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(CHEM 233) Column Chromatography Lab Report

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Lab Report 2 – Lecture notes 2.22


University of Illinois at Chicago


Organic Chemistry Laboratory I (CHEM 233)

Lab 2: Chromatographic Methods: Separation of Dyes and Spinach
Pigments by Column and TLC.
Zeba N. Siddiqui (Partner: Keti Berberi)
September 19, 2014
Methods and Background
Two experiments were performed to test the separation of pure compounds from a mixture,
which is known as chromatography. Column Chromatography is one of the experiments and its
goal is to create an effective column chromatography setup and procedure that will separate
methyl orange from methylene blue. Variables of the procedure includes the amount of silica
used, the construction of the model, and the composition of the mobile phase. Thin-Layer
Chromatography (TLC) is the second experiment. Its primary goal is to develop mobile phase
composition. This will separate five pigments: chlorophyll a, chlorophyll b, pheophytin a,
carotenes, and xanthophylls. Pigments of spinach leaves will be used with the TLC plate and an
understanding of polarity, retention time, and retention factor is necessary to complete the
analysis of the results.
Separation of the compounds is due to the affinity to polarity of the solvent to the substance. A
stationary phase is a solid, or liquid, that does move through the TLC plate, but allows liquids to
flow. Mobile phase, however, is a liquid, or gas, that flows through the stationary phase.
Compounds with a higher affinity towards the stationary phase (polar) will interact with the
solid, preventing movement. Compounds that lean towards the mobile phase (nonpolar/ less
polar) will interact with the liquid, which allows movement to flow through the stationary phase.
Column Chromatography is the technique used to purify compounds from mixtures using a
pipette column. This method is used for larger sample sizes. Dye mixture is added to a solvent, a
separation is visible after an adequate amount of time is given, an example of the setup is shown



 (CHEM 233) Column Chromatography Lab Report

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