Experiment 3: Thin-layer Chromatography and Column Chromatography

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Name: AGAWIN, Aaron D. Instructor: Engr. Edsel B. Calica, Ph.D.
Section: 2CHEA Date Expt. Discussed: 09/19/2020
Group No.: 1 Date Report Submitted: 12/19/2020
Experiment 3: Thin-layer Chromatography and Column Chromatography
ABSTRACT
The TLC and Column Chromatography experiments' objectives were to determine the
effects of the polarity and dielectric constant of solvent mixtures and eluents on the separation of
component mixtures, to isolate red peppers' components through Column Chromatography, and
to determine a sample commercial analgesic's active ingredient. TLC and Column
Chromatography were used throughout the experiment. Reagents such as anhydrous sodium
sulfate, Hexane, Acetone, silica gel, isopropyl alcohol, and distilled water. All of the experiments
showed that a component's displacement is directly proportional to the polarity of the component
and solvent or eluent. The first virtual TLC experiment showed that an ideal solvent for separation
is a mixture of less polar and more polar solvent due to differences in affinity between the silica
gel and solvent mixture. The second virtual TLC experiment showed that Spinach leaf contains,
in order of polarity, Carotene, Xanthophyll, Chlorophyll A, and Chlorophyll B. The virtual Column
Chromatography experiment showed that spinach leaves' dark pigment has less polar Beta-
Carotene and more polar Chlorophyll. The actual TLC experiment confirmed that the sample
commercial analgesic most likely contains Acetaminophen as an active ingredient as the sample
commercial analgesic yielded a spot that coincides with the spot of Acetaminophen. Further
investigations on the high polarity of the solvent mixture in the second virtual TLC experiment, the
possibility of other compounds in the actual TLC experiment, and the yellow and orange color
bands' identities in the actual Column Chromatography experiment are needed.
1. INTRODUCTION
Chromatography is a method for separating a mixture’s components based on their
distribution between immiscible stationary and mobile phases. It comprises two types –
Preparative and Analytical. The former is for separation of large quantities of compounds while
the latter is their analysis. The mobile phase flows with the analyte through a stationary phase
that adsorbs the analyte. The analyte’s attraction between the stationary and mobile phases
depends on the physical and chemical properties of the analyte and stationary and mobile phases,
such as intermolecular forces and polarity. Silica (SiO2•xH2O) and alumina (Al2O3•xH2O) are
typical stationary phases. They have sites for hydrogen bonding and dipole-dipole interactions
with another compound from the hydroxyl groups and bonded Oxygen atoms shown in Figure 1,
resulting in the high polarity of silica and alumina. Hence, they will attract a polar analyte relative
to a nonpolar or less polar solvent or solvent mixture [1].
Figure 1. hydrogen bonding and dipole-dipole
interaction sites in Silica (left) and Alumina (right)

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Thin Layer Chromatography
TLC is an analytical chromatography method that deposits powdered stationary phases like
silica or alumina on thin glass, aluminum, or plastic films. However, most separated components
are colorless [2]. Hence, there are destructive, semi-destructive, and non-destructive visualization
methods for separated components to be visible. Destructive visualization methods include
chemical stains such as Acetaldehyde, Vanillin, Permanganate, PMA, Iron chloride, and
Bromocresol green. The most common semi-destructive visualization method is through an iodine
chamber. Non-destructive methods use short-waved (254nm) or long-waved (365nm) UV light to
glow a TLC plate pre-coated with a phosphor such as Zinc sulfide. Phosphors are fluorescent and
phosphorescent substances that glow in UV light. Lisa Nichols discussed the principles and
mechanisms of various visualization methods in this section [3]
The UV light visualization method is most applicable for substances that strongly absorb
UV light, such as aromatic compounds and conjugated systems. The separated components that
absorb the short or long-wave UV light will appear dark on the TLC plate. Lightly outlining the dark
spots with a pencil is necessary because a phosphor pre-coated TLC plate loses its fluorescence
when it is not under UV light. Some compounds aside from the pre-coated phosphor commonly
glow bright purple o blue under UV light in rare instances, as shown in Figure 2. However, they
are most common in highly conjugated systems. Performing a destructive visualization method
with a chemical stain reactive with the analyte after exposing the TLC plate in UV light is preferable
as most other substances besides aromatic compounds and conjugated systems are not active
in UV light.
Figure 2. Fluorescein (Fl) and rhodamine B (Rh) under a) Visible light, b) Short-wave UV, c)
Long-wave UV
Semi-destructive visualization methods comprise of an Iodine chamber. This method is
mostly common in visualizing aromatic compounds. It exposes the developed TLC plate to Iodine
vapor by adding iodine crystals to a TLC chamber or a chamber with powdered silica or alumina.
After placing a developed TLC plate and capping the chamber, the Iodine sublimes and reacts
with the analytes to produce iodine complexes, appearing as yellow-brown spots. However, this
complexation reaction is reversible because the Iodine will evaporate from the TLC plate and
leave the original analytes behind. Hence, it is theoretically possible to use other visualization
methods, although the analyte may also vaporize during air exposure.

