Chromatographic Methods: Separation of Dyes and Spinach Pigments by Column and

TLC
Riyushi Mahadik
February 5, 2014
Methods and Background
The purpose of this lab was to get familiar with techniques of column chromatography and thinlayer chromatography. Also, this lab helps to understand the relationship between solvent
polarity (dielectric constant) and eluting power in chromatography. The other purpose was to
identify the effect of functional groups on retention time (elutropic series). The methods, column
chromatography and thin-layer chromatography, used in this lab helps to observe the effects of
polarity of the solvent, eluting power, and adsorbent in the separation of dyes and spinach
pigments. These procedures include separation of mixtures of solutions into its components. The
column chromatography is used for separation in the dye containing 1: 1 methylene blue and
methyl orange. The thin-layer chromatography is used for separation of spinach pigments.
Increasing adsorption on polar stationary phases
RCO2H > ROH > RHN2 > RRC=O > RCO2R > ROR > C=C > R-X
Figure 1: Elutropic series for polar stationary phases.
In chromatography there are two phases that helps the mixture to separate in to components.
Mobile phase is the phase with a liquid or a gas that is passes through the stationary phase. The
mobile phase in this lab will be the solvents. The stationary phase is the fixed solid or liquid
phase. In this lab, the polar stationary phase is the silica gel. The components bind with of theses
phase in order to create the separation of pigments. The polarity also comes in the play when the
stationary phase is polar and the mobile phase in non-polar, the polar components of the mixture
bind to the stationary phase and the non-polar components would move down the mobile phase.
This hence proves the idea of like dissolves like. Based on the Figure 1 above that describes
the elutropic series, the functional groups that are more polar are the ones that have greater
affinity for the polar stationary phases. In both of the experiments, silica gel was used which is a
polar stationary phase and dissolves the polar components of the dyes to the stationary phase
better. Thus, the indication of the results would be that polar components of the mixture would
stay up in the column chromatography and more polar substances in thin-layer chromatography
will stay at stationary phase longer period of time. In order to calculate the time the component
of the mixture spends in a particular phase, the concept of retention time can be applied.
Retention time is the time corresponds to the time spent in the stationary phase. The more time
the component spent in the stationary phase, the less time spent in the mobile phase and thus, the
retention time of that component is more. Retention factor is the calculation of a distance
travelled by the substance over the distance traveled by the solvent. This predicts that the higher
Rf values are for the non-polar substances because they would travel the most in polar stationary
phases.
Mobile phases used in the experiments should be picked conscientiously because there should be
arrange of polar and non-polar mobile solvents in order to better justify our results. This means
the eluting power of each of the solvent system should be considered. The definition of Eluting

power is that the ability of a mobile phase to move a substance through a stationary phase. So, if
the stationary phase is polar like Silica gel, the non-polar mobile phases will have less Eluting
power to move a polar substance.
In the experiment of column chromatograph, the separation of dyes (1:1 methyl orange and
methylene blue) is observed. The column pipette has a polar stationary phase (Silica gel) and
several solvent systems mobile phases are used range from polar to non-polar solvents. The
column consists of the layers of cotton, sand, silica gel, and sand from bottom to top. Cotton is
used to avoid the leaking of the Silica gel out of the column. The sand is used to even out the
surface layers and avoid any problems in the flow. The mobile phases used in Column
chromatography are methanol, acetonitrile, acetone, dichloromethane, and hexane. Out of all of
the solvents acetonitrile is more polar that other solvents, thus indicating long retention time and
a small retention factor for the polar substances.
The thin-layer chromatography is used to analyze the separation of spinach pigments according
to their polarities. The pigments in the spinach are chlorophyll b, chlorophyll a, pheophytin b,
pheophytin a, xanthophyll, and carotene. Different solvent compositions will be used to
determine which solvents efficiently separates more pigments on a TLC plate. Different ratios of
Acetone and Hexane will be used as the solvents to separate the pigments. Acetone is more polar
than hexane, thus, the retention factors of the various components in spinach leaves will be based
on the affinity of polar components of the mixture to the polar stationary phase. This experiment
is slightly different from the column chromatography is that the capillary action moves
components upward depending on affinities for the mobile phase.
Thin layer chromatography determines the separation of six pigments in spinach leaves. But
none of the TLC plates had more than four pigments distinguished on it which means the data is
not significantly right. The most of the pigments observed were chlorophyll a, chlorophyll b
xanthophyll and carotenes. But in one of the plate, there were 4 distinct bands light green, dark
green, dark yellow, and light yellow from top to bottom. The possible experimental errors could
include dilution of the spinach mixture could lead to the less concentrated pigments.
Pigment polarity:
Least Polar: Carotene < Pheophytin a < Pheophytin b < Chlorophyll a < Chlorophyll b <
Xanthrophyll :Most Polar
Experimental Procedures
Column Chromatography
The five column pipettes were prepared with the layers of cotton, sand, silica gel and sand.
Firstly, a ball of cotton was inserted with copper wire. Then, about 1 cm of sand was added to the
cotton. The columns were filled with of silica gel in a hood. Again, a layer of 1cm sand was
added to the columns. Four drops of 1:1 methyl orange and methylene blue were added to the
columns. The columns were numbered for different solvent system. For example, number one

