𝑐!"!

(#) (ppm)
Stage 2 Chemistry 2022

Deconstruction and Design:

Factors Affecting Thin Layer
Chromatography (TLC)

A Design Practical investigating the factors that affect

the separation of components during the Thin-Layer
Chromatography of spinach plant pigments

Folio 2: Deconstruct and Design

148999T 148999T 1
What Factors Affect the Separation of Plant Pigments During Thin-Layer Chromatography?
An assessment of the potential factors affecting the separation of plant pigments during thin-layer chromatography has
been shown below, with justifications highlighted to provide evidence of deconstruction.

Elimination of Unfeasible Variables

Rather than evaluating all potential independent
variables, visibly unfeasible factors to investigate
were eliminated using the adjacent four-way Venn
Diagram. For example, the angle of inclination of the
beaker was identified as difficult to manipulate, as
the majority of incline angles would enable the
solvent to simply flow out of the vessel. Several
variables were observed to meet all four criteria, and
therefore were selected for further investigation.
However, literature reported no relationship
between submersion time and 𝑅# , so this variable
was abandoned. Moreover, plates of varying lateral
thickness were unavailable, so were thus not used.

Consideration of Independent Variables

Temperature: As temperature increases, both analyte and solvent particles gain kinetic energy, which is used to establish
interactions between the respective particles and the stationary phase. However, the increase in adsorption strength with
temperature has been reported as variable between compounds. Thus, separation will either increase or decrease with
temperature. However, maintaining any temperature besides room temperature would be difficult due to the open system
of the TLC setup.

TLC Plate Dimensions: Larger TLC plates possess greater surface area, and therefore more sites for adsorption per unit
volume. Hence, a greater number of interactions will occur per unit area, and the strength of solute absorption will increase.
Thus, it is likely that the distance traversed by both the analyte and mobile phase will be impacted. However, the largest
plate available was relatively small, meaning manipulating plate size would transform TLC to an overly small scale (cutting
plates to make them smaller); thus this was not considered viable for the parameters of a classroom experiment.

Eluent Polarity (Chosen Independent Variable): A solvent’s polarity index impacts the strength of an analyte’s solubility
within it, and therefore the degree of separation. Polar analytes will dissolve more readily in polar eluents, and vice-versa.
Thus, for a given analyte, the degree of separation change with eluent polarity. There are two methods of potentially
manipulating eluent polarity; using different pure samples of solvents, such as acetone or hexane, or mixing multiple solvents
together to create a mixture. The former method is more convenient, as it requires less preparation and avoids the complex
calculations for determining polarity (the polarity measures of common organic solvents are readily available online), however
gives the experimenter less control over the range and spread of density index values. The second method, while more
complicated to prepare, provides a greater level of control while enabling predetermined polarity measures to be used. This
method will ultimately cater to a more evenly defined trend, and was therefore selected. Eluent polarity can be quantified
using a calculated dielectric constant, and the use of electronic technology in combination with known constituent dielectric
constants (available online) will facilitate the simple measurement of this independent variable throughout.

148999T 2
Flowchart of Proposed Design
A flowchart (see right) was constructed
1
to evaluate and deconstruct certain
chromatography design parameters.
Certain factors, including the stationary
2
phase material, measure of polarity,
3
mobile phase (eluent) components,
and selected analyte to be measured,
were investigated. The direction of
4
decision making has been indicated
using shaded cells and bolded arrows.

Justification of Proposed Design

1 Stationary Phase: Normal-phase Chromatography (NPC) was favoured over Reverse-phase Chromatography (RPC),
due to the relative abundance of NPC compatible apparatus. The most common stationary phases materials for NPC
are silica (SiO2) and alumina (Al2O3). Both substances can be converted into very fine particles, thereby enabling efficient
separation. Ultimately, silica was selected because of its cost-effectiveness and high availability in a classroom context.

2 Polarity Measure: Scientific literature discussing the relationship between polarity and 𝑅# has been reported as linear
when considering species’ proportions from a mole-based approach. Similarly, applying a volume fraction or mass
fraction quantity for polarity of a mixed solvent has shown little justifiable correlation, and therefore catered to the
production of a multitude of unrelated non-linear trends. Therefore, both molar percentage and dielectric constant were
chosen as most appropriate. The dielectric constant of a mixed solvent in itself encompasses the molar percentage,
while also incorporating a quantification of polarity on the basis of polarisation tendencies, and is therefore a more
complete substantiaton measure of a mixed solvent’s overall polarity.

3 Mobile Phase: For the polarity of the mobile phase to be altered, the binary composition eluent must be comprised of
a polar and less-polar component. The miscibility of non-polar and polar solvent components was identified as a
difficulty; components of differing polarities have a tendency to be immiscible. Acetone was identified as an appropriate
polar solvent, due to its miscibility with non-polar silvents. Being a small molecule with a predominate hydrocarbon
chain, acetone is freely soluble in non-polar organic solvents, despite its polar carbonyl group. Therefore, acetone was
identifiied as an suitable polar constituent. Subsequently, hexane was chosen as the non-polar solvent constituent for
a trifactor of favourable characteristics; the hydrocarbon is cheap, relatively safe, and largely unreactive. Both liquids
were tested for miscibility, and thereafer concluded as suitable eluent constituents.

4 Chosen Analyte: The spinach plant contains a number of pigments, including Chlorophylls, Pheophytins, Carotene,
and Xanthophyll. Both Chlorophylls possess a green hue, and are therefore difficult to differentiate on a small-scale TLC
plate; they were therefore not chosen. Carotene was deemed unsuitable on the basis of its lack of hydroxyl groups; its
incapacity to form dipole-dipole or hydrogen bonds with a polar eluent means that it will likely be unaffected by
increasing eluent polarity. Three Xanthophyll species are present in spinach leaves, yet they all boast similar 𝑅# values
and identical colours, making them again difficult to differentiate. Therefore, Pheophytin A, which is distinct in colour
(grey) and of a relatively unique 𝑅# , was selected as an appropriate analyte to measure.

