Can Chromatography Separate A Pigment?
Teacher: Mae Eubanks-Love
School: Flower High School
Title: Can Chromatography Separate A Pigment?
Suggested Grade Level: High School
Time: 35 minutes
State Goals and Chicago Academic Standards
Most biomolecules are charged due to the presence of ionic groups as part of their molecular structures. Ionic groups are parts of molecules that can donate or accept protons (denoted by H+), each of which carries a single positive charge. Proton donors are called acids while proton acceptors are called bases. A measure of the concentration of protons in a solution is called the pH. The pH scale ranges from 114 units.
Water (H2O) is a solution that contains an equivalent number of proton-acceptor ions (OH) and protons (H+). Because the numbers of the two particles are equivalent, water has a pH of 7, which is called neutral pH. If there is a relatively high concentration of protons in a solution, the pH number is below 7 and the solution is called an acid. Some common acids found in the home are vinegar and lemon juice. If there is a relatively low concentration of protons (and therefore a relatively high concentration of proton-acceptor molecules), the pH number is above 7 and the solution is called a base. Some common bases found in the home are drain cleaners, baking soda, and ammonia.
Biomolecules can be separated from each other on the basis of the differences of their molecular charges with ion-exchange chromatography. Ion-exchange chromatography involves the use of a matrix made of a material that is either positively charged (for removing negatively charged biomolecules) or negatively charged (for removing positively charged biomolecules). The matrix reacts with the charged biomolecules and remove them from a solution. The biomolecules that are the same charge as the matrix stays in solution; they do not react with the matrix. Remember, opposites attract, and like charges repel.
In this exercise, two water-soluble pigments of differing molecular charges (the purple dye is positively charged and the yellow dye is negatively charged) are dissolved in water, which has a neutral pH (about 7). The two dyes are mixed together then separated with an ion-exchange matrix.
Make a prediction as to whether the ion-exchange matrix is positively or negatively charged based on the type of pigment bonded to the matrix. Record prediction on the student worksheet.
1. Separate a mixture of pigments with ion exchange chromatography
2. Predict whether the ion-exchange matrix is positively or negatively charged based on the type of pigment bonded to the matrix
Beaker with purple pigment
Beaker with yellow pigment
Empty plastic beaker
Tube with ion-exchange matrix and cap
Two transfer pipettes
Rack or beaker to hold tube
Ask students to
1. Write their names on the tube containing the ion-exchange matrix.
2. Use the pipette to transfer 6 mL of the purple pigment (A) to the empty beaker. Squeeze the bulb of the pipette and very slowly draw the liquid up to the line just below the bulb. This is approximately 1 mL. (Do this 6 times for 6 mL)
3. Use another pipette to transfer 6 mL of the yellow pigment (B) to the same beaker with the 6 mL of the purple pigment (from step 2).
4. Swirl the beaker carefully to mix the two pigments together. Notice the change in color.
5. Use one of the pipettes to transfer 6 mL of the purple + yellow (now green) mixture to the tube containing the ion-exchange matrix.
6. Place the cap tightly on the tube and shake vigorously for approximately 15 seconds.
7. Set the tube in a rack or beaker and wait several minutes for the pigments to separate.
8. If the ion-exchange matrix floats on the top of the solutions, gently swirl and tap the tube, allowing it to settle. Place the tube in a rack and allow it to sit for several hours.
Assess students on their class participation, their predictions, and the completion of the questions on the worksheet.
State Goals and Chicago Academic Standards
State Goal 12/Chicago Academic Standards C
Chicago Framework Statements 1, 4, 6
Conceptual StatementChicago Program of Study
The most commonly used unit of concentration is molarity (moles/liter). Molarity is used to express the degree of acidity/basicity of an aqueous solution in terms of hydronium ion concentration.
A solution is a homogeneous mixture consisting of a solvent and a solute. It is described in terms of molarity (the number of moles of solute per liter of solution).
Auto-ionization occurs in water when water reacts with itself to form hydronium (H3O+) and hydroxide (OH-) ions. The addition of an acid or base to a solution causes a shift in the equilibrium of the solution. pH is a measure of hydronium ion concentration.
Various theories explain the observed properties of compounds classified as acids and bases. An Arrhenius acid produces hydrogen (H+) and hydronium (H3O+) ions in aqueous solution. The Bronstead-Lowry theory defines an acid as a proton donor. An Arrhenius base produces hydroxide (OH-) in an aqueous solution. The Bronstead-Lowry theory defines a base as a proton acceptor.
Neutralization describes the reaction of an acid and base to produce a salt and water. Titration is an experiment procedure used to determine the concentration of one reactant from the amount and concentration of the other. A buffer solution resists change in pH.