column was filled with methanol up to the brim twice. Number two column was filled with
acetonitrile up to the brim twice. Number three column was filled with acetone up to the brim
twice. Number four column was filled with dichloromethane up to the brim twice. Number five
column was filled with hexane up to the brim twice. The solvents were collected in test tubes.
The observations were recorded that included identification of different colored bands.

Figure 2. Setup for column chromatography

Thin-layer Chromatography
Extraction
Two fresh leaves of spinach were grounded in a mortar with a pestle until they were
homogenous. Then, 10 ml of methylene chloride was added to the mixture which was mixed
well. Then, the apparatus for vacuum filtration was set up which included air tube connected to
Erlenmeyer flask and to the vacuum chamber. Then, a ceramic funnel with No. 1 filter paper was
fixed on the mouth of the flask. The vacuum chamber was turned on while the mixture of
spinach extraction was added for filtration. The filtrate was transferred into a separatory funnel.
Then 5 mL of H2O was added in the separatory funnel which was then swirled gently until there
was a separation of bottom organic layer and aqueous layer. The organic layer was drained into a
clean flask and the remaining aqueous layer was discarded. While doing this experiment, the first
trial was not done correctly. So, instead the remaining sample of spinach was used and added
more solutions (5 ml of methylene chloride and 5 ml of water) to it. A little amount of Na2SO4
was added to the solution (filtrate) until the solid clumped together.
Thin-layer Chromatography Preparation

Six TLC plates were numbered and a line was drawn approximately 1 cm from the bottom with a
dot on it. The open end of a capillary tube was immersed into the filtrate which was then applied
on to the spot on TLC plate. The filtrate was allowed to evaporate to identify the left spots. This
process was done 10-15 times while a green spot was identified easily on TLC plate. Then, six
beakers were set up that had different solvents for the experiments. TLC plate one was immersed
into 0 ml Acetone and 10 ml Hexane. TLC plate two was immersed into 2 ml Acetone and 8 ml
Hexane. TLC plate three was immersed into 4 ml Acetone and 6 ml Hexane. TLC plate four was
immersed into 6 ml Acetone and 4 ml Hexane. TLC plate five was immersed into 8 ml Acetone
and 2 ml Hexane. TLC plate six was immersed into 10 ml Acetone and 0 ml Hexane. The
beakers were covered with watch glass to eliminate excess air flow into the system. The TLC
plates were taken out from the beakers after the eluent reached the top of the TLC plate. Then
retention factors were calculated by taking the distance the pigment traveled divided by the
distance the eluent traveled.

Figure 3. Setup for thin-layer chromatography

Data Acquisition

5
4
3
2
1
Figure 4. Observing the separation of dyes in solvent systems in Column Chromatography

Figure 4. Observing the separation of dyes in different solvent systems in TLC

Data 1: Observations of Separation of Two Dyes in Column Chromatography with Different
Solvent Systems
Trials

Solvent System

Dielectric
Constants
(Er)

Polarit
y

Observations

Methanol

32.6

This was very quick, and distinct

separation with very dark yellow eluent,
but blue band stayed on top.

Acetonitrile

37.5

Similar results to methanol. The eluent in

the test tube was yellow in color.

Acetone

20.7

The yellow dye traveled down the test

tube and took ling time. While the blue
band stayed on top, the large yellow
portion was seen in the Silica gel.

Dichloromethane

9.1

Blue color at the top and yellow color was

observed throughout the stationary phase.
The eluent collected in the test tube was
colorless. Large green band in the middle

Hexane

This was very slow separation and the two

dyes barely separated. The eluent in the
test tube was colorless suggesting that
none of the dyes eluted out from the
column.