148999T 3
Aim
To investigate the effect of varying eluent polarity (measured through its dielectric constant, 𝜖) on the retardation factor (𝑅# )
of the plant pigment Pheophytin A (to be hereafter referred to as simply ‘Pheophytin’) during normal-phase thin-layer
chromatography.

Hypothesis
As eluent dielectric constant (𝜖) increases, the mean retardation factor of Pheophytin A will increase linearly, according to
the principles of competitive adsorption (to be outlined in Background Theory).

Independent Variable
The polarity of the eluent, given by the dielectric constant (𝜖) of binary composition hexane-acetone mixtures, calculated
using the mole fractions of each constituent. These standard solutions will be made by the experimenter to ensure accuracy.

Dependent Variable
The mean retardation factor (𝑅# ) of the polar compound Pheophytin A, obtained through thin-layer chromatography. A
sample containing the pigment will be extracted from the leaves of Spinacia oleracea using a methanol eluting agent.

Controlled Variables
Polarity of TLC Plate: The stationary phase is in constant competition with the mobile phase to interact with the analyte.
The capacity for a stationary phase to adsorb the analyte is dependent on its intermolecular interactions with analyte
particles, and therefore its own polarity – thus the plate’s polarity must remain constant. The strengthening or weakening
of intermolecular forces that accompanies a change in TLC plate polarity would therefore alter the analyte’s capacity to be
displaced on the plate’s surface, and therefore its capacity to be carried by the solvent front. 𝑅# would consequently change.
To maintain consistent stationary phase polarity throughout, identical TLC plates will be used throughout. These should be
sourced from the same manufacturer in all instances, and be scanned for imperfections (e.g. scratches) that render them
unidentical, prior to the conduction of the experiment.

Binary Eluent Constituents: The eluents to be used in each trial will be binary mixtures, consisting of acetone and hexane.
Both constituents will be used throughout all trials, to minimise the effects of unintended reactions (therefore any unintended
reactions will occur in all trials). For instance, some basic compounds, such as sodium hydroxide, react with silica under
TLC conditions; therefore, no compounds with alkaline tendencies will be used. Not only will both hexane and acetone not
chemically react with silica, but they will not react with each other, preventing unintended interactions from occurring in all
trials and distorting the interactions of the stationary and mobile phases. Thus, silica and acetone were used throughout.

Temperature: Dielectric constant (𝜖) is inversely proportional to temperature (see Variables section of main report), and
therefore temperature dependent. However, the approximation 𝜖$% will be used in all trials, under the assumption that room
temperature remains ~25ºC. Subtle fluctuations in room temperature may alter the eluent’s 𝜖 value, therefore making the
mathematical 𝜖 approximations either lower or higher than their experimental values. This variable will be kept constant by
using an electronic water bath, to ensure the chamber and its contents remain at exactly 25ºC throughout. Modification to
design: This variable was ultimately uncontrolled, as no water bath was available for use. This may present a potential
source of random error that may have hindered the reliability of the investigation.

148999T 4
Materials
Material Qty. Material Qty. Material Qty.
Electronic Water Bath 1 Beaker (200 mL) 6 TLC Plate 6
Spinach Leaves
Retort Stand 1 Micropipette 6 100 g
(Eluent)
Boss Head 1 Watch glass 6 Hexane (C6H14) 15 mL
Clamp 1 Forceps 1 Acetone (C3H6O) 15 mL
Metal Ruler 1 2B or HB Pencil 1 Distilled Water As needed
Volumetric Flask (250 mL) 6 Electronic Weighing Balance 1 Filter Paper 6 sheets

Method (with Justification)

Preparation of Solvents
- Solvents A (exclusively hexane) and F (exclusively acetone) will be taken from a pre-prepared stock solution
- Solvents B-E will be made up by combining the two stock solutions (acetone and hexane) in the following ratios;
Solvent Hexane Acetone Ratio (H:A) Solvent Hexane Acetone Ratio (H:A)
B 1 ml 4 ml 1:4 C 2 ml 3 ml 2:3
D 3 ml 2 ml 3:2 E 4 ml 1 ml 4:1

Thin Layer Chromatography

- Two horizontally equidistant marks will be made at the origin of the TLC plate (~2 cm from the base). A 2B or HB pencil
will be used for this process so that pigments from ink will not interfere with readings from the intended sample.
- Samples will be spotted onto both of the two marks at the origin of the TLC plate.
- The beaker will be filled to ~3 cm with Solvent A, and filter paper will line the edges, minimising the edge effect.
- The prepared TLC plate will be placed into the beaker. The watch glass will then be placed atop the beaker.
- The beaker will be submerged in a water bath (preheated to 25ºC), to ensure a consistent temperature across trials.
- After two minutes, the TLC plate will be removed and patted dry with a paper towel to prevent further capillary action.
- An 𝑅! value will thereafter be calculated, using a metal ruler to make appropriate measurements. Metal was preferred
over wood to eliminate the calibration errors associated with thermal expansion/contraction.
- Steps 1-6 will be repeated with Solvents B, C, D, and E. Five solvents will be used to cater to a defined trend.

Evaluation of Safety and Ethical Considerations

Acetone vapour has been reported to cause dizziness, headaches, and damage to the skin, if in contact with the human
body. To mitigate this safety risk, safety goggles and lab coats will be worn for the entirety of the experiment. Moreover,
broken glassware may pose a risk if in contact with bare skin. Therefore, any broken glassware will be removed immediately
to reduce the risk of cuts. As no live organisms are involved in this experiment, the investigation remains ethically sound. All
chemicals will be disposed of appropriately by a lab technician, after being placed into an organic waste bin.

