CHEM

212 Report Sheet 2019-2020

Column Chromatography

Group Experiment 3 – Individual Lab Report

Last Name: Vance
First Name: Torean
Lab Partner(s): Zack
TA Name: Anthony Izzotti
Date Lab Performed: 2019-09-26
Group: ☐ C
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Comments: If you performed your lab on a day other than your
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CHEM 212 Report Sheet 2019-2020

Objective (3 marks):
The purpose of this lab was to isolate three pigments (β-carotene, chlorophyll-a, and
chlorophyll-b) from raw spinach leaves via use of flash column chromatography. It was also to
determine the Rf values in a comparative TLC plate and to examine the λmax of the various
pigments to analyze and further understand differences in color.

Introduction (3 marks):
Pigment extraction is a fundamental process that involves many key techniques for a
developing chemist’s lab ability: grinding substance, proper use of organic solvents, liquid-
liquid separation, decanting a liquid, gravity filtering, pigment concentration, and UV-visibility
spectroscopy. Because this process is thorough and involves so many techniques, mastering
this basis sets up a student well for future laboratory success.

This lab used flash chromatography –or column chromatography– as well as Thin Layer
Chromatography (TLC) to separate the desired pigments, and to better analyze each
individual chemical. The process of mixture separation is important to understand, because
most compounds found in nature are rarely, if ever, in a pure state: especially with organic
compounds. In this lab using silica gel, a polar substance, and a en eluent composed of a
petroleum ether and acetone the chemist was able to separate the pigments in the raw
spinach based on their polarity. The hydrocarbon, β-carotene, filtered down the column the
fastest, while the chlorophyll with an extra, polar aldehyde group, chlorophyll-b, was the
slowest to descend the column.

Usage of the TLC plate after this filtration was another important aspect to understand for
this lab. The TLC plate’s purpose was to calculate the Rf value of each isolated concentrated
pigment to determine the relative distances each pigment travelled through the solvent.
Using the same pigments one used them to undertake UV-visibility spectroscopy that allowed
one to better understand how wavelength affects color.


Results (12 marks):

Table 1. Column and Corresponding TLC Plate Observations
Wavelength of
Colour Rf value (8:2 TLC) Pigment Identity
Max. Abs (nm)
Fraction 1 Yellow 1.0 β-carotene 448
Fraction 2 Green 0.15 Chlorophyll-a 428
Fraction 3 Blue 0.06 Chlorophyll-b 428

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CHEM 212 Report Sheet 2019-2020

Figure 1. TLC Plates

Sample Calculation for Solvent 8:2:
Distance Chlorophyll-a (spot 2) travelled: 0.9 cm
Distance from starting line to solvent line: 5.9cm
Rf= Distance spot travelled/ Distance from starting line to solvent
Rf = 0.9cm/ 5.9cm = 0.15


Discussion (12 marks):

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CHEM 212 Report Sheet 2019-2020

1. Discussion of physical properties: Comment on the effectiveness of column

chromatography, and rationalize Rf values and the order of elution of the compounds from
the column.
The column chromatography was very effective in separating the different pigments. When
separating the pigments from spinach through the silica gel, the observed β-carotene
pigment was filtered out with eluent the fastest; this gives the impression of separating from
chlorophyll-a and chlorophyll-b which followed in consecutive order close to each other, but
further from the β-carotene. The β-carotene was easier to isolate (and worked more
effectively) because it separated faster and had a much greater apparent quantity, whereas
chlorophyll-a and chlorophyll-b were hard to distinguish where the values between another
ended and thus isolation was less effective.

The mechanism behind the order of elution of the compounds is due to the differing
polarities of the compounds present. β-carotene is the least polar among the three pigments
(as it lacks the polar porphyrin ring) so it was least attracted to the polar silica gel and thus
was ‘washed’ down with the less polar –comparative to the silica gel– eluent the fastest.
Correspondingly, chlorophyll-a and chlorophyll-b are both more polar than β-carotene and
thus took longer to be filtered out with eluent as they were more attracted to the silica gel.
Chlorophyll-b, being the most polar compound (because of the presence of an Aldehyde),
took the longest and thus appeared at the top of the column. Thus the Rf values make sense
as the chlorophylls (b being smaller than a) were the smallest and β-carotene, which moved
the fastest had the largest.