Plate
Numbe
r

Data 2: Retention of each pigment from Thin-Layer Chromatography

Eluant
Pigments Observed
Distance
Distance
(Acetone/
Traveled by
Traveled by
Hexane)
Substance
Solvent (cm)mL
(cm)-dx
ds

Rf Value
(dx/ds)

0:10

Green spot at the starting

point

4.9

0 cm/4.9 cm=
0

2:8

Green spot moved from the

starting point

1.1

5.7

1.1 cm/5.7
cm= 0.193

4:6

Three bands were seen: light

greens, dark green and yellow

Green: 2.4

5.3

2.4 cm/5.3
cm= 0.453

Light green:
2.3

2.3 cm/5.3
cm= 0.433

Yellow: 2.2

2.2 cm/5.3
cm= 0.415
4

6:4

Four bands: light greens, dark

green, dark yellow, and light
yellow

Green: 3.5

3.9

Light green:
3.4

3.4 cm/3.9
cm= 0.872

Dark Yellow:
3.3

3.3 cm/3.9
cm= 0.846

Light Yellow:
3.1
5

8:2

10:0

Light Green and Dark Green

pigments

Green pigment

Green: 4.6

3.1 cm/3.9
cm= 0.795
4.5

Light Green:
4.5
4.5

3.5 cm/3.9
cm= 0.897

4.6 cm/4.5
cm= 1.02
4.5 cm/4.5
cm= 1

4.5

4.5 cm/4.5
cm= 1

Relevant Equations
Retention Factor: Rf = (distance traveled by substance)/ (distance traveled by solvent)
Table of Pigments and their Colors:
Pigments

Colors

Xanthrophyll
Chlorophyll a
Chlorophyll b
Pheophytin a
Pheophytin b
Carotene

Yellow
Green
Light Green
Dull Gray
May not be visible
Light Yellow

Table 3: Retention of each pigment from Thin-Layer Chromatography

Pigment Retention Factors (Rf)
Plate
Number

1
2
3
4
5
6

Mobile Phase
Composition
(Acetone/
Hexane) (ml)
0:10
2:8
4:6
6:4
8:2
10:0

Chlorophyll
a
0.193
0.453
0.897
1.02
1.00

Chlorophyll
b
0.433
0.872
1
-

Pheophytin
a& b
-

Xanthophylls

Carotenes

0.415
0.846
-

0.795
-

Pre-lab Questions
Pre-lab- Column Chromatography
1. Circle the most polar compound in each pair of molecules.
a. Ester is more polar than ether because ester has a C=O
b. Carboxylic acid is more polar than alkane chain because Carboxylic acid has
C=O.
c. Alcohol is more polar than amine because the alcohol has OH group.
d. Water is more polar than H-X because Oxygen in water has two lone pairs of
electrons
e. Carboxylic acid is more polar than the alcohol group because Carboxylic acid has
C=O.
f. Amine is more polar than ether because amine has NH bond.
2. What general conclusion can you draw about heteroatoms such as N, O, Cl and polarity,
a. Nitrogen is more electronegative than O and Cl. Thus the polarity will be
N>O>Cl.

3. Place a square around each molecule in 1 that would elute first in a normal phase column.
What is the relationship between the polarity and retention time for normal phase column
chromatography?
a. Higher the polarity, higher will be the retention time. Less polar molecules will
elute out first in a polar stationary phase.
4. Sketch your proposed column chromatography setup in your laboratory notebook,
including stationary phase, cotton plug, sand layers, column, etc. Label relevant item and
briefly describe its function or purpose.
a. The column consists of the layers of cotton, sand, silica gel, and sand from bottom
to top. Cotton is used to avoid the leaking of the Silica gel out of the column. The
sand is used to even out the surface layers and avoid any problems in the flow.
The stationary phase is the Silica gel. At the top of the sand, the mixture will be
added.

5. Determine what solvents are available for use in the hood, Decide on at least five solvents
systems that you intended to try and rank them based on their polarity.
a. (Most Polar)Acetronitrile > Methanol > Acetone > Dichloromethane > Hexane
(Least Polar)
Conclusion with Post-lab Questions
The main goal of this lab was to understand the techniques of column and thin-layer
chromatography. The experiment of column chromatography helps establish the relationship
between the polarity and eluting power. Polar solves such as methanol and acetonitrile separated
dyes into two different components easily than non-polar solvents such as hexane and
dichlotromethane. In the case where the dyes were separated, the blue color being the most polar
was seen on the top of the stationary phase and yellow eluent was out of the column being the
most non-polar component. The greater affinity of polar componnets with the polar stationary