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Figure 3. a) Iodine chamber with silica gel, b-d) Inserting and jostling TLC plate with developed
TLC plate, e) Developed TLC plate with iodine-complex chemical stains
There are many destructive visualization methods with chemical stains, requiring
knowledge of the analytes’ functional groups for choosing a reactive chemical stain. Their
difference from the semi-destructive Iodine vapor is that the colored spots retain as the chemical
reactions are almost irreversible, leaving a few original analytes. Chemists either spray or dip the
developed TLC plate with a chemical stain that produces a colored product with the analyte. It is
common to heat the developed TLC plate after exposure to a chemical stain to accelerate the
chemical reaction. However, some analytes might not react with the chemical stain, yielding
colorless spots. Thus, the chosen chemical stain must react with all of the compounds to be
analyzed. Examples of chemical stains are p-Anisaldehyde, Vanillin, Permanganate,
Phosphomolybdic Acid (PMA), Iron (III) Chloride, Bromocresol Green. A summary of
visualizations methods mentioned is shown in Figure .

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Figure 4. TLC Visualization Methods a) UV Light Lamp, b) Iodine Vapor, c) p-Anisaldehyde, d)
Vanillin, e.) Permnaganate, f) Phosphomolybdic Acid (PMA), g) Iron (III) Chloride, h)
Bromocresol Green
p-Anisaldehyde and Vanillin stains are applicable for nucleophiles such as alcohols and
amines and numerous aldehydes and ketone. However, they are inapplicable for alkenes,
aromatics, esters, and carboxylic acids. Their developed plates must be warm for a light pink to
dark pink background to appear. Both are light-sensitive and highly acidic. Hence, they must be
kept in a refrigerator and wrapped in aluminum, and handled with gloves. P-Anisaldehyde
approximately has six months of shelf life, slightly shorter than other chemical stains. It is also
initially colorless and turns light pink then dark pink, becoming more impotent but often still usable
as it darkens. Meanwhile, Vanillin is initially light yellow and darkens over time, becoming
discardable when it turns blue.
Permanganate stain has permanganate ions (MnO4-) that are reactive with alkenes and
alkynes through addition reaction, often immediately changing color with them. It is originally deep
purple and turns yellow upon reaction. Some chemists consider it as a universal stain as it can
also oxidize many oxidizable functional groups like aldehydes. However, heating until the
background starts to yellow and not brown, as it signifies overheating, is sometimes needed to
visualize some functional groups, often improving the contrast between the spots and the
background. Note that it should be handled with gloves as it is corrosive and can stain skin brown.
Figure 5. Potassium Permanganate reagent, Mechanism of addition reaction of Alkenes and
Permanganate ions

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Phosphomolybdic Acid stain (PMA) can visualize various compounds such as alcohols,
alkenes, alkyl iodides, and numerous carbonyl compounds, and is considered by some chemists
as an universal stain. PMA (Mo6+) is initially yellow-green and reduces to Molybdenum blue
(Mo5+ or Mo4+) upon oxidizing another compound. It needs robust heating to develop spots, but
is overheated when the background darkens. Its spots are indistinguishable in color as they often
appear green or blue. Note that it is light-sensitive and should be kept in a jar covered in aluminum
foil. It is also expensive but has a long shelf life of more than five years.
Figure 6. a) Phosphomolybdic Acid (PMA) reagent, b) PMA’s Molecular Formula
Iron (III) Chloride stain can specifically visualize phenols and some carbonyl compound
with high enol content. The Fe3+ ions from Iron (III) Chloride forms the faint blue complexes with
phenols as shown in Figure. However, the complexes’ structures are still debatable. Note that
chemists promptly record observations of the developed plate’s colors with this chemical stain as
their colors can rapidly disappear, yet it has an advantage of a long shelf life of more than five
years.
Figure 7. a) Iron (III) Chloride reagent, b) Phenol-Fe3+
complex
Bromocresol Green stain precisely visualizes acidic compounds in a solution lower than
pH 5 with its Bromocresol Green acid-base indicator that turns yellow below pH 3.8 and blue
above pH 5.4. A spotted acidic compound shifts the equilibrium towards the Bromocresol green’s
yellow form.

