Chromatography Lab # 4 Chromatography is a method for separating mixtures based on differences in the speed at which they migrate over or through a stationary phase which means that a complex mixture will separate into the chemicals from which it was made. During the process of chromatography a mixture of various components enters a chromatograph apparatus and the different components are flushed through the system at different rates. These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated adsorption/desorption acts that take place during the movement of the sample over the stationary (immobile) bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column. In any chemical or bio processing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods that industries can use to accomplish these goals and chromatography is the easiest and most common for a number of reasons. First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right stationary phase, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins. The three most common types of chromatography are paper, thin layer and gas. paper chromatography (PC) o stationary phase is liquid soaked into a sheet or strip of paper o mobile phase is a liquid solvent o some components spend more time in the stationary phase than others o components appear as separate spots spread out on the paper after drying thin layer chromatography (TLC) o stationary phase is a thin layer of adsorbent (Al 2 O 3 or SiO 2, usually) coating a sheet of plastic or glass o as with paper chromatography, components appear as spots on the sheet o may require UV light to see spots on sheet gas chromatography (GC) o sample mixture is injected into a long tube (the column) o mobile phase is an inert gas that sweeps the sample down the tube o stationary phase lining the tube selectively adsorbs or dissolves components o the stationary phase is a solid or very syrupy liquid o silicone polymers (like Silly Putty) are often used as stationary phases in gas chromatography o a detector responds to separated components as they leave the tube
We will be focusing on paper chromatography for both parts of this lab. Paper chromatography is an analytical technique used for separating and identifying mixtures that are or can be colored. A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper near the base of the plate. The sample is absorbed onto the paper, which is the stationary phase. The paper is dipped into a suitable solvent (mobile phase), such as alcohol or water, and is placed into a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent and due to differences in their attraction to the fibers in the paper. The more soluble the component the further up it goes. Paper chromatography can take anywhere from several minutes to several hours. Part A- Chromatography of Markers Lab Problem: Materials: Procedure: 1) Obtain three markers, one of each type (permanent, dry erase, magic). They can not be the same color. 2) Obtain three strips of chromatography paper. 3) In the center of each strip about 10mm from one end, place a dot of the marker to be tested. The dot should be SMALL about 2mm in diameter and dark enough to be visible. 4) Arrange three test tubes into a test tube rack. 5) Using a pipette, place approximately 1 ml (or one full squirt full) of distilled water into each test tube. 6) Carefully insert the chromatography paper into the test tube dot end down. The paper must be touching the water BUT the dot can not be touching the water. If possible it should not be touching the wall of the test tube either. 7) Allow the test tube to remain undisturbed until the water line (the solvent front) is about 1 inch from the top of the paper. 8) Carefully remove the chromatograph from the test tube without spilling any solvent. 9) Lay the chromatograph onto a paper towel. 10) Observe what colors were produced by the migration of pigments by the solvent and record this on your data table.
11) Using a metric ruler, measure the distance the solvent traveled from the colored dot. This means measure from the dot to the water line. Record this value in your data table under solvent distance. 12) Using a pencil, draw a line at the top of each pigment color spot. Take a ruler and measure the distance from the colored dot at the bottom to the line of each color spot. Record the distance for each color into one of the boxes under Color Distance in the data table. Be sure to write which color that distance was for. 13) Calculate the R f values for each color and record these R f values on the data table. (R f = distance solute moved/distance solvent moved) 14) Empty your test tubes and repeat the steps for the same exact markers but with isopropyl alcohol as the solvent. Data Table 1 Marker # (color?) Solvent Solvent Distance (mm) Color Distance (mm) And Color R f for each!
Draw your chromatographs below: (Please label everything and show all the color pigments) Questions: 1) What color markers were mixtures from your experiments? Which markers were pure substances? 2) Which solvent worked best as the mobile phase for each of the markers? Why do you think that solvent worked the best? 3) Why does the spot need to be above the level of the solvent when the chromatograph is placed in the solvent?
Part B: Candy Chromatography Problem: Materials: Procedure: 1) Cut a piece of chromatography paper into an 8cm by 8cm square. 2) Draw a line with a pencil about 1 cm from one edge of the paper. 3) Make 6 dots with the pencil equally spaced along the line, leave about 0.5 cm between the edge of the paper and the first (and last) dots. 4) Below the line, use the pencil to label each dot for the different colors of candy. 5) Obtain a well plate and one of every color of the candy. Put 1 pipette full of sulfuric acid (be careful acid burns) into one well for each color. 6) Drop the candy into the sulfuric acid and wait for the color to dissolve off the candy. 7) Take a pipette and pull a little bit of the colored liquid up. Put a really small dap of the colored liquid onto corresponding labeled dot on your chromatography paper. Use a light touch so that the dot of the color stays really small. Make sure you can see the colored dot! 8) Repeat step 7 for every color but use a new pipette every time. 9) When the paper is dry, fold it half so that it stands up on its own (with the fold vertical and the dots are at the bottom). 10) Next obtain a 100ml beaker and pour about 1 pipette full of ammonia into the beaker. (Ammonia has a really strong smell beware!) Basically the level of solution should be low enough so that when you put the chromatography paper in the dots will be above the liquid level. 11) Add the chromatography paper to the 100ml beaker and watch the movement of the solvent. 12) When the ammonia is about 1cm from the top of the paper, remove the paper and lay it flat on a piece of paper towel. 13) Using a metric ruler, measure the distance the solvent traveled from the line at the bottom. Record this value in your data table under solvent distance. 14) Using a pencil, draw a line at the top of each pigment color spot. Take a ruler and measure the distance from the line at the bottom to the line of each color spot. 15) Calculate the R f values for each color and record these R f values on the data table. (R f = distance solute moved/distance solvent moved)
Data Table Color Candy Solvent Distance Color Distance (write each color in the box) R f Draw your chromatograph below: (Please label everything and show all the color pigments)
Questions: 4) From a comparison of the Rf values, what conclusions can be made about the similarities or differences in the dyes used in each of the candies? 5) Obtain the candy package (before you leave my room) and determine if the food coloring on the package matches up to your findings. Which dye matches which candy that you worked with? 6) If you were to do this experiment again, what would you do differently and what is one very detailed error that affected your lab?