquids (8). A vertical glass tube is packed with an
adsorbing material, in this case silica, and the sample is poured into the column and is continuously
washed through with a solvent (elution) (8,9). While the original procedure called for alumina, that
chemical is not provided in Chem 1010 labs (1). Silica is an adsorbing material and is insoluble in water
(10). Cotton, hexane, silica, and acetone will all be used to replicate column chromatography (1,3).
Column chromatography works because different components of the sample are adsorbed to different
extents and move down the column at different rates (8). Because different pigments have different
polarities, the polar pigments (chlorophyll a and b) will move through the column slower than the
nonpolar pigments when hexane is the solvent (11). The best way to obtain chlorophyll a and b is by
using a mix of acetone and hexane, because hexane is nonpolar and acetone is polar, and chlorophylls
are more solvent in acetone (12). The best way to obtain carotenoids is by extraction with hexane,
because hexane is a nonpolar solvent (13). The collection of the eluate as it passes out from the column
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

in fractions is the best way to obtain the separated pigments (8). Some limitations of the column
chromatography is that chlorophyll a and b will be very close together when they separate in the
column, and it can be easy for the two samples to bleed into one another (1). The column must be
continuously washed through with the elution, but the elution will be changed four times to elute the
eluate (1). This change could be too slow or two quick, messing with the concentrations of the eluate. A
variable that might affect the results is that the experimenters must watch for the change in the eluate
(1). If the change is not noticed quick enough, especially with chlorophyll a and b, the results could be
affected. This method was chosen because it was done successfully in a peer-reviewed source (1). The
changes made to the column were implemented after research on what each of the changes were (i.e.,
Alumina to silica, etc.). At the end of the column chromatography, there should be four 2-dram vials
with different colored pigments in them (1). Those four 2-dram vials will be placed in the UV-Vis for
analysis of absorbance (3). Success will be determined based on the number of different colored dram
vials, and UV-Vis absorbance readings for each dram vial.

Part 2: UV-Vis Spectrophotometer

UV-Vis spectrophotometer will be used to determine the absorbance of the extracted pigments (3). In
turn, that absorbance will be used to determine concentration via Beer’s Law (14). The UV-Vis
spectrophotometer measures the ultraviolet spectrum (15). Specifically, it measures the intensity of the
radiation (light) absorbed by the sample as a function of wavelength (5). When looking at an object, the
color you see is the color that is reflected off the object instead of absorbed (7). Each color of the
spectrum has a specific wavelength (7). Absorbance is needed to determine concentration because the
purity of the pigments is unknown. The absorbance of the pigments should be measured between 0.3
and 0.85 (3). If absorbance is below 0.3, then the plant extract is too diluted (3). If absorbance is above
0.85, the plan extract needs to be more diluted (3). This is a limitation to using UV-Vis because the
absorbance is dependent on the concentration of the solution (3,14). If the extracted pigments are
mixed, the UV-Vis will incorrectly read the absorbance (16). Since absorbance is necessary for
concentration, the experiment will produce incorrect results (16). The data will be analyzed by
determining the concentration of the pigments from the absorbance. Using the UV-Vis was determined
from the sources provided. While four of the sources talked about separating the pigments, only one
source talked about finding the absorbance levels of each pigment (3). It also outlined the wavelengths
at which each sample will absorb the best (3). Each pigment will have their own absorbance spectrum
with a specific peak (3). When placed in the UV-Vis, and that peak will be used to identify what the
pigment is. Additionally, it talked about what the absorbance means if it is too high or too low and gave
the range for acceptable absorbances (3). The UV-Vis was the best instrument provided that would
allow the relationship between absorbance and concentration to be observed. The use of Beer’s law will
help to explore the relationship between the two more (14).

Procedure
Materials:
Silica
Ring Stand Clamp
Column Sand
Acetone
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

Hexane
Mortar and Pestle
Cotton
3 6 Inch Pasteur pipets and bulbs
2 10 ml Graduated Cylinder
2 50 mL beakers
5 10-dram vials
4 2-dram vials
Plant leaves (spinach)
UV/VIS Spectrophotometer

Part 1- Hexane Extraction

1. Tear 10g of fresh plant leaves and grind them using a mortar and pestle for two minutes (1)
2. Add acetone until covering plant material. Continue to crush plant leaves for 2-5 minutes (1)
3. Decant and discard the acetone (1)
4. Add 15 mL hexane and continue to grind the mixture for five minutes and pipette the solution
into a 50 mL beaker. (1)
a. Repeat extraction process 2-3 times until you get a good amount
5. Wait for hexane and water to separate in beaker and pipet the hexane layer into a suitable
container (modified from 1)
Part 2- Preparing the Column (modified from source 2)
1. Plug the Pasteur pipet with cotton in the narrowest part of the column
2. Attach the Pasteur pipet, or the column, to the ring stand clamp
3. Weigh around 2 mm of sand on analytic balance and pour into column, or pour into column until
the sand reaches main body
4. Fill the column 2/3 of the way with silica, leaving about an inch of space at the top of the column
(9)
5. Weigh around 2 mm of sand on analytic balance and pour into column
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

