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Lecture 8

CHEM 233 Lecture Notes - Lecture 8: Watch Glass, Pheophytin, Methyl Orange

Course Code
CHEM 233
Driver Tom

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Chromatographic Methods: Separation of Dyes and Spinach Pigments by Column and
Riyushi Mahadik
February 5, 2014
Methods and Background
The purpose of this lab was to get familiar with techniques of column chromatography and thin-
layer 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 > RR’C=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
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
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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 ¾ 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
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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
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.
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