Chemistry 151 Lab 4: Chromatography Last updated Dec. 2013 Introduction Mixtures, both homo- and heterogeneous, can be separated (or resolved) into substances by physical means. Common examples of physical separations include filtration (separation by size) and distillation (separation by boiling point). This lab examines another separation technique that s commonly used in science labs: chromatography. Chromatography usually involves taking a mixture (often referred to as the mobile phase) and passing it through a stationary phase. The mobile phase is usually in the liquid, gas, or aqueous phase while the stationary phases is typically a liquid or solid. Depending on the type of chromatography being performed, there can be various factors that affect how well the mixture in the mobile phase separates (if it separates at all), but the most common factor is how the components of the mixture interact with the stationary phase. For example, if a chemical in a mixture interacts strongly with the stationary phase, it will take longer to pass through another that only interacts weakly. There are many different types of chromatography, some of which can involve elaborate (and usually expensive) instrumentation. In this lab, however, we will use a very simple form known as paper chromatography. As the name implies, the stationary phases is simply a piece of paper (albeit one designed specifically for this technique). The mixtures being analyzed are usually applied directly (spotted) at one end of the paper, which is then placed in a solvent that serves as the mobile phase. The solvent will travel from one end of the paper to the other just like water rising up a paper towel dissolving the mixtures and carrying them across the paper. If a proper solvent is chosen as the mobile phase, the mixture will resolve as it passes through the stationary phases, allowing one to identify its components. The movement of each component in the mixture can be described in terms of its retention factor (Rf), which is simply a ratio of how far the component traveled relative to the how far the solvent did (often referred to as the solvent front). R f = distance traveled by component distance traveled by solvent front If, for example, a component in a mixture traveled the same distance as the solvent, its R f would be 1.0. If it only travels half the distance of the solvent, it would have an R f of 0.5. The retention factor can be a useful way to quantitatively express how well a mixture resolves in a particular chromatogram. The more resolved the components of a mixture are, the greater the difference between their R f values. In this lab, you will analyze mixtures of six common FD&C (food, drug, and cosmetic) dyes: blue 1, blue 2, red 3, red 40, yellow 5, and yellow 6. First you will prepare a set of known mixtures and use these to determine which of three solvents act as the best mobile phase. This solvent will then be used to help you identify the dyes present in a set of unknown mixtures To help you identify the components of your mixtures, each chromatogram you prepare will contain the mixtures being analyzed as well as the individual dyes. Once the mixtures are resolved, you will compare the retention factors of their components with those of the individual dyes. However, since most of these dyes have their own distinct color, that can also be used in your analysis.
Procedure Part I - Choosing a Solvent 1. Get three beakers, ranging from 250-600 ml in size (they don t all have to be the same size). Using masking tape, label each with one of the solvent systems being studied: deionized water, rubbing alcohol, and 0.10% table salt. 2. Using a graduate cylinder, measure 7 ml of each solvent and transfer it to its respective beaker (for 600 ml beakers, use 10 ml). Cover each beaker with Parafilm to prevent evaporation as you prepare the chromatograms. 3. Take a 7.5 cm x 13.5 cm piece of chromatography paper and, using a pencil, draw a line approximately 1 cm from the bottom of the long end of the paper. This will be your origin line. 4. Make a series of 10 marks along the line you drew. They should be at least 1 cm apart and the ones on either end should be at least 1.5 cm from the edge of the paper. 5. Label the markings as shown in Figure 1 below. 1cm B1 B2 Y5 Y6 R3 R40 B1 R40 B1 Y5 R3 Y6 B2 Y5 1.5cm 1cm Figure 1 6. Repeat steps 3-5 twice more, giving you three identical chromatograms. 7. Place 2-3 drops of each of the dyes you ll be using in a well plate (label wells using masking tape if needed). Take your plate and 10-12 toothpicks back to you work area. 8. To spot your chromatogram, dip a toothpick in a dye for a few seconds to soak up some of the liquid then gently press the toothpick on the paper at the origin line. Make sure your spots are concentrated enough that they won t disappear as they move up the paper (most will stretch out as they travel), but don t make the spots too large or else the spots may overlap as the chromatogram develops. For your mixtures (the last four marks), spot the second dye directly over top of the first. 9. Roll each chromatogram long-ways into a cylinder. Staple the ends so the edges are touching, or as close as possible, but not overlapping (see Figure 2). 10. Remove the Parafilm and place a chromatogram into each solution, making sure the spots aren t submerged into the solution and that the paper doesn t touch the sides of the beaker. 11. Replace the Parafilm and allow the solution in each beaker to rise up the chromatogram. When the mobile phase is about 1.5 cm from the top of the paper, remove it, unroll it and to allow it to dry*. As they re drying, the solutions will continue to creep up the paper for a couple of minutes. Once it s stopped moving, use a pencil to mark the solvent front and allow the paper to finishing drying. Note the solvent system used in the top right corner.