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Figure 8. a) Bromocresol green’s colors (left = low pH, middle = reagent, right = high pH),
b) Bromocresol green’s chemical structures
Its spots with carboxylic acids are fairly visible while its spots with phenols are hardly
noticeable as shown in Figure. The developed plate theoretically does not need heat for spots to
appear. However, heat can increase the contrast between the spots and the background.
Figure 9. a) p-cresol stained with p-Anisaldehyde appears as red spot, b) p-cresol stained with
Bromocresol green is faint (indicated by arrow)
The retardation or retention factor (Rf), the ratio of the analyte’s distance from the origin
line and the eluting solvent’s and eluent’s distance from the origin line, quantifies the analyte’s
attraction between the stationary and mobile phases, shown in EQ 1 and EQ 2.
Where
Figure 10. Developed TLC Plate
TLC analyzes the analyte’s purity. A pure analyte yields one spot while a component
mixture yields several spots. The Rf of each component quantifies its identity when comparing to
the Rf of the component’s reference standard.
Rf1 =
x1
xt
(EQ 1) Rf2 =
x2
xt
(EQ 2)
Rf1 Component 1’s retention factor
Rf2 Component 2’s retention factor
x1 Distance from origin to component 1
x2 Distance from origin to component 2
xt Distance from origin to solvent front
Solvent front
Component 1
Component 2
Origin

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Over-the-counter (OTC) analgesics usually contain active ingredients such as
Acetaminophen, Aspirin, caffeine, and phenacetin, as shown in Figure 11.
Figure 11. a) Acetaminophen, b) Aspirin, c) Caffeine, d) Phenacetin
Column chromatography
Column chromatography is a preparative chromatography method that purifies an impure
mixture loaded through a column packed with silica or alumina adsorbent. An eluent is an organic
solvent or solvent mixture that flows through the column, absorbing the mixture. Elution is the
reversible absorption and adsorption process that depends on the polarity and molecular structure
of the eluent and adsorbent. This process is similar to TLC but is different in the principle of elution
as TLC relies on capillary action while Column Chromatography relies on gravity and pressure
applied to the column [4].
Table 1. Dielectric constants of common solvents.
Eluent Dielectric constant (𝜀)
Dichloromethane 8.93
Ethyl Acetate 6.0
Hexane 1.99
Methanol 32.6
Toluene 2.38
Table 1 shows the dielectric constants of some common eluents. The dielectric constant
(𝜀), the material’s stored energy in an electric field, quantifies the solvent’s polarity. 𝜀 is directly
proportional to charge stability, which is directly proportional to polarity. The eluents’ polarity
determine the components’ speed across the column. Hence, more polar solvents move organic
components faster than less polar solvents. Note that there are multiple sources for different
dielectric constants that have slightly different values for each compound. Hence it is best to use
only one source of dielectric constants that contain all of the compounds to be analyzed as these
values pertain to similar conditions and experimental procedures.
Different polarities give different degrees of separation as compounds can separate or
stay together in specific eluent mixtures, highlighting the importance of choosing a specific eluent
or eluent mixture. An eluent that is too polar can displace all of the compounds at once with little
to no separation while an eluent that is too nonpolar cannot displace nor separate the compounds
at all [5].
The sum of the products of each solvent’s 𝜀 and concentration corresponds to the solvent
mixture’s 𝜀 as shown below.
εmixture =
Vsolvent 1
Vmixture
εsolvent 1 +
Vsolvent 2
Vmixture
εsolvent 2
(EQ 3)

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Where
εmixture Dielectric constant of eluent mixture
Vsolvent 1 Volume of solvent 1
Vmixture Volume of eluent mixture
εsolvent 1 Dielectric constant of solvent 1
Vsolvent 2 Volume of solvent 2
εsolvent 2 Dielectric constant of solvent 2
Chlorophyll pigments produce unripe peppers’ green color and decompose as they ripen.
Violaxanthin and Lutein then replaces the peppers green color into yellow, alongside beta-
carotene which gives an orange color. Capsanthin and Capsorubin then produce mature peppers’
red color. Meanwhile, spinach leaves also contain pigments such as Chlorophyll A and B,
Xanthophylls, and Carotenes. These compounds differ in polarity, the basis of their separation,
similar to the OTC analgesics’ active ingredients. Note that some of the beforementioned
compounds range in color and their relative polarities is the best analysis of their identity
Figure 12. Pigments in red pepper and spinach leaves a) Violaxanthin or Xanthophyll, b) Lutein,
c) Beta-Carotene, d) Capsanthin, e) Capsorubin, f) Chlorophyll A, g) Chlorophyll B
Different experiments were performed with different aims; namely isolate red peppers’
components in color bands through silica column chromatography with three increasingly polar
eluents, to determine the active ingredient in a sample commercial analgesic.
The experiment’s goals were to
• To simulate the TLC method for organic compound mixture analysis
• To determine the effects of the polarity of organic compounds and solvent mixture on TLC
analysis
• To simulate plant pigments’ isolation through TLC and Column chromatography
• To determine the effect of the eluent’s polarity on the organic compounds obtained through
column chromatography
• To calculate the dielectric constants of the eluents separating the red pepper’s pigments on
column chromatography