Data Presentation – Proposed Table of Results

Trial 𝜖 𝑑"#$"%& 𝑑'(")'(*&+%(-) 𝑑'(")'(*&+%(/) 𝑅! ⋅ 1 𝑅! ⋅ 2 𝑅! ⋅ Avg

148999T 5
Reference List
1. Ahmad, 1996, Solvent Polarity as a Function of R f in Thin-Layer Chromatography of Selected Nitro Functions,
https://www.researchgate.net/publication/268407396_Solvent_Polarity_as_a_Function_of_R_f_in_Thin-
Layer_Chromatography_of_Selected_Nitro_Functions, Accessed 2022
2. Baranowska, 2004, Retention–Mobile Phase Relationships for Methylxanthines and Biogenic Amine Metabolites
in Adsorption Thin-Layer Chromatographic Systems,
https://link.springer.com/content/pdf/10.1556/JPC.17.2004.3.14.pdf, Accessed 2022
3. Chemistry LibreTexts, 2020, Cohesive and Adhesive Forces in Capillary Action, ,
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_M
odules_(Physical_and_Theoretical_Chemistry)/Physical_Properties_of_Matter/States_of_Matter/Properties_of_Liq
uids/Capillary_Action, Accessed 2022
4. Furió, 1996, Difficulties with the Geometry and Polarity of Molecules: Beyond Misconceptions,
https://pubs.acs.org/doi/abs/10.1021/ed073p36?casa_token=lsN4IgG2wJ4AAAAA:Ci7yTDBkOApR02q_VexB0
YQuxxvnjAcpZ3qB5v-FKQkKERi2SSoU9-2xDUfA4yeDt7Szt0s95j6aoAoq9w, Accessed 2022
5. Griffiths, 1979, Correlations among solvent polarity scales, dielectric constant and dipole moment, and a means
to reliable predictions of polarity scale values from cu,
https://www.sciencedirect.com/science/article/pii/S0010854500821098, Accessed 2022
6. Hines, 2016, Separating Plant Pigments, https://slideplayer.com/slide/9022838/, Accessed 2022
7. Iczkowski, 1961, Electronegativity, https://pubs.acs.org/doi/pdf/10.1021/ja01478a001, Accessed 2022
8. Juniata, not dated, Thin Layer Chromatography, https://more.juniata.edu/academics-classes/science-in-
motion/labs-and-equipment/media/chemistry-files/TLC%20of%20Veggies.pdf, Accessed 2022
9. Kusmita, 2015, Identification, Isolation and Antioxidant Activity of Pheophytin from Green Tea (Camellia sinensis
(L.) Kuntze), Accessed 2022
10. Lumbessy, 2021, Identification of chlorophyll pigment on Gracilaria Salicornia seaweed
https://iopscience.iop.org/article/10.1088/1755-1315/681/1/012017/pdf, Accessed 2022
11. Moldoveanu, 2013, Mobile phases and their properties,
https://www.sciencedirect.com/topics/chemistry/dielectric-constant, Accessed 2022
12. Pierce, 1981, Selection of solvents for thin-layer chromatography by means of a simple ranking system based on
dielectric constants,
https://www.tandfonline.com/doi/pdf/10.3109/00498258109045323?casa_token=k5lXydsurpIAAAAA:b5E5FJvC
N-zOK6IjpmKtvxAG1I6e5GlZiwcIj8cHQOPOEXWgoYZJKlzrndTpjfvd3WBofY9nkHzgC54, Accessed 2022
13. Rathore et. al, 1993, Thin-layer chromatographic behaviour of carbamate pesticides and related compounds,
https://www.sciencedirect.com/science/article/pii/002196739380566Q, Accessed 2022
14. Snyder, 1968, Principles of Adsorption Chromatography; the separation of non-ionic organic compounds,
http://library.nuft.edu.ua/ebook/file/Snyder1968.pdf, Accessed 2022
15. Soczewinski, 1971, Application of the law of mass action to thin-layer adsorption chromatography systems of the
type of electron donor solvent, https://link.springer.com/content/pdf/10.1007/BF02314900.pdf, Accessed 2022
16. Uchiyama, 1983, https://www.sciencedirect.com/science/article/pii/S0021967301881176,
https://www.sciencedirect.com/science/article/pii/S0021967301881176, Accessed 2022

148999T 6
Stage 2 Chemistry 2022

Design Practical:
Chromatography

Aim: To investigate the effect of varying eluent polarity

(measured through its dielectric constant, 𝜖) on the
retardation factor (𝑅! ) of the plant pigment Pheophytin
within normal-phase thin-layer chromatography.

Folio
148999T2: Design Practical 148999T 7
Introduction
Polarity
Electronegativity is a measure of an atom’s tendency to attract bonding pairs of electrons (Iczkowski,
1961). In a covalent bond between two equally electronegative atoms, the net electron position will be
(on average) halfway between the two atoms. In the case of a bond between atoms of differing
electronegativity (polar bonds), the electrons will be found closer to the more electronegative atom (𝛿 ! )
(Furió, 1996). Molecules with asymmetrically distributed polar bonds are considered ‘polar molecules’.
Dielectric constant (𝜖) is a unitless value often used to quantify polarity (Appendix 1.1) (Griffiths, 1979).

Figure 1.1 The distribution of bonding electron pairs in polar (right) and nonpolar (left) covalent bonds.

Thin-Layer Chromatography
Thin-layer chromatography (TLC) is a technique used to separate mixtures into components of varying
polarity. The method is often applied analytically, to monitor the development of a reaction, and
preoperatively, to purify small samples (Paul, 1967). TLC involves a stationary phase, mobile phase
(eluent), and sample to be analysed (analyte) (Figure 1.2). The mobile phase travels up the TLC plate
by capillary action (Appendix 1.2), often carrying analyte particles with it (Snyder, 1968).

Figure 1.2 A simple illustration of the typical setup for Thin-Layer Chromatography (TLC).

148999T 8
An overview of the main steps within TLC is illustrated below.

