Chromatography

Introduction

1-propanol.


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Chromatography is a method of separating the chemical components of a mixture based on the differential solubilities of the components in two solvents or phases, a stationary phase and a mobile phase.  Many different types of chromatographic methods are in practice; two major types are partition chromatography, where the stationary phase is a thin film of water adsorbed on the surface of a inert matrix, and adsorption chromatography, where the stationary phase is a solid adsorbent.

Paper chromatography is a type of partition chromatography in which the stationary phase of the system is water permanently bound to the cellulose fibers of chromatography paper.  The mobile phase is usually a relatively nonpolar solvent that is allowed to migrate through the paper.  A number of different nonpolar solvents can be used, each solvnet likely giving slightly different results.  In this week's experiment, 70% 1-propanol is the mobile phase (fig. 1).  The system, then, is designed to compare the solubility of solutes in water to their solubility in a more nonpolar solvent. Thus a simple method like paper chromatography could be used to measure amino acid solubility in nonpolar solvents and produce an amino acid hydrophobicity scale.


The basic paper chromatography system.


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How it Works

Small samples of the substances being studied are applied near one end of the chromatography paper and allowed to dry. The end of the paper containing the dried samples is then submerged into a shallow pool of the mobile phase, and the mobile phase is given time to migrate up the paper. 

As the mobile phase migrates, it reaches the dried samples. At that point, each sample is partitioned between the two phases to various extents, depending on their solubility in each of the two solvents.  The more soluble a sample is in the nonpolar mobile solvent, the greater its tendency to migrate with the mobile solvent.  The more soluble a sample is in water, the greater its tendency to remain stationary in the aqueous phase. Thus, the amount of sample migration along the chromatography paper ultimately depends on differences in solubility of each sample in water versus the nonpolar solvent.


Relative mobility is the ratio of the distance the experimental solute migrates to the distance the solvent migrates along a chromatographic medium.


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Collecting the Data.

The raw data collected from the chromatogram is migration distance data, usually measured in mm or cm. From this raw data, an index of solubility, known as relative mobility, is calculated.  Known as the retention factor (Rf) among chemists, relative mobility is the ratio of the distance the experimental solute migrates to the distance the solvent migrates along a chromatographic medium:

Rf = Dsample / Dsolvent

where Dsample is the migration distance of the sample and Dsolvent is the migration distance of the mobile phase.

Since the Dsample is never greater than the Dsolvent, the Rf is always a value between 0.0 and 1.0.  The greater the Rf, the greater the relative solubility in the nonpolar mobile phase. Using this as a measure of hydrophobicity, the greater the Rf, the greater the hydrophobicity.