Chromatography Notes

Chromatography

Introduction

• Chromatography is a technique used to separate mixtures.
• Paper chromatography is the simplest form and relevant to the AP exam.
• The principle involves separating materials based on differences in their attractions to different phases.

Paper Chromatography

• When ink on paper gets wet, it separates into component colors, illustrating the principle of chromatography.
• A mixture (analyte) is deposited on chromatography paper (stationary phase).
• The bottom edge of the paper is lowered into a liquid (mobile phase).
• The liquid wicks up the paper, carrying the components of the mixture.

Mobile Phase

• The mobile phase is the solvent that moves up the paper.

Stationary Phase

• The stationary phase is the paper itself.

Analyte

• The substance being separated.

Experiment Setup

• A dot of green marker (analyte) is placed on chromatography paper (stationary phase).
• A pencil line is drawn as a reference; Ink should not be used as it will interfere with the analyte.
• The paper is suspended in a beaker with the bottom edge immersed in the mobile phase.
• The mobile phase is a 70% isopropanol (rubbing alcohol) solution.
• The liquid level must be below the pencil line to avoid washing the dyes off the paper.

Process

• The alcohol wicks up the paper, and the mobile phase interacts with the analyte.
• If the analyte is a mixture, its components will separate.
• Components more attracted to the paper (stationary phase) stay closer to the reference line.
• Components more attracted to the alcohol solution (mobile phase) travel farther up the paper.
• The separation is due to differences in attractions between molecules and the two phases.

Observations

• The green marker separates into green and yellow colors, demonstrating the principle.
• If the dot moves without separating, the analyte is not a mixture.

Chromatography Diagram

• Paper clip and wooden splint hold paper in place.
• A nonpolar solvent (e.g., hexane) may be used as the mobile phase.
• Paper is polar due to cellulose (hydrogens bonded to oxygens).
• Multiple dyes move to different locations.
• If a dye is a mixture, it will separate into multiple spots.
• The unknown dye is compared to known dyes to determine its identity.

Retention Factor ($R_f$)

• The retention factor ($R_f$)is used to compare spots when solvents don't reach the same position.
• $R_f = \frac{\text{distance spot moves}}{\text{distance solvent front moves}}$
• The solvent front is the location where the mobile phase stops.
• If two materials have the same $R_f$ value, they are likely the same material.
• $R_f$ is a unitless value.

Example

• If a dye moves half the distance the solvent moves, its $R_f$ value is 0.5.
• If an unknown dye has the same $R_f$ value as a known dye, they are likely the same.

Polarity and Dye Movement

• Polar materials dissolve in polar materials; nonpolar materials dissolve in nonpolar materials.
• Dyes that move farther with a nonpolar solvent are less polar.
• Dyes that stay closer to the polar paper are more polar.

Example Question

• Which dye (A, B, or C) is the least polar?
• Answer: Dye C is the least polar because it moves the farthest with the nonpolar solvent.
• Explanation: Nonpolar dyes are more strongly attracted to nonpolar solvents and least retained by polar paper.

Explanation of deducing dye A presence in the unknown sample

• The unknown sample moves to a position that is midway between the origin and the solvent front, & so does dye A.
• This also means that dye A has a retention factor that is close to 0.50 on the chromatogram with three dyes and the unknown has a retention factor close to 0.50.

Advanced Chromatography Techniques

• Thin Layer Chromatography (TLC)
• High Performance Liquid Chromatography (HPLC)
• Gas Chromatography (GC)

Gas Chromatography (GC)

• A liquid sample is vaporized and separated into components.

High Performance Liquid Chromatography (HPLC)

• The sample is not vaporized, but components are separated based on attractions to different materials.
• Both HPLC and GC separate based on differences in attractions between materials and mobile/stationary phases.

Gas Chromatography-Mass Spectrometry (GC-MS)

• GC is often coupled with mass spectrometry to identify components.
• Components are heated, moved through a magnetic field, and broken into masses.
• Mass spectrometry determines the materials in the mixture and their relative amounts.

Visual Observation

• Visual observation of paper chromatography is a good starting point because HPLC and GC occur inside closed chambers.
• The visual experience helps understand the principles.

Experiment Observations

• The green marker is separating into yellow and blue components.
• The yellow component is stubborn and very attracted to the polar paper.
• The blue component is attracted to the relatively lower polarity mobile phase.
• Given enough time, the two materials should completely separate.