Sabrina Schmidtke Partnership for Environmental Education and Rural Health Protein Chemistry Laboratory Texas A&M University

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

Separate Analyze Identify Purify Quantify ComponentsMixture Illustration of Separating a Mixture

Real Life Examples of the Uses for Chromatography Pharmaceutical Company Hospital Law Enforcement Agency Environmental Agency Manufacturing Plant

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

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?

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?

Illustration of Chromatography MixtureComponents Separation Stationary Phase Mobile Phase

Liquid Chromatography Gas Chromatography Paper Chromatography Thin-Layer Chromatography Types of Chromatography

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)

Paper Chromatography Experiment What Color is that Sharpie?

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.

beakers 5 covers Distilled H 2 O Isopropanol Graduated cylinder Transfer pipettes 5 strips of filter paper Sharpie pens Pencil Ruler Scissors Tape Safety glasses Materials List

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.

Preparing the Isopropanol Solutions Label the beakers with a Sharpie Pen – 0%, 20%, 50%, 70% and 100% Using the graduated cylinder, transfer pipettes, distilled H 2 O, 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

Isopropanol Solutions Isopropanol Solutions for Paper Chromatography 0%20%50%70%100% 100% Isopropanol 0 ml3 ml7.5 ml10.5 ml15 ml Distilled H 2 O 15 ml12 ml7.5 ml4.5 ml0 ml Total Volume 15 ml

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

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

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

Observing the Chromatograms Concentration of Isopropanol 0%20%50%70%100%

Black Dye Concentration of Isopropanol 0%20%50%70%100% - Dyes separated – purple, yellow 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

Green Dye Concentration of Isopropanol 0%20%50%70%100% - Dye separated – blue and yellow - Blue – Soluble in concentrations of isopropanol >20% - Yellow – Soluble in concentrations of isopropanol >0%

Red Dye - Dyes separated – red and yellow Concentration of Isopropanol 0%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%

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

Alternative Experiments Food Colors 100% Water

Alternative Experiments Flower Pigments 0%20%60%100% Concentration of Isopropanol

Alternative Experiments Kool-Aid 20%40%60%80% Concentration of Isopropanol