Topic: CHEMICAL DYNAMICS--SEPARATIONS

Source: A seminar presented in Central Illinois Saturday Science Education Seminars for Elementary Teachers

Presenter: Michael J. Welsh Chemistry Department Illinois State University Normal, IL 61790

Funded by: Scientific Literacy Center Illinois State Board of Education 1990-1993

Abstract: Chemical Dynamics--Separations includes examples of safe chemistry in the elementary classroom that can be done with over-the-counter materials. The task of analysis will be the focus of the activities presented here. In order to analyze material, a chemist must be able to separate a sample into its component parts. Chemists use a technique called chromatography to accomplish much of the needed separation. While the terminology presented here must be simplified for elementary children, the attempt is made to clarify introductory knowledge and offer suggestions on how to teach the topics using an activities approach. The activities presented are appropriate for students in grades 1-6. The concepts will be valuable for grades 7-12, and should be developed accordingly.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

INTRODUCTION

Chemists have many varied and different tasks. However, the tasks can be summarized into three broad categories. Chemists classify materials and reactions, develop chemical syntheses to create new materials, and do extensive chemical analysis to determine the quality and maintain control of manufacturing the materials they have developed.

Clearly a chemist's job may be subdivided and categorized in much more detail. Students should be exposed to a more detailed list of tasks as they continue their education. However, these divisions provide the basis for a fundamental understanding of the chemist's job.

The task of analysis will be the focus of our workshop. In order to analyze material, a chemist must be able to separate a sample into its component parts. Chemists use a technique called chromatography to accomplish many of the needed separations. We will carryout three experiments to illustrate two types of chromatography: thin layer (paper) and column chromatography.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

Table of Contents

Introduction to Column Chromatography and Thin Layer............4 Chromatography

Introduction to Paper Chromatography............................5

Introduction to Column Chromatography...........................6

Lesson 1 Radial Paper Chromatography: Creating a..............7 Flower Pattern

Lesson 2 Thin Layer (Paper) Chromatography: Separation........8 of Food Dyes in M&M Candy

Lesson 3 Liquid Chromatography: Separation of Grape and.......9 Lemon-Lime Kool-Aid Food Dyes

Sources for Supplies...........................................11

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

INTRODUCTION TO COLUMN CHROMATOGRAPHY AND THIN LAYER CHROMATOGRAPHY

Chromatography is defined as a method for the separation of a mixture into its components by distribution between a mobile phase and a stationary phase. Column chromatography and thin-layer chromatography (tlc) are related in that the stationary phase in both cases is a finely divided solid, and the mobile phase is a liquid. In each case the mixture to be separated is applied to the stationary phase, either at the top of the chromatographic column or near the bottom end of the tlc plate, and mobile phase solvent is allowed to move along the column or tlc plate, carrying the components of the mixture with it. The rates at which the various components move along the path through the stationary phase will vary, depending upon their solubility in the mobile phase, and the strength with which molecules of the different substances are adsorbed on the surface of the stationary phase particles.

In tlc the stationary phase material is coated as a thin layer on an inert support such as a glass plate or sheet of plastic or aluminum. A sheet or strip of the support material which is coated in this way is called the "thin-layer chromatographic plate" or, simply, the tlc plate. Tlc is most commonly used as a very sensitive analytical technique, capable of analyzing samples at the microgram scale. However, if plates of larger size are used, and if the coating of stationary phase material is thicker, it may be possible to separate quantities on a small preparative scale, up to 100 mg or more.

Column chromatography is nearly always applied in the separation of mixtures on the preparative scale. In this case the stationary phase material is usually packed into a glass tube equipped with a stopcock, the mixture is applied to the top of the column, and mobile phase material is allowed to pass down the column under the influence of gravity, with fractions collected as the liquid flows out through the stopcock. The fractions are then analyzed (often by tlc), and material contained in various fractions is recovered by evaporation of the mobile phase solvent. Column chromatography is routinely used for quantities as small as 100 mg or less, up to multi-gram quantities, limited only by the size of the chromatographic column available.