Figure 1.3 An illustration of the basic processes involved in Thin-Layer Chromatography

Competitive Adsorption
Adsorption is defined as the temporary attachment of particles to a surface, such as the TLC plate. In
Normal-Phase Chromatography, a polar stationary phase (typically made from silica, SiO2) is used in
combination with a comparatively non-polar mobile phase (Paul, 1967). Silica gel (SiO" ∙ 𝑥H" O) contains
a network of silicon-oxygen bonds, hydroxyl (O-H) bonds, and water molecules on its surface.
Consequently, strong intermolecular forces form between the polar TLC plate and other polar analytes,
enabling adsorption to occur (Figure 1.4) (Moldoveanu, 2013).

Figure 1.3 An illustration of analyte adsorption (left) compared to no adsorption (right)

148999T 9
As the solvent front rises up the TLC plate, the eluent attempts to interact with analyte particles. It is
therefore in competition with the adsorbent stationary phase to retain the analyte. If an analyte particle
exhibits a stronger interaction with the stationary phase, it remains adsorbed to the surface (Rathore,
1993). If the analyte exhibits stronger interactions with the mobile phase, however, it is ‘desorbed’, and
dissolved into the eluent (Juniata, not dated). Therefore, competitive adsorption is dictated by an
analyte’s relative affinity to both the stationary and mobile phases.

Figure 1.4 A depiction of the competitive adsorption that occurs within Thin-Layer Chromatography

Polar eluents often possess the capacity for dipole-dipole, hydrogen, or ion-dipole bonding with polar
analyte species. For instance, Pheophytin possesses a highly polar heterocyclic chlorin ring (Figure
1.5), so can hydrogen bond with polar eluents, including
acetone (Lumbessy, 2021). These interactions are much
stronger than the comparatively weak dispersion forces
exhibited by non-polar eluents, and therefore have a greater
capacity to displace adsorbed analyte particles (Parmawati,
2020).

Figure 1.5 The chemical (skeletal) structure of the plant pigment Pheophytin A

148999T 10
Therefore, as eluent polarity increases, the tendency for a polar analyte to become displaced on the
surface of the stationary phase increases (Paul, 1967). Thus, the equilibrium between mobile-stationary
phase interactions with analyte particles is disturbed, and a greater number of analyte particles are
dissolved into the eluent (Baranowska, 2004). Due to the direct relationship between polarity and an
eluent’s affinity to interact with a certain analyte, the competitive adsorption model predicts this increase
in dissolved analyte particles to be approximately linear when testing on eluents of ‘mild’ polarity
(Snyder, 1968). Particles dissolved into the eluent are carried up by the mobile phase further, and
therefore migrate a greater distance (Pierce, 1981). This distance can be quantified using a retardation
factor.

Retardation Factor (𝑅& )

After separation, individual eluent components appear as ‘spots’. A spot’s retardation factor (𝑅# ) is
defined as the ratio of its distance travelled, compared to the distance travelled by the solvent front
(LibreTexts, 2022). 𝑅# is calculated using the formula shown below, and is dependent on the polarity
of both the stationary and mobile phase, as well as that of the analyte (Snyder, 1968).

𝑑(analyte)
𝑅# =
𝑑(eluent)

𝑅# Retardation factor, unitless

𝑑(spot) Distance travelled by constituent ‘analyte’, cm
𝑑(solvent) Distance travelled by solvent front (eluent), cm

Figure 1.6 An equation illustrating the formula from which 𝑅# is calculated

To explore the effect of eluent polarity on Pheophytin separation during thin-layer chromatography, TLC
will be undertaken on a sample of plant pigments (sourced from the spinach plant). Eluent polarity will
be altered by using eluent mixtures of acetone (𝜖 = 20.7) and hexane (𝜖 = 1.88). The proportions of
both eluent components will be altered, to create eluent mixtures of varying dielectric polarity. The
mean 𝑅# value for the polar pigment Pheophytin will be measured in each instance, to investigate the
relationship between eluent polarity (𝜖) and retardation factor (𝑅# ).

148999T 11
Hypothesis
As the eluent dielectric constant (𝜖) increases, the mean retardation factor of Pheophytin will increase
linearly, according to the principles of competitive adsorption.

Variables
Independent
The polarity of the eluent, given by the dielectric constant (𝜖) of binary composition hexane-acetone
mixtures, calculated using mole fractions of each constituent.

Dependent
The mean retardation factor (𝑅# ) of the polar compound Pheophytin, obtained through thin-layer
chromatography on a solution extracted from the leaves of Spinacia oleracea.

Controlled
Stationary Phase Polarity
The stationary phase is in constant competition with the mobile phase to interact with the analyte. The
capacity for a stationary phase to adsorb the analyte is dependent on its intermolecular interactions
with analyte particles, and therefore its own polarity. The strengthening or weakening of intermolecular
forces that accompanies a change in TLC plate polarity would therefore alter the analyte’s capacity to
be displaced on the plate’s surface, and therefore its capacity to be carried by the solvent front. 𝑅#
would consequently change. To maintain consistent stationary phase polarity throughout, identical TLC
plates from the same manufacturer were used in all trials.

Figure 1.6 An illustration of the relative affinities of two different stationary phases for an analyte

148999T 12
Binary Eluent Constituents (Acetone and Hexane)
The eluents used in each trial were binary mixtures, consisting of acetone and hexane. Both
constituents were used throughout all trials, to minimise the effects of unintended reactions. For
instance, some basic compounds, such as sodium hydroxide, react with silica under TLC conditions.
Not only do both hexane and acetone not chemically react with silica, but they do not react with each
other, therefore preventing unintended interactions from occurring in all trials and distorting the
interactions of the stationary and mobile phases.

Uncontrolled
Temperature
Dielectric constant (𝜖) is temperature dependent (Moldoveanu, 2013). The approximation 𝜖"$ was used
in all trials, under the assumption that room temperature remained ~25ºC. Subtle fluctuations in room
temperature may have significantly altered the eluent’s 𝜖 value (Figure 1.7), therefore making the
mathematical 𝜖 approximations either lower or higher than their experimental values. This variable could
have been mitigated by the use of an electronic water bath, to ensure the chamber and its contents
remained at exactly 25ºC throughout. However, temperature fluctuations were likely minimal.