Part 3- Separating the Pigments

1. Label each 10-dram vial with a sharpie as one of the five following names:
a. Waste, beta-carotene, xanthophylls, chlorophyll a, and chlorophyll b
2. Saturate the column using 100% hexane
3. Add 1.0 mL prepared pigment extract to column (2)
4. Start elution using 100% hexane, being careful not to let the solvent level to drop below the top
layer of sand (2)
5. Use the clean 10-dram vial labeled beta-carotene to collect the band of color until drops
become colorless (2)
6. Switch out previous 10-dram vial with the 10-dram vial labeled waste to collect colorless drops
7. Change the eluting solvent to a 90:10 hexane: acetone mixture (9 mL hexane and 1 mL acetone)
to elute the xanthophylls (2)
8. Use the clean 10-dram vial labeled xanthophylls to collect the xanthophyll layer, this layer
should be yellow (2)
9. Once the xanthophyll layer is eluted, change the eluting solvent to 20:80 hexane: acetone
mixture (2 mL hexane and 8 mL acetone) (2)
10. Use the clean 10-dram vial labeled chlorophyll a to collect the chlorophyll a layer, this layer
should be blue-green color (2)
11. Once the chlorophyll a layer is eluted, use the clean 10-dram vial labeled chlorophyll b to collect
the chlorophyll b layer, this layer should be bright green (modified from 2)
12. Once all color bands have been collected from the column, place the 10-dram vial labeled waste
under the column to let the column run dry
13. Transfer the four pigments eluted into 4 2-dram vials. Place at least 2 mL of liquid into each.
Label.
Part 4- Using UV-VIS for light absorbance (modified from source 3)
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

1. Zero the UV-VIS.

2. Place the Chlorophyll A dram vial into the UV-VIS. Record the absorbance level.
a. Chlorophyll A absorbs light most strongly at A428 and A661 nanometers (3). Finding
absorbance level at these two wavelengths will allow the ability to estimate the
concentration of the solution (More at 428 nm).
3. Place the Chlorophyll B dram vial into the UV-VIS. Record the absorbance level.
a. Chlorophyll B absorbs light most strongly between A400 and A500 nanometers (3). Finding
absorbance level between these two wavelengths will allow the ability to estimate the
concentration of the solution (Closer to 450 nm).
4. Place the Xanthophyll dram vial into the UV-VIS. Record the absorbance level.
a. Xanthophyll absorbs light most strongly between A400 and A500 nanometers (3). Finding
absorbance level between these two wavelengths will allow the ability to estimate the
concentration of the solution (Closer to 450 nm).
5. Place the Beta-carotene dram vial into the UV-VIS. Record the absorbance level.
a. Beta-carotene absorbs light most strongly between A400 and A500 nanometers (3). Finding
absorbance level between these two wavelengths will allow the ability to estimate the
concentration of the solution (Closer to 450 nm).
6. Use the Beer-Lambert's Law and absorbance level to determine concentration of each dram vial.

Anticipated Results
It is expected that at least four different pigment molecules are extracted and isolated from a plant
source. Two of those pigments are expected to be two types of chlorophyll while the other two are
expected to be two types of carotenoids. If the outcome is successful, the first band to pass through the
column should be beta-carotene, which is anticipated to be a yellow-orange color. After changing the
eluting solvent, the next band of color to pass through the column should be the xanthophyll, which is
anticipated to be a yellow color. Changing the eluting solvent once again, the next two bands of color to
pass through the column should be chlorophyll a and chlorophyll b, which are anticipated to be a blue-
green color and bright green color respectively. Beta-carotene should be the least polar component of
the plant source since it should elute first and chlorophyll b should be the most polar component since it
should elute last.

Safety Analysis
Silica is slightly hazardous because it can cause significant irritation. It causes respiratory tract irritation
and may cause eye and skin irritation. Work with Silica may be carried out outside of the fume hood.
Nitrile gloves and appropriate protective eyeglasses or chemical safety goggles in addition to face
protection when working with silica to avoid contact with eyes, skin, and clothing. Any leftover Silica
should be disposed of in the proper waste container. Silica is a colorless to white solid that has a melting
point 1710°C and a boiling point of 2230°C. (17)

Acetone is slightly hazardous because it may be harmful if swallowed or inhaled. Due to it being
extremely flammable, its vapor might cause flash fire, breathing in vapors can cause drowsiness and
respiratory tract irritation. It is extremely flammable; thus, it should be kept away from open flames.
Work with Acetone may be carried out in the fume hood. Nitrile gloves and appropriate protective
eyeglasses or chemical safety goggles in addition to face protection when working with Acetone to avoid
Student Names: Amanda Haner, Kaitlyn Bergeron, Kemal Rifky Lab Section: AX05
Date: 9/13/2021

contact with eyes, skin, and clothing. Contact with eye, skin, and clothing should be avoided. Any
leftover Acetone should be brought to the TA for safe disposal. Acetone is a clear colorless liquid that
has a melting point -94°C and a boiling point of 56.5°C. (18)

Hexane is hazardous because it is extremely flammable, thus it should be kept away from open flames. It
may cause skin irritation or corrosion. Contact with eye can cause irritation or serious eye damage. Work
with Hexane may be carried out in the fume hood. Nitrile gloves and appropriate protective eyeglasses
or chemical safety goggles in addition to face protection when working with Hexane to avoid contact
with eyes, skin, and clothing. Contact with eye, skin, and clothing should be avoided. Any leftover
Hexane should be brought to the TA for safe disposal. Hexane is a colorless liquid that has a melting
point -95°C and a boiling point of 68°C. (19)

References:

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2. Johnston, A., J. Scaggs, C. Mallory, A. Hasket, D. Warner, E. Brown, K. Hammond, M.M.
McCormick and O.M. McDougal. A green approach to separate spinach pigments by column
chromatography. Journal of Chemical Education (2013) 90:796-798.
3. Lichtenthaler, H.K. and Buschmann, C., Chlorophylls and carotenoids: Measurement and
characterization by UV‐VIS spectroscopy. Current Protocols in Food Analytical Chemistry (2001)
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