Tlc plates may be prepared in the laboratory by a variety of techniques. These usually involve the preparation of a slurry of the stationary phase material, such as finely divided silica gel (SiO2) or alumina (Al2O3), in an appropriate solvent, and the coating of the surface of a clean glass plate with a uniform layer of this slurry. When the solvent has evaporated the silica gel or alumina remains as a layer on the surface of the plate. "Binders" such as plaster of Paris are sometimes incorporated with the silica gel or alumina to enhance the durability of the layer, and indicators may be included to make it easier to locate the positions of components on the plate after it has been developed.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

INTRODUCTION TO PAPER CHROMATOGRAPHY

Paper chromatography is related to thin-layer chromatography. While the experimental techniques are similar to thin-layer chromatography, the principles are more like those of extraction. Paper chromatography is actually a liquid-liquid partitioning technique, rather than a solid-liquid technique. For paper chromatography, a spot is placed near the bottom of a piece of high-grade filter paper (Whatman No. 1 is often used). Then the paper is placed in a developing chamber. The development solvent ascends the paper by capillary action and moves the components of spotted mixture upward at differing rates. The cellulose itself does not function as the stationary phase. Cellulose absorbs water from the atmosphere, especially from an atmosphere saturated with water vapor. Cellulose can absorb up to about 22% of water. It is this water absorbed on the cellulose that functions as the stationary phase. Since the water is the stationary phase, the components in a mixture that are most highly water-soluble are held back and move most slowly.

THE Rf VALUE

Thin-layer (paper) chromatography conditions include:

1. Solvent System 2. Absorbent 3. Thickness of the adsorbent layer 4. Relative amount of material spotted

The ratio of the distance the compound travels to the distance the solvent travels is called the Rf value. The symbol Rf stands for "ratio to front," and it is expressed as a decimal fraction:

Rf = distance traveled by substance____ distance traveled by solvent front

When the conditions of measurement are completely specified, the Rf value is constant for any given compound, and it corresponds to a physical property of that compound.

Since filter paper is manufactured with good uniformity, Rf values can often be relied on in paper chromatographic work. However, Rf values are customarily measured from the leading edge (top) of the spot, not from its center as in thin-layer chromatography.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

INTRODUCTION TO COLUMN CHROMATOGRAPHY

Silica gel and alumina are also the most common stationary phase materials used in column chromatographic separations. The main difference between the materials used in tlc and column chromatography is in particle size. The very fine particle size used in tlc is usually not suitable for column chromatography, because the rate of flow of mobile phase material through the very finely divided material is too slow, at least under the influence of gravity alone. An important modification of column chromatography utilizes mechanical pumps or gas pressure to speed up the flow rate of mobile phase solvent, allowing one to use a smaller particle size. This is desirable, since the smaller the particle size the greater the effective surface area of a particular weight of stationary phase material. This is important since the interaction of the stationary phase material with the compounds being separated is a surface phenomenon.

The column is prepared or "packed" in a glass tube having a stopcock at the bottom. For a small column a burette or similar tube may be used. The quantity of stationary phase material used will vary depending upon the difficulty of the separation, but a good rule of thumb is to use about 25 g of stationary phase solid for each gram of mixture.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

LESSON 1 RADIAL PAPER CHROMATOGRAPHY: CREATING A FLOWER PATTERN

Our first example of paper chromatography will be a simple experiment using water soluble marker to create flower like patterns on filter paper. The activity will be based on the following article in the Journal of Chemical Education with a few modifications. We will only investigate the second part of the article, creating a flower pattern on filter paper.

Becker, R., Ihde, J., Cox, K., and Sarquis, J. (1992). Making Radial Chromatography Creative Chromatography. Journal of Chemical Education, Vol. 69, No. 12, Pg. 979-980, December.

Modifications

1. Instead of poking a hole in the filter paper, fold the paper in half. In the center of the paper punch out a semi-circle with a paper punch.

2. Instead of using a plastic cup for the water, use a small Al foil pie tin.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

LESSON 2 THIN LAYER (PAPER) CHROMATOGRAPHY: SEPARATION OF FOOD DYES IN M&M CANDY

Our second example of paper chromatography will involve the separation of food dyes in M&M candies. A portion of the population is allergic to the dye tartrazine, F, D and C yellow #5 and people need to be careful when eating foods that contain the dye. Are yellow M&M candies the only ones with yellow #5 is them? For a person allergic to yellow #5, which candies can he or she eat?

The activity will be based on the following article in the Journal of Chemical Education with a few modifications.

Kandel, M. (1992). Chromatography of M&M Candies. Journal of Chemical Education, Vol. 69, No. 12, Pg. 979-980, December.

Modifications

1. Instead of scraping the dye off the candies with a toothpick, I have soaked 12 candies of each color in 5 ml of the 0.1% NaCl solution used in developing the chromatograph. This gives a more concentrated solution.

2. Draw the line for the color spots 2 cm from the bottom edge.

3. Using the thin end of a flat toothpick dab each candy extract 5 times on the paper, drying after each application. To be able to see the dyes separate clearly you need a fairly concentrated spot of candy extract. Don't blot the toothpick with paper towel, it is not necessary.