Dielectric Constant as a Function of Temperature

23

22

21
Dielectric Constant

20

19

18

17
0 5 10 15 20 25 30 35 40 45 50 55
Temperature (ºC)

Figure 1.7 An illustration of the inverse proportionality between dielectric constant and temperature.
The original relationship proposed by (Moldoveanu, 2013) provided temperature values in Kelvin,
however temperature values have been converted to ºC for ease of comparability.

148999T 13
Materials
Chemical Quantity
Propan-2-one (C3H6O); alternatively known as acetone 10.0 ml
Hexane (C6H14) 15.0 ml
Distilled water (H2O); for control trial 5.0 ml
Distilled water (H2O); for rinsing As required
Spinacia oleracea (spinach) solution 10.0 ml

Glassware Quantity
Mason jar 1
Organic waste container 1
Capillary spotter 1
Test tube (10 ml) 2
Measuring cylinder (10 ml) 5
Watch glass 6

Miscellaneous Quantity
Filter paper (circular sheet) 3
Silica TLC plate 6
HB pencil 1
Ruler 1
iPad equipped with digital measurement software 1
Cement mat 1

148999T 14
Procedure
Preparation of Binary Composition Solvents
1. 1.0 ml of propan-1-one and 4.0 ml of hexane were measured in independent measuring cylinders
2. The contents of each measuring cylinder were combined in a test tube, and lightly agitated. A watch
glass was subsequently placed over the test tube to minimise evaporation of volatile constituents
3. Steps 1-2 were repeated a further four times to create binary composition solvents according to
the following ratios (See Figure 3.1)

Figure 3.1 A table illustrating the volumes of the various reagents (hexane and acetone) used to
construct the binary composition solvents used within the experiment. A simplest-form ratio has also
been given to facilitate easier interpretation of the solvent’s overall polarity.

Solvent 𝑉%&'()& (ml) 𝑉(*&+,)& (ml) 𝑉%&'()&/(*&+,)&

A 1.0 4.0 1:4

B 1.5 3.5 3:7
C 2.0 3.0 2:3
D 2.5 2.5 1:1
E 3.0 2.0 3:2

Thin Layer Chromatography

4. A line was drawn 20.0 mm from the base of each of the five TLC plates, using a HB pencil
5. Another line was drawn 10.0 mm from the top of each TLC plate
6. 2.0 ml of analyte (Spinacia oleracea solution) was drawn using a capillary tube, and spotted onto
the origin of one plate drawn in the previous step. Two ‘spots’ of analyte were spotted onto the
TLC plate
7. 2.0 ml of Solvent A was transferred into the mason jar
8. The prepared TLC plate was placed perpendicularly to the bottom surface, into the reaction vessel.
The lid was then sealed, preventing the escape of volatile solvents
9. Once the solvent front had reached the designated line, the TLC plate was removed from the
reaction vessel
10. An 𝑅# value for Pheophytin (in each of the two spots) was then calculated, using the iPad application
Measure
11. Steps 1 to 10 were repeated for Solutions B, C, D, and E

148999T 15
Identification and Management of Safety and Ethical Risks
Safety Considerations
Acetone Exposure
Acetone has been known to initiate headaches, dizziness, confusion, nausea, vomiting, effects on the
blood, passing out and possible risks of a coma. Therefore, exposure to acetone poses a significant
safety risk. This potential risk was minimised by wearing personal protective equipment (PPE), namely
safety goggles and lab coats, throughout the experiment, and by placing the lid on the acetone
container in between trials.

Broken Glassware
Several pieces of glassware, including glass test tubes, measuring cylinders, and mason jars, were
used in this experiment. Broken glassware may cause injury if it punctures the skin. Accordingly, any
broken glassware was disposed of immediately, and extensive cleaning of the laboratory occurred after
the experiment was completed to remove residual glass that may cause injury.

Fluid Spills
Within the experiment, liquid, including the spinach extract, acetone, and hexane solutions, as well as
distilled water, were used. These liquids, if spilled, may cause a slipping hazard, leading to potential
injury. Therefore, paper towel was made readily available throughout the experiment’s entirety and all
spills were cleaned immediately after occurring, to minimise this safety risk.

Ethical Considerations
No living organisms were used within this experiment, which was overall ethically sound. The mixture
containing Pheophytin was obtained ethically from the spinach plant (Spinacia oleracea) All organic
waste was disposed of appropriately, in an organic waste container. This was collected by the lab
technician and safely removed.

Due to acetone’s reported ability to interfere with the brain if ingested, participants were granted the
opportunity to temporarily leave the room if necessary. This ensured that their physical safety was
prioritised. Hence, three members were allocated to each group to enable this rotational participation
to occur.

148999T 16
Results
Qualitative Data
Below are simplified illustrations of the chromatograms obtained in the experiment, for the trials using
solvents A to E.

Figure 5.1 An illustration of the TLC plates obtained during the experiment, isolating only Pheophytin.
Distilled water was used as a test, and no separation occurred due to the strength of the secondary
interactions between water and silica molecules.

It was also observed that eluents of higher polarity took greater periods of time to reach the designated
line for the final solvent front. This is likely because, as polarity increases, the strength of the interactions
between the mobile phase and analyte increases, thus slowing the rate of movement of the solvent
front. This observation evidence that the solutions of greater dielectric constant are indeed more polar.

148999T 17
Sample Calculations
Mole Fraction of Binary Eluent
The binary eluent mixtures were made from acetone and hexane, which are polar and non-polar,
respectively, and therefore have different dielectric constants. Using a mole fraction, rather than
alternative measures such as volume fraction or mass fraction, has been documented as favourable,
due to its capacity to account for parameters such as partial pressure. While partial pressure is more
applicable to gases, the proportion of eluent in the gaseous phase during TLC has led scientific protocol
to favour mole fractions in chromatographic investigation . Thus the mole fractions of each created
mixture needed to be determined.

