Introduction to chromatography

Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Separate
Analyze
Identify
Purify
Quantify
Components
Mixture

Uses for Chromatography

Chromatography is used by scientists to:

Analyze – examine a mixture, its components, and their relations to one another
Identify – determine the identity of a mixture or components based on known components
Purify – separate components in order to isolate one of interest for further study
Quantify – determine the amount of the a mixture and/or the components present in the sample

Uses for Chromatography

Real-life examples of uses for chromatography:

Pharmaceutical Company – determine amount of each chemical found in new product
Hospital – detect blood or alcohol levels in a patient’s blood stream
Law Enforcement – to compare a sample found at a crime scene to samples from suspects
Environmental Agency – determine the level of pollutants in the water supply
Manufacturing Plant – to purify a chemical needed to make a product

Definition of Chromatography

Detailed Definition:

Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.

Terminology:

Differential – showing a difference, distinctive

Affinity – natural attraction or force between things

Mobile Medium – gas or liquid that carries the components (mobile phase)

Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase)

Simplified Definition:

Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Explanation:

Compound is placed on stationary phase

Mobile phase passes through the stationary phase

Mobile phase solubilizes the components

Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)
Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)
Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

Types of Chromatography

uses charge,
uses pores, and
uses covalent bonds to create the differential affinities among the mixture components for the stationary phase.

Principles of Paper Chromatography

Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.

Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.

Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Paper Chromatography Experiment

What Color is that Sharpie?

Overview of the Experiment

Purpose:

To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography.

Time Required:

Prep. time: 10 minutes

Experiment time: 45 minutes

Costs:

Less than $10

6 beakers or jars
6 covers or lids
Distilled H2O
Isopropanol
Graduated cylinder
6 strips of filter paper
Different colors of Sharpie pens
Pencil
Ruler
Scissors
Tape

Materials List

Preparing the Isopropanol Solutions

Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers:

0%, 5%, 10%, 20%, 50%, and 100%

Preparing the Chromatography Strips

Cut 6 strips of filter paper
Draw a line 1 cm above the bottom edge of the strip with the pencil
Label each strip with its corresponding solution
Place a spot from each pen on your starting line
Developing the Chromatograms
Place the strips in the beakers
Make sure the solution does not come above your start line
Keep the beakers covered
Let strips develop until the ascending solution front is about 2 cm from the top of the strip
Remove the strips and let them dry

Developing the Chromatograms

Observing the Chromatograms

Concentration of Isopropanol

0%
20%
50%
70%
100%

Black Dye

Concentration of Isopropanol

0%
20%
50%
70%
100%

Dyes separated – purple and black
Not soluble in low concentrations of isopropanol
Partially soluble in concentrations of isopropanol >20%

Blue Dye

Concentration of Isopropanol

0%
20%
50%
70%
100%

Dye separated – blue
Not very soluble in low concentrations of isopropanol
Completely soluble in high concentrations of isopropanol

Alternative Experiments

Test different samples:

Other markers, pens, highlighters
Flower pigments
Food Colors

Test different solvents:

Other alcohols: methanol, ethanol, propanol, butanol

Test different papers:

Coffee filters
Paper towels
Cardstock
Typing paper

TEKS Standards

Student conducts laboratory investigations using safe, environmentally appropriate, and ethical practices.
Student uses scientific inquiry methods during laboratory investigations.
Student uses critical thinking and scientific thinking and problem solving to make informed decisions.
Student knows how to use a variety of tools and methods to conduct science inquiry.
Student knows that substances have physical and chemical properties.

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