2. Rationalize the decision to use 8:2 petroleum ether/acetone as the solvent system for the
column.

8:2 Petroleum ether/ acetone was the optimal solvent system for the column because it was
the best ratio of less polar (petroleum ether) to more polar (acetone) compounds. Since the
pigments are all relatively nonpolar, the higher concentration of the acetone (higher polarity)
there is in the solvent, the faster each pigment will descend down the column of (polar) silica
gel: because the less relative attraction the compound has to the Silica gel. In order to
balance the time in which the lab could be completed there had to be a balance between
efficiency at optimally separating the pigments while not creating a pace of descent that did
not allow optimal isolation of pigments. 7:3 petroleum ether/acetone would have been too
fast and ineffective and completely isolating the pigments. 9:1 petroleum ether/acetone
would not have been optimal for timing (too slow), because of how much slower it would
make the more polar chlorophylls descend down the column.

3. Column chromatography is also very useful for separating mixtures of organic compounds
that are not coloured. Briefly explain how you would use column chromatography for the
separation of a colourless mixture.

Because it is very difficult for a chemist to distinguish between seemingly identical solution
being eluted it is important to have solutions to this problem. One is the use by adding small
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CHEM 212 Report Sheet 2019-2020

quantities of irreversibly fluorescing compounds such as berberine for silica, which would
allow for chromatographic separation because the region inside the column that contains the
adsorbed substance would appear as a dark band, which would be an effective visual
method.

Otherwise, one should slow down the rate at which eluent separates the desired compounds
(i.e a less polar solvent system), and then use of acid stain on the collected TLC plates we can
determine when there is a change in compound through constant monitoring via TLC plate
samples. This will allow the chemist (through a labour intensive process) when to stop
collecting one compound at start with the next, and yet still be able to effectively isolate
(approximately) the different desired chemical compounds.

4. Rationalize the observed colour of each isolated pigment (β-carotene, chlorophyll-a, and
chlorophyll-b) based on structure.

Based on molecular structure, each pigment will have a specific UV-visible spectrometry.
From the wavelengths of light that the pigment absorbs, one can determine what color each
isolated pigment is. β-carotene absorbs light the most at a wavelength 448nm which
corresponds to blue/purple, and thus we see the color on the opposite side of the light
spectrum which is orange/yellow. Chlorophyll-a and chlorophyll-b both had a greatest
absorbance of light at a wavelength corresponding to violet and thus shows a reflection of
blue/green. While in our data both chlorophyll-a and chlorophyll-b both had the same λmax
color that we perceive is more than just the a reflection of the wavelength of peak
absorbance but also the whole absorbance spectra of that particular molecule. Chemically
speaking, we would expect β-carotene to absorb the lowest energy (longest wavelength) light
because it has the most conjugated bonds and thus the smallest HOMO to LUMO gap of
electron promotion (which has an energy gap corresponding to the energy of the light
absorbed to promote the electron). We would expect chlorophyll-b to have a slightly longer
wavelengths on average as it has an aldehyde functional group (compared to the methyl
group of chlorophyll-a) which adds an additional conjugated bond, which explains its possible
reason for its blue-ish color. Because chlorophylls have many less conjugated bonds than the
structure of β-carotene, it is reasonable that their color is so vastly different.

End of Counted Page Limit (5 Pages)

References (-2 marks if not included):
Armarego, W. L. F. Purification of Laboratory Chemicals, 6th ed.; Elsevier Science &
Technology: Oxford, 2009.

Brockmann, H. Chromatography of Colourless Substances and the Relation between
Constitution and Adsorption Affinity. Discussions of the Faraday Society 1949, 7, 58.

Reusch, W. Visible and Ultraviolet Spectroscopy. UV-Visible Spectroscopy.