*At this point, you ll probably realize that one solvent is definitely not the one you ll be using for Part II. You won t need to wait for this one to reach the top of your chromatogram. 12. Measure the retention factors of each spot and determine which solvent resolved your mixtures the best. Measure each distance from the origin line to the top of the spot. Figure 2 Part II Identifying the Dyes in an Unknown Mixture 1. Prepare a fourth chromatogram, similar to ones made in Part A, and spot it with the six individual dyes plus two unknowns that will be assigned by the instructor, giving you a total of eight spots. 2. Use the solvent you decided worked best in Part I to resolve your chromatogram. Do not use the same solution you used to develop the previous chromatogram. Dispose of it and measure another 7 ml. Also make sure you use new toothpicks to avoid contaminating your samples. Write your and your partner s names on the four chromatograms and attach them to one of your reports (or attach two to yours and two to your partner s; doesn t matter). If you ve already tossed your chromatograms from Part I in the trash, I hope you ve learned a lesson about reading the entire procedure before starting a lab. Waste Disposal All waste can be poured down the drain with running water.
Name: Part I - Choosing a Solvent Data Section: 1) Solvent Front Water Rubbing alcohol 0.10% table salt Starting time Ending time Time elapsed, min Distance moved by solvent, cm 2) FD&C dyes Water Rubbing alcohol 0.10% table salt Distance, cm R f Distance, cm R f Distance, cm R f Blue 1 Blue 2 Yellow 5 Yellow 6 Red 3 Red 40 B1/Y5 mix: Blue 1 Yellow 5 B1/R40 mix Blue 1 Red 40 R3/Y6 mix: Red 3 Yellow 6 B2/Y5 mix Blue 2 Yellow 5 3) Which solvent system did the best job resolving the dyes? Explain.
Part II Identifying the Dyes in an Unknown Mixture 1) Unknowns & 2) Solvent system 3) Distance traveled by solvent, cm Time elapsed 4) FD&C dyes Distance, cm R f Blue 1 Blue 2 Yellow 5 Yellow 6 Red 3 Red 40 5) Unknowns Unk # Color Distance, cm R f spot 1 spot 2 spot 3* spot 4* Unk # spot 1 spot 2 spot 3* spot 4* *If applicable. Otherwise, leave blank 6) Identification of unknowns Unknown # Dyes present: Unknown # Dyes present:
Name: Section: Post-Lab Questions 1. What problems might you have encountered if the following had happened. a) The solvent moved up the paper too quickly. b) The chromatogram was curled into a cylinder unevenly, causing it to tilt slightly when placed in the beaker. c) The origin line was drawn with an ink pen. 2. The following data was collected: Retention factors B1 B2 Y5 R3 R40 Solvent A 0.89 0.15 0.30 0.22 0.66 Solvent B 0.85 0.10 0.38 0.23 0.60 Solvent C 0.87 0.18 0.35 0.21 0.54 a) Which solvent resolves the five dyes the best? Explain. b) A piece of chromatography paper contains four spots: B1, Y5, R40, and a B1/Y5 mixture. Using your answer from 2a, sketch the predicted chromatogram (use circles to represent the dyes, as seen in the last pre-lab question, not the oblong streaks you saw in your actual chromatograms). solvent front origin line
3. Two-dimensional chromatography is a technique where the mobile phase is passed through the stationary phases twice, the second pass being perpendicular to the first. a) Using your answer from question 2a, sketch a chromatogram that was spotted with a mixture of B1, B2, and R3 (again, using circles to represent the dyes). solvent front origin line initial spot b) Sketch the predicted chromatogram if the paper from question 3a was turned 90 degrees and placed into the solvent a second time. solvent front 1 st origin line initial spot 2 nd origin line
Name: Section: Pre-Lab Questions 1. Define each of the following: a) Mobile phase b) Stationary phases c) Solvent front d) Origin line 2. Explain why each of the following are important when preparing a chromatogram? a) Not have the spots too close to each other on the origin line. b) Keep the origin line above the surface of the solvent c) Remove the chromatogram before the solvent travels the entire length of the paper.
3. A chromatogram was prepared using a procedure similar to Part I of this lab. solvent front origin line A B C D E A/B C/D a) Measure the distance traveled by the solvent (cm preferred, but any unit will do). b) Measure distance traveled by substances A-E (in the same unit as the solvent front). A B C D E c) Determine the retention factors for substances A-E A B C D E Show you work for calculating the retention factor of A d) Calculate the retention factors for the two spots in each mixture (A/B and C/D) A B C D e) If you had a sample that was believed to contain two or three of the five substances measured above, could the solvent used to prepare the above chromatogram be used to identify the components of your unknown mixture? Why or why not?