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• To determine the relationship between the eluents’ dielectric constants and the polarity of the
compounds separated with column chromatography
2. MATERIALS AND METHOD
2.1 REAGENTS AND APPARATUS
Isolating spinach leaf pigments by Column Chromatography required dry spinach leaves,
and reagents such as anhydrous sodium sulfate, Hexane, Acetone, silica gel. It also required
apparatus such as weighing scale, mortar and pestle, filter funnel, spatula, hair dryer, column
stand, wood applicator, rotary evaporator, and heating bath.
Analyzing spinach leaf pigments by TLC required spinach leaf paste and reagents such as
isopropyl alcohol and distilled water. It also required apparatus such as fine capillary tube,
chromatographic chamber, Whatman filter paper strip (20*2 cm2
), pencil, and scale.
2.2 EXPERIMENTAL PROCEDURE
Isolation of Spinach Leaf Pigments by Column Chromatography
Preparation of the crude extract
Ten grams of dry, stemless, and veinless spinach leaves were weighed. They were crushed and
grounded with a mortar and pestle. 10mL of 50:50 hexane and acetone mixture was added and
mixed into a fine paste. The filtrate was transferred into a conical flask with a funnel plugged by a
small cotton piece.
Removing the traces of water present in the crude
2-3 spatula of anhydrous sodium sulfate was added to the crude extract to remove traces of water.
Sodium sulfate was then filtered off with a funnel.
Adsorbing the Crude filtrate to Silica gel
The crude extract was added with 3g silica gel. It was gently heated with a hairdryer until the silica
gel flowed freely.
Packing the Column for Chromatography
A cylindrical glass column was plugged with a small cotton piece and was mounted on the stand.
25g fresh silica gel and 100mL Hexane were added in a 250 mL beaker and was stirred with a
glass rod into a silica slurry, poured into the Column wherein a conical flask was placed below it.
The stop cock was opened to drain the excess hexane solvent and was closed when the solvent
was just above the settled silica gel.
Loading the Crude material on to the Column
The adsorbed crude material was transferred into the solvent layer above the silica gel in the
packed Column.
Elution using Hexane
The Column was eluted with Hexane until the yellow β- carotene ran down the Column, collected
in a conical flask.
Elution using Acetone

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The Column was eluted with Acetone until the green pigments moved down the Column, collected
with another conical flask.
Removal of Solvent
The yellow and green pigments were concentrated with a rotary evaporator. The pigments left
behind in the round-bottomed flask were transferred into watch glasses with a spatula.
Ten grams of dry, stemless, and veinless
spinach leaves were crushed and
grounded with a mortar and pestle.
10mL of 50:50 hexane and acetone
mixture was added and mixed into a fine
paste.
The filtrate was transferred into a conical
flask with a funnel plugged by a small
cotton piece.
2-3 spatulas of anhydrous sodium sulfate
was added to the crude extract to remove
traces of water and was filtered off with a
funnel.
The crude extract was added with 3g
silica gel and was gently heated with a
hairdryer until the silica gel flowed freely.
A cylindrical glass column was plugged
with a small cotton piece and was
mounted on the stand.
25g fresh silica gel and 100mL Hexane
were added in a 250 mL beaker and was
stirred with a glass rod into a silica slurry,
poured into the Column wherein a conical
flask was placed below it.
The stop cock was opened to drain the
excess hexane solvent and was closed
when the solvent was just above the
settled silica gel.
The adsorbed crude material was
transferred into the solvent layer above
the silica gel in the packed Column.
The Column was eluted with Hexane until
the yellow β- carotene ran down the
Column, collected in a conical flask.
The Column was eluted with Acetone until
the green pigments moved down the
Column, collected with another conical
flask.
The yellow and green pigments were
concentrated with a rotary evaporator,
while the pigments left behind in the
round-bottomed flask were transferred
into watch glasses with a spatula.

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Figure 13. Schematic Diagram of the Isolation of Spinach Leaf Pigments by Column
Chromatography
Figure 14. Column Chromatography set-up
Analysis of Spinach Leaf Pigments by Thin Layer Chromatography
Preparation of TLC plate
Two pencil lines 4cm from an end and lengthwise from the center of a Whatman filter paper was
drawn. The intersection point was named point P.
Spotting of mixture
The mixture was dropped at point P using a fine capillary tube and was air-dried. The process
was repeated at the same point.
Preparation of Chromatographic chamber
Equal volumes of isopropyl alcohol and distilled water were poured into a chromatographic
chamber mixed with a glass rod.
Elution of Spinach Leaf Pigments
The filter paper was vertically submerged in the chromatographic chamber 2cm above the solvent
mixture. The lid was closed afterward, and the solvent was left to rise to 15cm.
Analysis of Spinach Leaf Pigments
The solvent front was marked on the filter paper and was dried. Pencil marks were drawn on the
center of the pigments. The distance between the solvent line and origin line and between the
pigments and origin line were measured. The Rf values of the pigments were calculated.