Mass of Component
The mass of an eluent constituent, such as acetone, was calculated using its density and the measured
volume in the eluent. The density of acetone is a known value (0.784). A sample calculation has been
undertaken for Solvent A (1:4 volume fraction mixture).

𝑚 = 𝑉(*&+,)& × 𝑑(*&+,)& = 1 × 0.784 = 0.784 g (3sf)

Moles of Component
The compound’s molar mass was then used to calculate the number of moles of the compound
present in the eluent mixture. The molar mass of acetone was known to be 58.08 g. mol-1.

.0123452 0.234
𝑛= /0123452
= $3.03 = 0.0135 moles (3sf)

Mole Fraction
The mole fraction could be subsequently calculated, by simply expressing the number of moles of
acetone as a ratio of the number of moles of all constituents in the mixture.

𝑛(*&+,)& 0.0135
𝜑(*&+,)& = = = 0.307 (3sf)
𝑛%&'()& + 𝑛(*&+,)& 0.0304

The mole fractions for both acetone and hexane within each solvent is listed in Appendix 3.

148999T 18
Sample Calculations
Eluent Dielectric Constants
Dielectric constant (𝜖) was selected as an appropriate quantification of the polarity of the solvent
mixture. The dielectric constant for many known solvents is readily available in literature. Thus, these
values can be used as standards from which subsequent calculation can be undertaken. The dielectric
constant of the eluent mixture was calculated using the mole fraction of the sample.

5
𝜖. = L 𝜖 5 𝜑5
6

𝜖. Dielectric constant of the binary composition solvent, unitless

𝜖5 Dielectric constant of a solvent constituent, unitless
𝜑5 Molar fraction of a solvent in the mixture, unitless

The dielectric constant for the binary composition mixture ‘Solvent A’ has been expressed below.

𝜖. = 𝜖7897:;!6!9;< × 𝜑7897:;!6!9;< + 𝜖=<>:;< × 𝜑=<>:;<

𝜖. = (20.7)(0.69) + (1.88)(0.31) = 7.67 (3sf)

A table depicting the respective mixture dielectric constants for each of the five binary composition
solvents is shown below.

Table 5.1 A table depicting the dielectric constant of each of the five solvents used in the experiment

Solvent 𝑉%&'()& (ml) 𝑉(*&+,)& (ml) 𝜑%&'()&/(*&+,)& 𝜖

A 1.0 4.0 69:31 7.67

B 1.5 3.5 57:43 10.0
C 2.0 3.0 46:54 12.1
D 2.5 2.5 36:64 13.9
E 3.0 2.0 27:73 15.6

148999T 19
Sample Calculations (continued)
Retardation Factor (𝑹𝒇 )
𝑅# is defined as the ratio between the distance travelled by the analyte, and the distance travelled by
the eluent (mobile phase) during Thin Layer Chromatography. The following example calculates the 𝑅#
value for Pheophytin, for the trial using Solvent A.

@ABC:;D< B79C C8:F<GB "$.$

𝑅# = @ABC:;D< B9GF<;C C8:F<GB = 4H.$ = 0.51515 ≈ 0.515 (3sf)

Mean Retardation Factor (Mean 𝑹𝒇 )

To reduce the effect of random error, 𝑅# was measured for both spotted samples on each TLC plate,
so that an average of the two values could be used. The following example calculates the mean 𝑅# value
for Pheophytin, in the trial using Solvent A.

I6 (6) L I6 (")…LI6 ()) 0.$6$L0.$0$

Mean 𝑅# = )
= "
≈ 0.510 (3sf)

Quantitative Data
Table 5.2 A table illustrating the retardation factor (𝑅# ) and mean retardation factor obtained in the
experiment, when conducting TLC using five eluents of differing dielectric constant (𝜖).

Trial 𝜖 𝑑&NO&)+ 𝑑P%&,P%Q+5)(6) 𝑑P%&,P%Q+5)(") 𝑅# ⋅ 1 𝑅# ⋅ 2 𝑅# ⋅ Avg

A 7.67 49.5 25.0 25.5 0.505 0.515 0.510

B 10.0 49.5 32.5 33.0 0.657 0.667 0.662
C 12.1 49.5 38.5 39.0 0.778 0.788 0.783
D 13.9 50 43.5 44.0 0.870 0.880 0.875
E 15.6 51 47.0 47.0 0.922 0.922 0.922

148999T 20
Quantitative Data (continued)
The data represented in Table 5.1 was graphically represented on a scatter plot, therefore giving rise
to Figure 5.2.

Figure 5.2 A graph illustrating the relationship between the mean retardation factor (𝑅# ) of the pigment
Pheophytin and the eluent polarity, measured through its dielectric constant (𝜖). As both parameters
are unitless, units have been omitted from the axes of subsequent graphs.

Pheophytin A Retardation Factor as a Function of Eluent Polarity

1.0

0.9
y = 0.0531x + 0.1215
R² = 0.9847
0.8
Mean Pheophytin A Retardation Factor

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
Eluent Dielectric Constant

Figure 5.2 indicates a positive, linear correlation between the eluent dielectric constant (𝜖) and the
mean retardation factor (𝑅# ) of Pheophytin. The smallest and greatest dielectric constants (𝜖 = 7.67 𝜖
= 15.6) yield the smallest and greatest retardation factors (𝑅# = 0.510 and 0.922) respectively. The
values conform to a linear regression with low scatter about the line-of-best-fit, as substantiated by an
R2 value of 0.9847. The R2 value is somewhat tainted by an apparent deviation from linearity at 𝜖 =
15.6. Discussion surrounding this anomalous point will occur within the Evaluation of Procedures.

148999T 21
Quantitative Data (continued)
In light of the apparent deviation from linearity in Figure 5.2, results from trials using Solvent E, which
used an eluent of polarity 𝜖 = 15.6 were omitted. The reconstruction of the graph produced Figure 5.3,
which has been included below.