4. Double check the tartarzine (yellow food color) standard. The Kroger Company yellow food color is pure yellow #5. Some brands (McCormick, for example) of food color add a small amount of red to the yellow dye to make it more appealing. You have to test this before class.

5. The chromatograms are developed in a plastic container instead of a beaker. It is not necessary to tape the chromatography paper. However, the paper must not touch the sides of the developing container. This will cause the solvent front, 0.1% aqueous NaCl, to rise unevenly and make the dyes run together.

CHEMICAL STRUCTURES FOR BLUE 1, BLUE 2, GREEN 3, RED 3, RED 40, YELLOW 5, AND YELLOW 6 COULD BE ADDED FOR TEACHERS AND UPPER GRADE STUDENTS TO STUDY.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

LESSON 3 LIQUID CHROMATOGRAPHY: SEPARATION OF GRAPE AND LEMON-LIME KOOL-AID FOOD DYES

Background Information

Here we will use SEP-PAK C18 Cartridges to separate the dyes of concentrated grape and lemon-lime Kool-Aid. SEP-PAK C18 Cartridges are reverse phase columns and behave in an opposite manor to those discussed previously in the theory of column chromatography. With reverse phase columns, the more polar material comes off the column first.

Grape and lemon-lime Kool-Aid can be used to demonstrate separation by liquid chromatography. The red (F, D & C red #3) and blue (F, D & C blue #1) food dyes of grape Kool-Aid, and the yellow (F, D & C yellow #5) and blue (F, D & C blue #1) food dyes of lemon-lime Kool-Aid mixtures can be separated by using a C18 SEP-PAK Cartridge from Waters Associates. The SEP-PAK is reusable.

The Kool-Aid solutions are prepared by mixing a 2 quart package of each grape and lemon-lime flavored Kool-Aid (no sugar) with 25 ml of water. Two different mobile phases need to be prepared. Each mobile phase contains only isopropyl (rubbing) alcohol and water. The Kool-Aid solution is injected onto the SEP-PAK column with a syringe. Both mobile phases are injected onto the SEP-PAK column until a change in color in the eluant is detected. Each fraction containing a specific color is collected in a separate container. Each Kool-Aid flavor has two distinctly colored components. With a more refined liquid chromatographic method, more components might be isolated. However, the presence or absence of additional components is not relevant to this activity.

The purpose of this demonstration/experiment is to demonstrate a liquid chromatographic separation in a convenient and rapid manner. At the same time this separation is run, a large bore liquid chromatography column could be used to do the same separation. The time required to prepare the larger column and for the separation on the larger column can be compared to the SEP-PAK separation.

Procedure

1. Make two different mobile phases as follows:

a. Mobile Phase A: 1 part 70% isopropyl (rubbing) alcohol to 3 parts water.

b. Mobile Phase B: 1 part 70% isopropyl (rubbing) alcohol to 1 part water.

LESSON 3 Continued

2. The Kool-Aid solutions are prepared by mixing a 2 quart package of each grape and lemon-lime flavored Kool-Aid (no sugar) with 25 ml of water. Rinse the SEP-PAK column with Mobil Phase B before you use it. Inject about 0.10 ml of one of the Kool-Aid solutions onto the SEP-PAK column using the short end.

3. Rinse the syringe with distilled water to remove any trace of the Kool-Aid. Fill the syringe with Mobile Phase A and attach it to the SEP-PAK. Slowly depress the plunger on the syringe and collect the eluant. The rate of eluant should be about one drop per second. Note the color of the eluant. For the grape Kool-Aid the first eluant will be red (red #3), and for the lemon-lime Kool-Aid the color of the eluant will be yellow (yellow #5). When the eluant becomes clear, remove the syringe and fill it with Mobile Phase B. Replace the syringe onto the SEP-PAK and again depress the plunger. Collect the eluant from this mobile phase. The color of this portion (often called a fraction) is blue (blue #1) for both grape and lemon-lime Kool-Aid. Clean the SEP-PAK by rinsing it with distilled water and isopropyl alcohol.

4. The procedures above are only a first approximation to the separation of Kool-Aid on a SEP-PAK. You may find that you get better separations by varying the proportions of isopropyl alcohol and water.

CHEMICAL DYNAMICS--SEPARATIONS (Teacher Copy)

SOURCES

SEP-PAKS

Waters Division of Millipore 34 Maple Street Milford, MA 01757

800-252-HPLC

Chromatography Materials

FLINN Company P.O. Box 219 Batavia, IL 60510-0219

800-452-1261