1. GenChem 3 rd Class Chemical Separations Today’s Agenda:  Develop the problem of the day.  Perform first challenge: extract pigments.  Discuss the challenge of chemical separations.  Perform second challenge: determine the best solvent for the problem.  Develop a consensus on the best answer.  Learn how to write an introduction.  Discuss writing the report.

2. GenChem One of the central goals of Chemistry is to analyze chemical substances. Detection: Does the sample contain substance X? Identification: What is the identity of the substance in this sample? Central Goals and Questions Separation: How can the substances present in the sample be separated? Quantitation: How much of substance X is in the sample? Today we will investigate the SEPARATION of chemical substances

3. GenChem Chemists use their knowledge and understanding of the properties of substances to separate the different components of materials. Why is there a need to separate substances? Identification Characterization EliminationQuantitation Application Transformation

4. GenChem Separations D2L-graded pre-lab assignment

5. GenChem In particular, biological materials tend to be mixtures of hundreds of different substances. What do you know about the properties of substances that cause leaves and flowers to be colored? They absorb visible light of certain colors and reflect others.. Chlorophyll, for example, absorbs red and blue light. Carotene absorbs green and blue light.

6. GenChem Let′s think!. In your groups:  Discuss what properties of pigments in a leaf or a flower you could use to extract them;  Propose an experimental strategy to extract the different pigments in a plant; Chlorophyll Carotene

7. GenChem The solubility of one substance in another depends on the relative strength of the attractive forces between their particles AA, BB >> ABInsoluble, Immiscible AA, BB <= ABSoluble, Miscible What is going on? Could we use water or acetone or hexane to extract the pigments from spinach leaves? Demo

8. GenChem Why does simply sticking a leaf in a beaker of solvent not work? What do you know about the structure of leaves? Where are the pigments located? How can you get them out? Pigments are located in chloroplast walls inside cells. Solvent can’t get to them. Need to break down cell walls. Let′s think!

9. GenChem Natural Product Extraction Let′s think! In your groups review your responses to the following pre-lab question and come up with a consensus answer. Take the six-point "Procedure for BIological Sample Extraction" given in section 5-1 of your technique book and rewrite it as a procedure you can use to extract the photosynthetic pigments from spinach leaves. This procedure will need to contain the specific equipment and the amounts of chemicals you expect to use.

10. GenChem Extraction Tips Make sure your procedure accounts for the following. A description of how to make the 1:1 mixture of hexane and acetone including how much to make. Separation of the organic layer from the water layer. The recommended procedure is to use a transfer pipet to remove the dark green organic layer (top layer) into a small test tube, taking care to avoid collecting any of the milky, aqueous layer at the bottom of the mortar. Don’t rinse the mortar with acetone!

11. GenChem First Challenge Extract the pigments from spinach leaves. Available resources:  Acetone, hexane, spinach leaves & sand;  Mortar and pestle;  Glassware. Implement your procedure. Let′s explore! You have 15 minutes

12. GenChem Let′s think! Compare the color and depth of color of your pigment extract with those from other groups.  How do you explain the differences?  How dark should your extract be?  How can you make it darker? Concentration of the extract by reducing the solvent volume. Place it in a small beaker and set the beaker in a hood. Leave alone until the volume is reduced to about 1 ml. If it evaporates to dryness, add 1 ml of additional solvent.

13. GenChem Separating a Mixture  What do you think is in the extract?  What do you need to do next to determine how many pigments are in there?

14. GenChem Chromatography unequal Separation based on unequal attractions between the different components of a moving mixture an a stationary phase. Mobile phase Stationary Support Animation: http://www.youtube.com/watch?v=SdYb6GgBQ7s http://www.youtube.com/watch?v=SdYb6GgBQ7s

15. GenChem Imagine we inject a substance composed of molecules that have weak attractive forces with the STATIONARY PHASE MOBILE PHASE or ELUTANT BASED ON MOLECULAR INTERACTIONS PREDICT HOW WILL MOVE THROUGH THE TUBE

16. GenChem What if we injected molecules with strong attractive forces with the stationary phase? BASED ON MOLECULAR INTERACTIONS PREDICT HOW WILL MOVE THROUGH THE TUBE

17. GenChem Suppose we put a mixture of two solutes and one solvent (mobile phase) into our coated tube. What do you expect to happen? What will happen if more solvent is added?

18. GenChem With the proper selection of stationary and mobile phases you can separate materials based on intermolecular forces. Gas chromatography

19. GenChem Thin Layer Chromatography (TLC) TLC Plate Support and Stationary Phase Developing jar What is it?

20. GenChem Thin Layer Chromatography (TLC) Spotting Running  what’s the mobile phase?  what’s the stationary phase?  what makes the mobile phase move?

21. GenChem Thin Layer Chromatography Let′s think! In your groups review your responses to the following pre-lab question and come up with a consensus answer. Consider the 12-step procedure for performing a TLC that is given in chapter 6-1 of the technique book. Describe how you will use this procedure to determine the best solvent system to separate the components of a spinach leaf. Do not repeat any of the 12 steps. Instead, describe what additional steps you will need to take to solve the problem.

22. GenChem TLC Tips Make sure your procedure accounts for the following. Don’t waste chemicals! Don’t let dust, dirt, liquids (like water) and/or your fingerprints get on the TLC. Make sure they fit. Keep the spot small. Mark original spot location. Different strips may develop at different rates. Mark solvent front. Capillary tubes are GLASS WASTE. Put them in your notebook!