Figure 5.3 A graph illustrating the relationship between the mean retardation factor (𝑅# ) of the pigment
Pheophytin A and the eluent polarity, measured through its dielectric constant (𝜖). In an attempt to
increase the linearity of the relationship, Trial E (𝜖 = 15.6) was removed from Figure 5.2, and the line-
of-best-fit was recalculated.

Pheophytin A Retardation Factor as a Function of Eluent Polarity

(omission of ! = 15.6)
1.0

0.9
y = 0.0585x + 0.068
R² = 0.9971
0.8
Mean Pheophytin A Retardation Factor

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Eluent Dielectric Constant

Figure 5.3 again illustrates a positive, linear correlation between the eluent dielectric constant (𝜖) and
the mean retardation factor (𝑅# ) of Pheophytin. Evidently, however, the values conform to a linear
regression more closely, with negligible scatter about the line-of-best-fit and an increased R2 value (in
comparison to Figure 5.2) of 0.9971. The defined linear tendencies of the first four consecutive
datapoints confirms the relationship’s linearity; a logarithmic relationship would not cater to such
linearity.

148999T 22
Analysis of Trends
Justification of an Appropriate Trend
𝑅# exists on a continuous spectrum from 0.0 to 1.0 only, meaning the linear 𝜖/𝑅# relationship obtained
in this experiment appears implausible for eluents of extreme polarities. For instance, the equation of
the line-of-best-fit in Figure 5.1 indicates that an eluent mixture exceeding an acetone proportion of
66.52% would yield an impossible 𝑅# of >1.

A possible solution to this predicament is that as eluent dielectric constant approaches extremities, the
𝜖/𝑅# relationship takes the form of a sigmoid function (Figure 6.1). The results displayed in Figure 5.1
resemble Segment 3 of the proposed sigmoid model, which appears reasonable, given the mild polarity
of the binary solvents used. Although further experimentation with the extension of dielectric parameters
is required to emulate the complete 𝜖/𝑅# relationship, the results of the experiment nonetheless align
with the competitive adsorption theory presented in literature, thus supporting the hypothesis.

Figure 6.1 An illustration of the complete 𝜖/𝑅# relationship, based on the principles of the competitive
adsorption model. The relationship, which is rarely investigated in its entirety in literature, has been
separated into five segments, which each resemble a different regression; linear (1), exponential (2),
linear (3), logarithmic (4), and linear (5).

148999T 23
Application of a Sigmoid Function
The possibility of a sigmoid model was tested by applying a logistic regression to the obtained data.
This regression was compared to a quantification of Pheophytin separation by (Parmawati, 2020), that
implemented an acetone-hexane eluent of 𝜖.=.10.0. (Parmawati’s) 𝑅# was 10.87% smaller than the
value obtained from this experiment, indicating the experimental methodology as moderate-to-highly
accurate. However, although (Parmawati’s) experiment implemented a silica TLC plate, acetone-
hexane binary eluent, and Pheophytin analyte, the magnitudes of other chemical parameters such as
temperature and vessel saturation cannot be confirmed, thereby evidencing limitation to this proposed
evaluation of accuracy.

Figure 6.2 A sigmoid regression (equation shown in the bottom right corner of the graph) was applied
to the obtained data using Desmos digital graphing software. A regression of best fit with respect to
the obtained data values was therefore utilised.

148999T 24
While the mild percentage error between this experiment’s and (Parmawati’s) results rendered the
experimental methodology valid to Pheophytin isolation using a hexane-acetone eluent, comparisons
were drawn to independent investigations with varied parameters to establish validity with regards to
the effect of eluent polarity on chromatographical separation (Figure 6.3). The significant percentage
error that results from these comparisons (Figure 6.4) suggests that experimental values themselves
can only be applied to hexane-acetone binary mixtures. However, the sigmoid nonetheless supports
the competitive adsorption model, evidencing high validity for the parameters tested.

Figure 6.3 A comparison was drawn between values from independent experiments and the
retardation factors obtained in this experiment. The parameters and percentage error of the results
used to draw comparisons have been stated in Figure 6.4.

Figure 6.4 A comparison of independent studies to the sigmoid from this investigation

# Eluent ϵ R R(SAF<;) R R(A;C) % Error

1 Hexane/Acetone/Ethyl Acetate 7.49 0.590 0.463 -21.5%

2 Hexane/Acetone/Ethyl Acetate 7.93 0.730 0.505 -30.8%

3 Ethanol 25.3 0.810 0.946 +16.8%

148999T 25
Evaluation of Procedures
Random Error
Pheophytin Heterogeneity
Although the spinach solution was spotted onto the TLC plate using the same capillary spotter in each
trial, biological pigments such as Pheophytin are non-uniformly distributed within plants such as
Spinacia oleracea, meaning greater or smaller Pheophytin concentrations may have been used
between trials. As 𝑅# is thought to be dependent on analyte concentration (Harvard, not dated),
measured 𝑅# values may have been lower or higher than their true value, decreasing reliability. Using a
homogenous standard Pheophytin solution would minimise this random error.

Spot Detection Ambiguity

𝑅# was calculated as a ratio of the distance the analyte ‘spot’ travelled, in comparison to the distance
travelled by the solvent front. However, spot measurements were ultimately prone to random error; the
ambiguity surrounding spot detection may have generated larger or smaller 𝑅# values, thereby
decreasing precision and thus reliability. To minimise the inherent error within human measurement,
the chromatogram was digitally enlarged (Figure 7.1), allowing easier identification of the spot centre.
Upon identification, software was then used to measure the distance travelled by the spot, minimising
the effect of this error.

Figure 7.1 Digital software was used to identify and localise the centre of each spot. This process
reduced the ambiguity and therefore generated more representative 𝑅# values.