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Figure 15. Analysis of Spinach Leaf Pigments by Thin Layer Chromatography
3. RESULTS
Figure shows the results of the solvent polarity’s effect on organic compounds’ separation
in TLC. It shows that the organic compounds travel farther with increasing concentration of Ethyl
Acetate. The 100% Hexane and 100% Ethyl Acetate did not separate the organic compounds.
However, the former did not displace the organic compounds from the origin line while the latter
displaced them until the solvent front.
Legend
Figure 16. Solvent polarity’s effect on organic compounds separation in TLC
Hexane 100% 75% 50% 25% 0%
Ethyl Acetate 0% 25% 50% 75% 100%
Table 2. Virtual TLC experiment - Spinach leaves’ components
Distance Travelled Rf
Less polar
component
More polar
component
Two pencil lines 4cm from an end and
lengthwise from the center of a Whatman
filter paper was drawn, then the
intersection point was named point P.
The mixture was dropped at point P using
a fine capillary tube and was air-dried and
the process was repeated at the same
point.
Equal volumes of isopropyl alcohol and
distilled water were poured into a
chromatographic chamber mixed with a
glass rod.
The filter paper was vertically submerged
in the chromatographic chamber 2cm
above the solvent mixture, then the lid
was closed afterward, and the solvent was
left to rise to 15cm.
The solvent front was marked on the filter
paper and was dried, while pencil marks
were drawn on the center of the pigments.
The distance between the solvent line and
origin line and between the pigments and
origin line were measured, then the Rf
values of the pigments were calculated.

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Table 2 shows that the solvent traveled 3.5cm from the origin line, marking the solvent front.
Chlorophyll B (dark green), Chlorophyll A (light green), Xanthophyll (yellow), and Carotene
travelled 0.8cm, 1.6cm, 3.1cm, and 3.3cm, respectively. They yielded Rf values of 0.23, 0.46,
0.89, and 0.94, respectively.
Table 3. Virtual Column Chromatography experiment – Spinach leaves’ dark pigment
Color Eluent Compound
Yellow Hexane Beta-carotene
Green Acetone Chlorophyll
Table 3 shows that the spinach leaves dark pigment containing yellow and green color
bands, eluted by Hexane and Acetone. The former is Beta-carotene, while the latter is
Chlorophyll.
Figure 2 shows the results from a commercial analgesic’s TLC analysis. The components
- reference standards of Aspirin (C) and Acetaminophen (B), and sample of commercial analgesic
(A) traveled 6.2cm, 3.7cm, and 3.7cm from the origin, respectively. The solvent traveled 7.8cm
from the origin, marking the solvent front. Components C, B, and A yielded Rf values of 0.79,
0.47, and 0.47, respectively.
Reference Standard
Samples
Figure 17. Provided Data from previous TLC analysis of commercial analgesic
Table 4. Provided Data from previous red pepper column chromatography experiment
Solvent 3.5 -
Chlorophyll B (dark green) 0.8 0.23
Chlorophyll A (light green) 1.6 0.46
Xanthophyll (yellow) 3.1 0.89
Carotene (orange) 3.3 0.94
Component Analyte’s
distance from
origin (cm)
Solvent front’s
distance from
origin (cm)
Rf
Aspirin (C) 6.2 7.8 0.79
Phenacetin - - -
Acetaminophen (B) 3.7 7.8 0.47
Caffeine - - -
Commercial analgesic
(A)
3.7 7.8 0.47
Crude caffeine - - -
Eluent Dielectric constant (ε) Color band/s obtained
Hexane/dichloromethane 5.46 Yellow
Dichloromethane 8.93 Orange
Dichloromethane/methanol 20.77 Red-orange

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Table 4 shows the results from a previous red pepper column chromatography experiment.
The eluents Hexane/dichloromethane, Dichloromethane, and Dichloromethane/methanol have ε
values of 5.46, 8.93, 20.77, and eluted red pepper paste’s Yellow, Orange, and Red-orange color
bands, respectively.
4. DISCUSSION
Solvent polarity’s effect on organic compounds separation in TLC
The first virtual experiment analyzed a TLC plate coated with silica gel and spotted with a
mixture of less polar and more polar components. The solvent mixture for each run is different in
the concentration of Hexane and Ethyl Acetate. The higher the percent volume of Ethyl Acetate,
the higher the distance between the analytes and the origin. The less polar component travels
farther than the more polar component in the solvent mixtures as the more polar component is
more attracted to the polar silica gel than the partially polar Hexane and Ethyl Acetate mixtures
[6]. Ethyl Acetate is a more polar solvent than Hexane as the former has a Dielectric constant of
6.0 higher than Hexane’s 1.99 Dielectric constant.
100% Ethyl Acetate can displace the two components until the solvent front, indicating
that they are more attracted to Ethyl Acetate than Silica gel. Since both components are polar
and a polar solute’s degree of dissolution is proportional to the solvent’s polarity, Ethyl Acetate is
more polar than Silica gel. It has more sites for hydrogen bonding and dipole-dipole interactions.
Opposite to 100% Ethyl Acetate, 100% Hexane does not displace the more polar and less polar
components as Hexane is a nonpolar solvent and can only displace nonpolar components from
its lack sites for hydrogen bonding and dipole-dipole interactions that Silica gel has [7].
Virtual TLC experiment - Spinach leaves’ components
Equal volumes of isopropyl alcohol and water serve as the eluent. The two are polar
molecules, but water is much more polar. It solely contains hydroxyl groups, which are sites for
hydrogen bonding. In contrast, isopropyl alcohol contains besides hydroxyl group sites for
hydrogen bonding, polar carbon-oxygen bonds that serve as sites for dipole-dipole interactions.
Meanwhile, the carbon chains serve as sites for London dispersion forces whenever individual
isopropyl alcohol molecules become close to one another at any instant. Mixing these two eluents
produces an eluent mixture that is much more polar than the rest of the eluents. It has a dielectric
constant of 49.0, as shown in EQ 4, from another Dielectric constant table [8]
Rf =
1
2
(17.9) +
1
2
(80.1)
Rf = 49.0
(EQ 4)
Notice that even with a very polar eluent mixture, the silica gel still outcompeted the
isopropyl alcohol/water eluent mixture. The most polar component Chlorophyll B did not travel as
much was more attracted to the silica gel than the eluent mixture. However, some of the literature
in TLC analysis of Spinach leaves use an eluent mixture of 7:3 hexane and acetone mixture [9],
with a dielectric constant of approximately 7.526, much lower than the 49.0 dielectric constant of
the isopropyl alcohol and water eluent mixture. The literature on the Rf resolution of equal parts
isopropyl alcohol and water eluent mixture on spinach leaves’ components is limited. Hence,
further investigation of this topic is needed.

