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Sabrina SchmidtkePartnership for Environmental Education and Rural Health

Protein Chemistry LaboratoryTexas A&M University

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What is Chromatography?

Chromatography is a laboratory technique for separating components within mixtures.

• Mixture: a material composed of two or more elements or

parts.

• Component: a constituent, element, or part

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Separate

• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

Illustration of Separating a Mixture

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Real Life Examples of the Uses for Chromatography

• Pharmaceutical Company

• Hospital

• Law Enforcement Agency

• Environmental Agency

• Manufacturing Plant

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• Mobile Phase – gas or liquid that carries the mixture of components through the

stationary phase.

• Stationary Phase – the part of the apparatus that holds the components as they move

through it, separating them.

Components of Chromatography

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Chromatography separates the components of a mixture by their differential affinity to the mobile and stationary phases.

• Differential – showing a difference, distinctive

• Affinity – natural attraction or force between things

How Does Chromatography Separate Mixtures?

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• Mixture of components 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

How Does Chromatography Separate Mixtures?

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Illustration of Chromatography

Mixture Components

Separation

Stationary Phase

Mobile Phase

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• Liquid Chromatography

• Gas Chromatography

• Paper Chromatography

• Thin-Layer Chromatography

Types of Chromatography

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Principles Used in Paper Chromatography

• Capillary Action – the movement of a liquid through the spaces of a porous material or a small diameter tube against the forces of gravity.

• Solubility – the degree to which a material (solute) dissolves into a liquid (solvent). (Like dissolves like)

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Paper Chromatography Experiment

What Color is that Sharpie?

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Overview of the Experiment

Purpose:

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

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• 5 beakers• 5 covers• Distilled H2O• Isopropanol• Graduated cylinder• Transfer pipettes• 5 strips of filter paper• Sharpie pens• Pencil• Ruler• Scissors• Tape• Safety glasses

Materials List

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Safety Precautions

• Isopropanol – (Rubbing Alcohol) is an antiseptic in low concentrations and an irritant in high concentrations. If you get it on your skin or in your eyes, alert your teacher and rinse with water. Wearing safety glasses will help prevent isopropanol from being splashed into your eyes.

• Glassware – (beakers or graduated cylinders) if you break anything remain calm, alert your teacher, and follow your lab safety protocol to dispose of the glassware in an appropriate manner.

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Preparing the Isopropanol Solutions

• Label the beakers with a Sharpie Pen – 0%, 20%, 50%, 70% and 100%

• Using the graduated cylinder, transfer pipettes, distilled H2O, and the isopropanol, prepare 15 ml of the following isopropanol solutions and place in the appropriately labeled beaker – 0%, 20%, 50%, 70% and 100%

• Cover the isopropanol solutions after they are made

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Isopropanol Solutions

Isopropanol Solutions for Paper Chromatography

0% 20% 50% 70% 100%

100% Isopropanol

0 ml 3 ml 7.5 ml 10.5 ml 15 ml

Distilled H2O 15 ml 12 ml 7.5 ml 4.5 ml 0 ml

Total Volume 15 ml 15 ml 15 ml 15 ml 15 ml

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Preparing the Chromatography Strips

• Cut 5 strips of filter paper 5 cm x 8 cm

• Using a pencil, lightly draw a horizontal line 1 cm above the bottom edge of each strip

• Label each strip in the top right-hand corner with its corresponding solution

• Place a small spot from each pen on your starting line

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Developing the Chromatograms

• Place the strips in the appropriate beakers (start keeping time now)

• Make sure the solution does not come above your start line

• Keep the beakers covered• The isopropanol solution will start

moving up the strips immediately• Let strips develop until the ascending

solution front is about 2 cm from the top of the strip (the strips in the lower percentages will finish first)

• Remove the strips and let them dry

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Observing the Chromatograms

• Observe how: - some of the dyes are made up of multiple colors

- spots of one color separated in low concentrations of isopropanol vs. higher concentrations

- spots of different colors first started separating in different concentrations

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Observing the Chromatograms

Concentration of Isopropanol

0% 20% 50% 70% 100%

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Black Dye

Concentration of Isopropanol0% 20% 50% 70% 100%

- Dyes separated – purple, yellow and black

- Not soluble in low concentrations of isopropanol

- Partially soluble in concentrations of isopropanol >20%

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Blue Dye

Concentration of Isopropanol0% 20% 50% 70% 100%

- Dye separated – blue

- Not very soluble in low concentrations of isopropanol

- Completely soluble in high concentrations of isopropanol

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Green Dye

Concentration of Isopropanol0% 20% 50% 70% 100%

- Dye separated – blue and yellow

- Blue – Soluble in concentrations of isopropanol >20%

- Yellow – Soluble in concentrations of isopropanol >0%

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Red Dye - Dyes separated – red and yellow

Concentration of Isopropanol0% 20% 50% 70% 100%

- Yellow –soluble in low concentrations of isopropanol and less soluble in high concentrations of isopropanol

- Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20%

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Alternative Experiments

• Test different samples:– Other markers, pens,

highlighters– Watercolor paints– Flower pigments– Food Color Dyes– Kool-Aid– Skittles

• Test different papers:– Coffee filters– Paper towels– Cardstock– Typing paper

• Test different solvents:– Methanol– Ethanol– Propanol– Butanol

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Alternative Experiments

Food Colors

100% Water

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Alternative Experiments

Flower Pigments

0% 20% 60% 100%

Concentration of Isopropanol

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Alternative Experiments

Kool-Aid

20% 40% 60% 80%Concentration of Isopropanol