148999T 26
TLC Plate Uniformity
The commercial manufacturing model assumes a homogenous distribution of TLC plate components.
Consequently, the number of surface hydroxyl groups (and thus analyte binding sites) should be
proportional to the surface area of the plate itself. However, the plates were manually cut to the
dimensions [50x15 mm] using scissors, and subtle differences in plate surface area were observed.
These differences may have lowered or raised the number of binding sites on each plate, altering the
stationary phase’s binding capacity and thus 𝑅# , lowering reliability and increasing scatter. A general
model proposed by (Snyder, 1968) indicates a linear relationship between 𝑅# and the logarithm of the
adsorbent surface area. The use of mechanically cut plates to exact dimensions would have reduced
the effect of this random error.

Figure 7.2 The uneven manual cutting technique of the TLC plates generated plates of different
surface area. The above image shows the six TLC plates used within the experiment, and subtle
deviations in the overall plate shape can be seen.

148999T 27
Systematic Error
Multilayer Adsorption
Potential error pertains to multilayer acetone adsorption within the narrow pores of the stationary phase.
Small polar solvents (e.g. acetone) tend to form ordered structures in the proximity of solid surfaces
(Snyder, 1968). These structures can become ‘trapped’ within the narrow pores of the stationary phase
by the subsequent adsorption of analyte particles (Figure 7.3).

Figure 6.1 A diagram depicting the effect of multilayer adsorption on the composition of the eluent,
due to the removal of acetone from the interstitial solvent front.

This multilayer adsorption removes acetone particles from the interstitial solvent front, thereby
decreasing the interstitial eluent 𝜖"$ in all instances, reducing accuracy and therefore the validity of
obtained results. This systematic error is evidenced by the deviation from 𝜖"$ /𝑅# linearity at the greatest
eluent polarity, 𝜖"$ = 13.2 (refer back to Figure 5.2); multilayer adsorption only occurs with a sufficiently
polar eluent.

148999T 28
Evaporation of Volatile Solvents
Potential systematic error pertains to the evaporation of acetone during the experiment. While both
solvent constituents of the eluent (hexane and acetone) have low boiling points (56 and 69ºC,
respectively), acetone is highly prone to evaporation at room temperature, while hexane typically
requires greater temperatures (e.g. 45-50ºC) to become sufficiently volatile. As samples were prepared
and tested in ascending order of polarity, evaporation of acetone would be sequentially amplified,
reducing the accuracy of the assumed dielectric constants in comparison to their true values, thereby
decreasing validity. This error could be identified by repeating the experiment with less volatile solvent
constituents, and comparing results for inconsistencies.

Conclusion
The effect of varying eluent polarity on the separation of pigments was tested by conducting thin-layer
chromatography (TLC) on a sample containing Pheophytin A, and measuring the pigment’s mean
retardation factor. The results indicate a linear proportionality between eluent polarity (measured
through its dielectric constant) and Pheophytin retardation factor, thereby supporting the principles of
competitive adsorption. The findings are consistent with other investigations with similar parameters,
yielding a minimal percentage error of <11%. However, significant discrepancy was observed between
𝑅# values obtained from TLC with ternary eluents, and 𝑅# from studies conducted on other biological
pigments, including Chlorophylls, Xanthophylls, and Carotene. Thus, the experimental conclusions
appear valid only for the separation of Pheophytin A during TLC with an acetone-hexane binary eluent.

148999T 29
Appendix 1
Dielectric Constant (𝜖)
𝜖 is an entity that assigns compounds a particular value, on the basis of their molecular polarity.
Essentially, compounds with higher 𝜖 are more polar. A compound’s 𝜖 indicates the extent to which a
material can become polarised by imposition of an electric field on an insulator (Figure A1.1), and is
defined as the ratio of the permittivity of a substance to the permittivity of free space.

Figure A1.1 An illustration of the polarisation that dielectric constant measures.

The 𝜖 value of a substance can be determined experimentally, using the above capacitor setup,
however the 𝜖 values for common solvents are readily accessible in scientific literature. As this
quantification is a measure of the ratio of two entities, both of which have identical units (Farads per
metre, F.m-1), dielectric constant is therefore unitless (Figure A1.2).

𝜀BTUBC:;D<
𝜖=
𝜀R8<< B7:D<

𝜖 Dielectric constant, unitless

𝜀BTUBC:;D< Permittivity of substance, F m!6
𝜀R8<< B7:D< Permittivity of free space, F m!6

Figure A1.2 An equation illustrating the formula from which dielectric constant (𝜖) is calculated.
148999T 30
Appendix 2
Principles of Capillary Action
In their liquid state, eluent particles exhibit secondary interactions with other eluent particles. The
strength of these intermolecular forces is dependent on the polarity of the eluent constituents. For a
binary solvent of hexane and acetone, the greater the molar proportion of acetone, the stronger the
interactions. These secondary forces are known as cohesive forces.

When the TLC plate is submerged in the eluent mixture, eluent particles also interact with the particles
on the surface of the TLC plate. These forces are known as adhesive forces. As the TLC plate is typically
made of hydrated silica or alumina, both very polar substances, the adhesive secondary interactions
are typically stronger than the eluent cohesive forces. Therefore, eluent particles have a greater affinity
for the stationary phase than the mobile phase, and travel towards (and therefore adhere to) the TLC
plate. The particles continually move away from each other and towards the stationary phase, rising up
the plate. This process is known as capillary action.

Figure A2.1 An illustration of the cohesive and adhesive forces that enable capillary action to occur.

148999T 31
Appendix 3
Mole Fractions of Eluent Mixtures
The mole fractions of each component in each trial have been listed below.

Figure A3.1 The mole fractions for each component of the binary eluents used.

Hexane (ml) Acetone (ml) Hexane (mol) Acetone (mol) 𝜑 Hexane 𝜑 Acetone

4.0 1 0.0304 0.0134 0.692 0.307

3.5 1.5 0.0266 0.0202 0.567 0.432

3.0 2 0.0228 0.0269 0.457 0.542

2.5 2.5 0.0190 0.0337 0.360 0.640

2.0 3 0.0152 0.0404 0.272 0.727

148999T 32
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