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The order of increasing polarity of the spinach leaves’ components is Carotene,
Xanthophyll, Chlorophyll A, and Chlorophyll B. These are also the order of decreasing Rf.
Carotene is the least polar component as it is the only hydrocarbon from the four components. In
contrast, Chlorophyll A and Chlorophyll B are the two most polar components from having many
Carbon-Oxygen, Carbon-Nitrogen, and Magnesium-Nitrogen polar bonds. The difference
between the two is at a specific site wherein Chlorophyll B has an Aldehyde (-CHO) and
Chlorophyll A has a Methyl group (-CH3), making the former slightly more polar than the latter.
Xanthophyll is also polar but not as polar as Chlorophyll A and B. It has less polar covalent bonds
from its Carbon-Oxygen and Hydrogen-Oxygen bonds. The virtual laboratory experiment did not
mention its visualization method. However, it is safe to assume that it is either visualized under
visible or UV light as spinach leaves’ components are visible under visible light and can absorb
254nm UV light as the components are highly conjugated systems [9].
Virtual Column Chromatography experiment – Spinach leaves’ dark pigment
The second virtual experiment analyzes spinach leaves’ dark pigment separated into two color
bands. The eluents Hexane and Acetone are nonpolar and polar, respectively. Hexane is a
hydrocarbon that only has London dispersion forces as intermolecular forces [7].
In contrast, Acetone has dipole-dipole interactions and London dispersion forces from the
Carbon-Oxygen group and carbon chain, respectively. A polar eluent elutes polar compounds,
similar to a nonpolar eluent and nonpolar compounds. Beta-carotene is relatively less polar than
Chlorophyll. The former only has London dispersion forces for being a hydrocarbon, and the latter,
besides London-dispersion forces, has dipole-dipole interactions from Carbon-Oxygen, Carbon-
Nitrogen, and Magnesium-Nitrogen polar bonds. Hence, Hexane elutes the less polar yellow polar
color band Beta-carotene. In contrast, Acetone elutes the more polar green color band
Chlorophyll.
Provided Data from previous TLC analysis of commercial analgesic
The distance of the Aspirin, Acetaminophen, Commercial analgesic, and the solvent front
was measured from the origin. These data were treated with the Rf formula. Each distance from
the origin of the Aspirin, Acetaminophen, and Commercial analgesic is divided with the solvent
front’s distance from the origin. A sample of this treatment is shown in EQ 5 wherein the Rf of the
Commercial analgesic (A) is calculated.
Rf Commercial analgesic =
3.7
7.8
Rf Commercial analgesic = 0.47
(EQ 5)
Acetaminophen is more polar than Aspirin [10]. The former has two sites for hydrogen
bonding from the hydroxyl and amide groups. The latter only has one site for hydrogen bonding
from the carboxylic acid group. Both also have dipole-dipole interactions from the Carbon-Oxygen
and Carbon-Nitrogen bonds. Their polarities correspond to their Rf values as the more polar
component Acetaminophen’s Rf is 0.47, lower than Aspirin’s Rf of 0.79.
The first actual experiment analyzes a commercial analgesic’s relative composition by
comparing two reference standards of Aspirin and Acetaminophen. Past TLC analyses of Aspirin,
Phenacetin, Acetaminophen, and Caffeine shows spots with different Rf values, indicating that
their polarities are dispersed enough to separate from an analgesic combination drug. The
developed TLC plate is visualized with Iodine vapor. All of the compounds mentioned above form