phase led blue dye to stay in the stationary phase for longer period of time. Thus, overall the blue
dye had higher retention time and yellow dye with lower rentention time. Acetone was the case
that was medium polar that partially separted the dyes. This proves there is a direct retlationship
between the polarity of the solvents and the separation of the dyes in a polar stationary phase.
Less polar solvents have less ability to separate the components in a polar stationary phases,
whereas; more polar solvents has a higher ability to separate the components in a polar station
phases.
In thin-layer chromatography, the extract from spinach leaves was applied on to TLC plates
which were placed into beakers with different ratios of acetone and hexane. The polar stationary
phase was the silica gel plate and the mobile phase was the mixture of the two solvents, acetone
and hexane. In the observations, the two best results came from plate 3 and 4 having the ration
do 4ml acetone to 6ml of hexane and 6ml acetone to 4ml of hexane. Polar solvent was acetone
and non-polar solvent was hexane. This enables us to observe the relationship between the
polarity of solvents and the retention factors of the substances in the extract. The ratio of 4A:6H
had three bands in comparison to 6A:4H which had 4 bands that indicates the polar substances
can separate the pigments more distinctly than non-polar substances. Xanthophylls being more
polar than chlorophylls and carotenes had smaller retention factors as compared to the other
pigments that were observed. From the ratio of 6A:4H, the four bands indicates the pigments
Chlorophyll a (Rf = 0.897), Chlorophyll b (Rf = 0.872),Xanthophyll (Rf = 0.846) and Carotenes
(Rf = 0.795) that polar pigments have lower retention factor because they travel less. The polar
pigments have greater affinity to the polar stationary phase than less polar pigments. It was very
hard to observe pheophytins in any of the trials. As a result, in 0A:10H solvent ratio, the polar
pigment chlorophyll b didnt show on the plate because the mobile phase solvent (hexane) in less
polar which doesnt allow chlorophyll b to travel up. Similarly, as the concentration of hexane
decreased in the solvent mixture, less polar pigments such as carotene and pheophytins were not
observed on the TLC plate. From these results, we can conclude that polar attracts polar and
non-polar attracts non-polar
The challenges were faced in thin-layer chromatography. In thin-layer chromatography, the knob
of the separatory funnel was not working properly which wasted some of the extract. Then we
added more of water and methylene chloride in order to collect the sample. But when that sample
ran through TLC, the pigments were so light and it was hard to distinguish the pigments. The
actual reason of this could be that the extract was diluted a lot and the concentration was low
which didnt show lot of bands on TLC. Another challenge was to make sure that the extract
from spinach leaves doesnt evaporate. Initially, one of our samples evaporated leaving the
residue behind, so added more of methylene chloride to it which diluted our extract further
Post lab discussion
In the Column chromatography, Hexane, Dichloromethane and Acetone did not provide adequate
separation because they are less polar solvents which reduce their ability to separate the
compounds of a mixture. In contrast, methanol and acetonitrile separated the mixture adequately
because they were more polar solvents which enabled the components of the mixture to separate
into different bands in the column.

In successful conditions, different colored bands should be observed. Whereas in unsuccessful

conditions the colorful bands were not separated all along the stationary phase of the column.
Based on the results, Methylene blue is more polar than methyl orange because it always
appeared on the top of the stationary phase. The yellow dye eluted out faster with smaller
retention time, whereas; the blue dye didnt elute out from the column.
In thin-layer chromatography, Chlorophyll a appeared as a green spot on TLC plate whereas
Chlorophyll b appeared as light green spot. Chlorophyll b has an aldehyde group and
Chlorophyll a does not have one. This makes Chlorophyll b more polar than Chlorophyll a.
If we wanted to prevent the demetalation of chlorophylls, additions of 5ml of water could have
been avoided.
Copper and Zinc does not have H atom to donate to chlorophylls. As a result, Chlorophylls will
remain ionic (demetalated).
When 100% acetone was used the polar pigments such as Xanthophyll and Chlorophyll B were
observed on TLC plates with larger retention factors. When 100% hexane was used the non-polar
pigments such as carotene and Chlorophyll a were observed on TLC plates with smaller retention
factors. If polar solvents are used as a eluent, Xanthophyll will have smaller Rf values. The
structure of Xanthophyll have an OH group whereas carotenes dont have it. This makes
Xanthophyll more polar than carotenes. If chlorophyll b is less readily dematalated , it will make
the negatively charge atom weak bases than those in chlorophyll a because according to
Bronsted-Lowry, the base donates proton.
Based on TLC analysis, chlorophylls are more polar than pheophytins because the nitrogen
atoms in their structure are protonated which makes them less polar as they are no longer ionic.
Reference
Gilbert, John C., and Stephen F. Martin. Experimental Organic Chemistry. Cengage Learning,
Massachusetts, 2011, 5th Ed, pp. 179-184, 188-192.