CHEM 2123 Week 7 Column Chromatography Lab Report

Austin Community CollegeCHEM 2123 – Organic Chemistry I Lab
Online Learning Module
Column Chromatography
To isolate -carotene from spinach leaves using solid/liquid extraction and column
Students will learn to:
• Prepare a chromatography column
• Successfully perform a separation of a mixture using column chromatography
Please read the following material in “Laboratory Techniques in Organic Chemistry” (4th ed.) by
Mohrig, et al:

Chapter 19 – Liquid Chromatography (Sections 19.1 – 19.7)
In this experiment, students will become familiar with the basic techniques of column
chromatography. The word chromatography comes from the Latin words for color separation
and refers to a variety of techniques that are used to purify mixtures of substances. In all
chromatography methods, there is a stationary phase and a mobile phase. The nature of the
two phases depends on the particular chromatography method that is being used.
Austin Community College
CHEM 2123 – Organic Chemistry I Lab
Online Learning Module
In this experiment, the technique that will be used is column chromatography. Column
chromatography is typically used by chemists to separate a mixture of molecules based on
polarity and size. An impure sample is loaded onto a column consisting of either alumina or silica
(stationary phase) and eluted using a pure solvent or solvent mixture (mobile phase).
Components in the mixture separate from one another based on their relative affinities for the
mobile and stationary phases. In general, molecules that are more polar travel slower through
the column due to greater affinity for the stationary phase whereas less polar components
typically have a greater affinity for the mobile phase.
In this experiment, the mixture to be separated consists of the colored pigments in
spinach leaves: carotene, pheophytin a, pheophytin b, chlorophyll a, chlorophyll b, and
xanthophyll. This procedure will isolate only the B-carotene the structure is given below.
Silica gel will be used as the stationary phase and, initially, hexane will be used as the
mobile phase. The hexane should be sufficiently nonpolar to move the carotene through the
silica gel but should not cause the other colored pigments to move much. Once the column has
equilibrated and the carotene forms a band at the top of the column then the polarity of the
mobile phase can be increased in order to elute the carotene more quickly. The solvent used will
be a 10% acetone to hexane solvent. Ultimately, the success of the chromatographic separation
can be qualitatively measured by the separation of the original green mixture into one yellow
component. This will be analyzed via TLC to determine if B-carotene was the component
isolated by testing against a carotene standard and a crude pigment mixture of the original
spinach extract.
The other colored components in spinach are more polar than carotene due to the fact
that the other colored components contain polar atoms and some even contain ionic bonding.
This will mean the other pigments will be held more tightly by the silica gel than the carotene.
To elute the other colored pigments, a more polar solvent will be necessary.
Include NFPA ratings for silica gel, hexane, acetone, and magnesium sulfate.
Austin Community College
CHEM 2123 – Organic Chemistry I Lab
Online Learning Module
Watch the demonstration video to write a procedure for this experiment. Include a procedure
on the solid/liquid extraction of the colored pigments from the spinach leaves, a procedure for
the dry packing of the chromatography column, a procedure on the elution of the carotene from
the column, and a procedure of the analysis using TLC.
Take a screenshot of the column showing the separated carotene within the column and add the
screenshot to your lab report. Also take a screenshot of the developed TLC plate and determine
the Rf of the carotene from the carotene standard, the isolated carotene from the
chromatography column, and the Rf of each component from the crude extract of the spinach
pigment mixture. Include the screenshot and the calculated Rf values in your lab report.
1. Using the structure for carotene (in this handout), cut and paste or hand draw the
structure into the theory and conclusions section of the lab report. Be sure to give a
citation for the structure.
2. Utilizing the structure of carotene, from 1, indicate whether the component would be
polar or nonpolar overall. Indicate the functional groups and why the molecule is polar or
nonpolar based on functional groups. Relate this to the order it came off of the column.
3. Define extraction in general and relate it to this experiment. This should include
solid/liquid extraction. Define these terms with citations and then indicate how we used a
solid/liquid extraction. You should explain the purpose for each substance used in this
extraction. Be sure to indicate polarities as it relates to the components of the spinach
leaves and the extraction solvent. You should include structures of the extraction solvent
including citations.
4. Define chromatography in general and column chromatography specifically and also
relate it to the experiment. Include with this, stationary phase and mobile phase.
Indicate the relative polarities of the stationary and mobile phases. Include structures
for the stationary phase and mobile phase solvents.
5. Explain how separation of the pigments took place on the column utilizing intermolecular
forces of attraction. You should include the expected intermolecular forces for each
component, the mobile phase, and the stationary phase. Indicate why 2 different solvents
were used to elute the carotene.
Austin Community College
CHEM 2123 – Organic Chemistry I Lab
Online Learning Module
6. Explain what TLC is using a cited source. Explain how TLC was utilized in this
experiment and the purpose of the TLC.
7. Include structures of the other components present in the spinach leaves that
remained on the column, indicate their relative polarities and why they did not elute with
the carotene.
8. ETC
Enter the sources that you used and be sure to include either superscripts or the author’s name
in ( ) in the body of the paper to show where you used the source.
The following is an example of an investigative lab report. Most of the
experiments in Organic Chemistry I will follow this lab report format.
Everything shown in bold below is what should actually be written in your
Both your name and date should appear on separate lines on the right hand side of
the page.
Your Name
Title of the Experiment Centered in the Page
Experiment 2 Extraction and Evaporation. Separating the Components of
I. Introduction
The introduction should be in your own words and explain the purpose behind the
experiment. This will only require at most 10 sentences. The purpose can be found
in the reading section from the departmental handout.
II. Safety
This section should have the NFPA rating for each chemical presented in the
demonstration video. This will include the Health, Flammability, Reactivity, and
other hazard ratings. You can find these ratings by doing an internet search of the
III. Experimental Procedure
While watching the demonstration video for the experiment, write the procedure
stepwise, numbering your steps, in your own words. There should be enough detail
such that anyone who reads your report could do the experiment without the use of
a lab textbook.
IV. Observations and Results
This section will include things such as boiling range, melting point, color, time for
reaction, spectra and etc. This section will include results related to your
observations such as melting point and purity relationship, percent yield, percent
recovery etc.
V. Theory and Conclusions
This section should indicate how your results related to the purpose of the
experiment and what can be interpreted from your results. This section should
indicate how your results prove the theory behind the reaction or procedure. Most
of the information on the theory section can be found in your lecture book or in the
prereading section of the lab experiment as well as the reading sections from the
textbook, and research using the ACC library particularly AccessScience, KirkOthmer Encyclopedia of Chemical Technology, Van Nostrand’s Scientific
Encylopedia, and Gale ebooks. This section will be graded the hardest and
rambling results in grade deduction. You should be able to make your point in
VI. Citations
Any format is acceptable, I tend to use MLA.

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