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complexes with Iodine to form dark spots on a yellowish background. They either have phenyl
functional groups or are highly unsaturated. UV light is an alternative to Iodine vapor in visualizing
Aspirin and Acetaminophen. They absorb UV light to form dark spots on a green fluorescent
background [3]. There are also no discrepancies in the shapes and form of the spots. The
reference standard Acetaminophen and commercial analgesic have the same Rf value, indicating
that the sample commercial analgesic’s active ingredient is Acetaminophen if it can only contain
either of the beforementioned compounds.
However, Acetaminophen is in combination with other drugs in over 600 over-the-counter
commercial analgesics alone [11]. PMA and Permanganate stains are possible visualization
techniques besides Iodine vapor or UV light. They can visualize many more compounds than just
Acetaminophen and Aspirin from being universal stains. Note that they can visualize the two
compounds as PMA and Permanganate oxidizes them. However, the plates need heat to either
develop or improve their contrast relative to the TLC plate background. This procedure risks
overheating, but PMA and Permanganate stains are substantial in ensuring that as much of the
commercial analgesic’s compounds are visible. It is best to visualize the TLC plate with UV light,
marking the spots with a pencil, and stain it with PMA or Permanganate. Theoretically, the
samples visualized with Iodine vapor might revert to their original composition as Iodine vapor is
a semi-destructive visualization method. However, it is still possible for the samples to evaporate
during Iodine’s vaporization [3].
Provided Data from previous red pepper column chromatography experiment
In this actual column chromatography experiment, the eluents differ in dielectric constants
to separate color bands containing the pigments. The analysts determined the dielectric constants
of the mixtures. An example of this calculation for 1:1 ratio Hexane/Dichloromethane in EQ 6.
ε (Hexane and Dichloromethane) = (
2.5 mL
5mL
) (1.99) + (
2.5 mL
5mL
) (8.93) = 5.46
(EQ 6)
The yellow color band contains Beta Carotene, the orange color band contains
Violaxanthin and Lutein, and the red-orange pigment contains Capsanthin and Capsorubin [12].
These color bands and their pigments correspond to the increasing Dielectric constants of the
eluents, suggesting that these color bands increase in polarity. Beta Carotene is the first eluted
pigment, indicating that it is the least polar pigment. Its low polarity arises being the only
hydrocarbon from the rest [13]. Violaxanthin and Lutein are the next eluted pigments, suggesting
that they are more polar than Beta Carotene from dipole-dipole interactions and hydrogen bonding
from their Carbon-Oxygen and Hydroxyl groups, respectively. The last eluted pigments are
Capsanthin and Capsorubin, the most polar pigments from the rest. They have more sites for
hydrogen bonding and dipole-dipole interactions from having Hydroxyl and carbonyl groups.
Similar to the pigments, the structures of the eluents determine their polarities. Hexane is
the least polar eluent as it is a hydrocarbon with only London dispersion forces. Dichloromethane
is more polar than Hexane as besides London dispersion forces, it has dipole-dipole interactions
from the two Carbon-Chlorine bonds. The most polar eluent Methanol has hydrogen bonding and
dipole-dipole interactions from its hydroxyl group and carbon-oxygen bond. The order of eluents
also correspond to their polarities as using the most polar eluent in the first run might not lead to
separation of the color bands as all of them are attracted to the most polar eluent. Suppose
Dichloromethane/methanol eluent is the first eluent in this experiment. In that case, the color
bands will elute all at once without separation [5].

17
Beta Carotene often appears as an orange pigment, but it sometimes appears yellow [14].
Hence the yellow color band’s identification as Beta Carotene is the best assumption considering
that it should be the first eluted pigment from its low polarity. Conversely, Lutein is usually yellow
and sometimes orange [15]. Thus, it is best to assume that it is in the orange color band as Lutein
is more polar than Beta Carotene. Violaxanthin is usually orange [16], so it is definitely in the
orange color band because it is more polar than Beta Carotene.
5. CONCLUSIONS AND RECOMMENDATIONS
The virtual TLC experiment on the Solvent polarity’s effect on organic compounds
separation showed that a more polar solvent could displace the component’s mixture. In contrast,
a less polar solvent displaces less of the component’s mixture. It also showed that the ideal
solvent for separation is a mixture of the less polar and more polar solvents. The two solvents
alone do not separate the components.
The virtual TLC experiment on the Spinach leaves’ components showed that the order of
polarity of spinach leaf’s components is Carotene, Xanthophyll, Chlorophyll A, and Chlorophyll B.
There should be further investigation on the equal volume isopropyl alcohol and water eluent as
it is a much more polar eluent than an eluent that is separates efficiently – 7:3 hexane and acetone
mixture. It is best if the TLC plate is under another visualization method such as UV light as some
components might not be visible in visible light.
The virtual Column Chromatography experiment on the Spinach leaves’ dark pigment
showed that it contains less polar Beta-carotene and more polar Chlorophyll as yellow and green
color bands, respectively. The eluents’ order also corresponds to polarity as the non-polar Hexane
eluted Beta-carotene while the polar Acetone eluted Chlorophyll.
The actual TLC experiment on analyzing a commercial analgesic showed that its most likely
active ingredient is Acetaminophen. However, the experiment did not use a universal solvent such
as PMA or Permanganate stains. Hence, other active ingredients might be in the mixture. It is
recommendable that UV light and PMA or Permanganate stain is the visualization method.
The actual Column Chromatography experiment on red pepper showed yellow, orange, and
red-orange color bands. The yellow color band has Beta Carotene, the orange color band has
Violaxanthin and Lutein, and the red-orange color band has Capsanthin and Capsorubin. Beta-
carotene often appears orange, while Violaxanthin and Lutein often appear yellow. However, it is
reverse in the experiment. Further investigation by performing a qualitative analysis such as TLC
is recommendable to ensure the component’s identity.