23. GenChem Second Challenge Determine the solvent mixture that, using TLC, best separates the various pigments present in the spinach leaf extract.. Available resources:  Solvents;  TLC strips;  Glassware. Now that you have a feasible procedure, just do it! Let′s explore! You have 60 minutes

24. GenChem Compare the TLC plates developed with different solvent mixtures.  Begin by drawing pictures of the TLC plates in your notebook  What mixture is more efficient in separating the components of the mixture?  How many different components seem to be present in the original pigment mixture?  Identify the chlorophylls and carotenoids. What property can you use to do this? Let′s think!

25. GenChem Consider the spots on the various TLC plates. How could you identify each individual component? Propose an experimental strategy. By performing the TLC with known compounds and comparing the results. Let′s think!

26. GenChem What properties of the spots allows us to use them to identify the spots? Color and distance the spot moves. Let′s think!

27. GenChem The distance the spot moves depends on how far the solvent moves. It is the RATIO of the distance the spot moves to the distance the solvent moves that is constant for any given component and solvent. This ratio is known as the R f value. Let′s think!

28. GenChem R f retention factor 1.Create a formula for calculating R f values. 2.For each strip, calculate a R f value for each spot on the strip. 3.Make a table that lists, for each strip, the spot colors and the calculated R f values. Let’s apply

29. GenChem Report your results for the identity of the best solvent mixture. In your group:  Discuss how the results obtained by the different groups compare with each other? Which are most credible? Which are least?  What were the strengths and weaknesses of the method you used to identify the chromophores?  How could you avoid or reduce the difficulties that you encountered? Let′s think!

30. GenChem You’ll be looking at the behavior of gases and the kinds of measurements that can be made. Looking Ahead to Next Week Let′s think!

31. GenChem Discussion From your Technique Book: Discussion. Analysis of the data and presentation of conclusions regarding original hypothesis. This should include a defense of the conclusions citing the results and a discussion of the reliability of the data used.

32. GenChem Discussion Outline What were the major goals of today’s lab? 1.Determine the best solvent to use to separate the various components. 2.Determine how many components can be identified. For each goal, what were the results? 1.The experimentally determined mixture of Let′s think!

33. GenChem Discussion of a Goal For Each Goal: Interpret/analyze result. What is the result? (Cite data/give examples). What does it mean? Does it make sense? Explain the chemistry. Is it what was expected? Is it consistent with your observations? Comparisons to literature or peers. If something went wrong: What were you expecting and why? What went wrong? How do you know? How could you fix it next time? Let’s apply

34. GenChem Discussion: Goal 1 The best separation was achieved with a mixture of acetone and hexane consisting of one part acetone and three parts hexane. With pure acetone none of the pigments traveled very far and they did not separate. With pure hexane the pigments all traveled far, but also did not separate. The 1 to 3 mixture caused the pigments to end up further apart than for any other mixture with the chlorophylls traveling the farthest and the carotenoids the least. Based on these results it can be said that all the pigments had strong affinity for the hexane and limited affinity for the acetone. It also appears that the chlorophylls have a greater affinity for the hexane than the carotenoids. That the two chlorophylls tracked closely together implies they have very similar affinities for the hexane which also implies they have very similar structures. The same is true for the carotenoids.

35. GenChem Additional Goals If the experiment has more than one goal, you will discuss the results of each like we did for Goal 1. In addition to discussing the results of each goal, you also need to discuss data quality…

36. GenChem Discussion: Data quality In a paragraph, discuss the quality and reliability of your data: Discuss pros/cons/accuracy/precision of tools/techniques. Use technique book! Be specific and give examples. Suggest fixes for the problems you identify and additional experiments to improve findings. Don’t use generic “human error”. Any discussion of human error should focus on specific, known errors and their effect on the results. Let’s apply

37. GenChem Discussion: Data quality The ability to determine separation depended on the size of the spots on the TLC strips. For some of the strips the spots were so big that they ran together. This made it difficult to determine how well they were separated. This problem could be addressed by repeating the TLC and applying less of the leaf extract to the plate. It should also be noted that, based on the data presented in the results table, both the 1:3 acetone to hexane and 1:2 acetone to hexane solvent mixtures produced reasonable separations. While the 1:3 appeared to do a better job it is quite possible that a mixture between these two would be even better. More trials with mixtures between 1:2 and 1:3 would help resolve this uncertainty.

38. GenChem Discussion See Technique book pp. 68-69 for more help with writing a Discussion.

39. GenChem The Worksheet You will now complete a worksheet for this experiment. To be submitted BEFORE leaving class:  Raw data. A copy of all measurements and observations you collected in lab.  Calculations. Examples of the R f calculation.  Results. An organized presentation of the results obtained.  Discussion. A discussion of the results obtained, their credibility and their meaning. If you don’t finish the worksheet in class you are to submit it tomorrow to the wooden dropbox outside the TSO

40. GenChem Pointers 1.Organize all your measurements into tables. Include all calculated results along with measured quantities. 2.Give labeled drawings of the TLC strips including colors of the spots and any other pertinent observations. 3.Organize your table to “flow”. Your presentation should lead the reader to your conclusion. 4.You will be graded on credibility. Your presentation must be credible. Proper use of significant figures and labeling (units) strongly impacts credibility. Let’s apply