18
6. REFERENCES
[1] Nichols L. 2.3D: Separation Theory [Internet]. Chemistry LibreTexts. Libretexts; 2020 [cited
2020Dec19]. Available from:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book:_Organic_Chemistry_La
b_Techniques_(Nichols)/02:_Chromatography/2.03:_Thin_Layer_Chromatography_(TLC)/
2.3D:_Separation_Theory
[2] Chemistry LibreTexts. Thin Layer Chromatography [Internet]. Chemistry LibreTexts.
Libretexts; 2020 [cited 2020Dec19]. Available from:
https://chem.libretexts.org/Bookshelves/Ancillary_Materials/Demos_Techniques_and_Exp
eriments/General_Lab_Techniques/Thin_Layer_Chromatography
[3] Nichols L. 2.3F: Visualizing TLC Plates [Internet]. Chemistry LibreTexts. Libretexts; 2020
[cited 2020Dec19]. Available from:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Book:_Organic_Chemistry_La
b_Techniques_(Nichols)/02:_Chromatography/2.03:_Thin_Layer_Chromatography_(TLC)/
2.3F:_Visualizing_TLC_Plates
[4] University of Colorado Boulder. Column Chromatography [Internet]. Organic Chemistry at
CU Boulder. [cited 2020Dec19]. Available from:
https://orgchemboulder.com/Technique/Procedures/Columnchrom/Columnchrom.shtml
[5] University of Massachusetts Amherst. Thin Layer Chromatography [Internet]. UMass
Amherst. [cited 2020Dec19]. Available from:
https://people.chem.umass.edu/samal/269/tlc.pdf
[6] Massachusetts Institute of Technology. 8.3 – Thin Layer Chromatography (TLC) Guide
[Internet]. MIT Open Courseware. [cited 2020Dec19]. Available from:
https://ocw.mit.edu/courses/chemistry/5-301-chemistry-laboratory-techniques-january-iap-
2012/labs/MIT5_301IAP12_TLC_Handout.pdf
[7] University of Oregon. Solutions: Like Dissolves Like Solubility and Intermolecular Forces
[Internet]. Chemdemos. [cited 2020Dec19]. Available from:
https://chemdemos.uoregon.edu/demos/Solutions-Like-Dissolves-Like-Solubility-and-
Intermolecular-Forces
[8] Louisiana State University. Dielectric Constant [Internet]. Louisiana State University Macro
server at The Polymer Analysis Laboratory. [cited 2020Dec19]. Available from:
https://macro.lsu.edu/HowTo/solvents/Dielectric%20Constant%20.htm)
[9] University of California, Los Angeles. Isolation of Chlorophyll and Carotenoid Pigments from
Spinach [Internet]. UCLA College Chemistry & Biochemistry. 2016 [cited 2020Dec19].
Available from:
https://www.chem.ucla.edu/~bacher/CHEM14CL/Handouts/Spinach_Pigments%20hando
ut_Spring%202016.pdf
[10] University of Missouri. Aspirin, Acetaminophen, and Caffeine separation from Excedrin via
Column Chromatography [Internet]. Chemistry University of Missouri. [cited 2020Dec19].

19
Available from: https://chemistry.missouri.edu/sites/default/files/class-files/aspirin-
acetaminophen-and-caffeine-separation-from-excedrin-via-column-chromatography.pdf
[11] Keaveney A, Peters E, Way B. Effects of acetaminophen on risk taking. Social cognitive
and affective neuroscience. 2020 Jul;15(7):725-32.).
[12] Varelis P, Melton L, Shahidi F. Encyclopedia of Food Chemistry. Elsevier; 2018 Nov 22.
[13] Zaripheh S, Erdman Jr JW. Factors that influence the bioavailablity of xanthophylls. The
Journal of nutrition. 2002 Mar 1;132(3):531S-4S.
[14] Francis FJ. Safety of food colorants. InNatural Food Colorants 1996 (pp. 112-130).
Springer, Boston, MA.
[15] Abdel-Aal ES, Akhtar H, Zaheer K, Ali R. Dietary sources of lutein and zeaxanthin
carotenoids and their role in eye health. Nutrients. 2013 Apr;5(4):1169-85.).
[16] Wang F, Huang L, Gao B, Zhang C. Optimum production conditions, purification,
identification, and antioxidant activity of violaxanthin from microalga Eustigmatos cf.
polyphem (Eustigmatophyceae). Marine drugs. 2018 Jun;16(6):190.

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