1. How do we analyze a system? Separating Substances
Unit 1
How do we analyze a system?
Separating Substances
This experiment is part of the first unit of the course, which is focused on helping students understand how we go about separating and identifying different substances.

2. How do we separate substances?
Lab Session 3
How do we separate substances?

3. Why would we want to separate substances?
The Motives
Chemical thinking allows us to take advantage of the characteristic properties of substances to separate the different components of materials.
Why would we want to separate substances?
This was a pre-lab question. Through volunteer offerings collate their lists. Compare to the list that pops up in the red box.
Identification Characterization
Elimination Quantitation
Application Transformation

4. The Challenge
Imagine that you work for a clothing company interested in using natural pigments to dye fibers.
Your task is to devise the most efficient method to extract and separate the pigments in a colored natural product (chili see pods).
Present the challenge to the students. Ask them to brainstorm in their groups things we need to do to face the challenge (extract the pigments; select a separation technique (such as TLC); test different solvents to find the most efficient).
What would you propose to do to a) extract and b) separate the pigments?

5. How can we extract the pigments from a natural product? Why?
Share Ideas
How can we extract the pigments from a natural product? Why?
Simple immersion in a solvent vs. grinding?
Ask the students how they propose to extract the pigments.
Pigments are located in chloroplast walls inside cells. Solvent can’t get to them. Need to break down cell walls.

6. What solvent could work best to separate components? Why?
A Useful Model
What solvent could work best to separate components? Why?
Many chemical separations are based on differences in the strength of the interactions between particles of different substances.
The point of the simulator is to give students a visual image of what molecules are doing when they interact with each other. It is to provide the background for the conclusions to be drawn in the next slide. If you don’t see the simulator on the slide, click on the square on the top right corner to open it in a web browser.
Here’s how to use the simulator.
Set temp to 150 K. Set Number of Atoms A to about 10. Set Number of Atoms of B to about 10. All the force sliders should read 0. No intermolecular interactions.
Ask “What will happen when I remove the barrier?”
Move the volume/partition slider to 0.
Let them digest what happens. The point is they create a random mix.
Now pose the question.
“What if there are strong interactions between A’s and other A’s and B’s and other B’s, but not between A’s and B’s?”
Put the partition back. Clear the screen by moving the Number of sliders to 0, then put them back to 10.
Move the intermol force AA slider all the way to the right. Do the same with the Intermol. Force BB slider.
Again ask “What will happen when the barrier is removed?” After predictions are made, remove the barrier.
Finally, “What if the interactions between A and B are great, but A to A and B to B are small.
Again, return the partition, clear the atoms and return to 10 of each. Move the Force AA and Force BB sliders to zero and move the Force AB to its max value.
Once again ask for predictions and then remove the partition.
Finally, if you have trouble getting out of this slide, set the number of atoms to 0.

7. To Keep in Mind
The solubility of one substance in another depends on the relative strength of the attractive forces between their particles
AA , BB >> AB Insoluble, Immiscible
AA, BB <= AB Soluble, Miscible
In general,
LIKE DISSOLVES THE LIKE
Conclusion. If particles are attracted to their own kind, but not others, they group separately (immiscible). If they are attracted to others, but not their own kind they mix (soluble). They need to understand that in order to face their challenge, they will have to find out what solvent, or mixture of solvents, works best to extract the pigments from the chili pods.
Now. Move on to address the SEPARATION question. Have them share their answer about the last question in the slide
How do you propose to separate the pigments in the extract?

8. Separating Components
To separate the different components in your sample you will use Thin Layer Chromatography.
Separation based on unequal attractions between the different components of a mixture an a stationary phase.
Mobile phase
Stationary
Support
These slides provide a schematic representation of how chromatography works at the molecular level.
They should know this from the discussion prior to the lab.

9. Chromatography
With the proper selection of stationary and mobile phases you can separate materials based on intermolecular forces.
Gas chromatography
Chromatography is one of the most used techniques to separate substances. Make student aware of the amazing sophistication of modern chromatographic techniques.
Point out that the x-axis of the graph is time. Ask them to identify the component with the highest affinity for the stationary phase.

10. Thin Layer Chromatography (TLC)
TLC Strip
Support and Stationary Phase
What is it?
Developing jar
Involve students in the presentation of the basic idea about how to implement the technique they will use in this lab.

11. Thin Layer Chromatography (TLC)
What’s the mobile phase?
What’s the stationary phase?
What makes the mobile phase move?
Running
Involve students in the presentation of the basic idea about how to implement the technique
Spotting

12. Chromatography
In TLC, the selection of the solvent used as the “mobile” phase should be made very carefully to induce the best separation possible.
The substances most attracted to the solvent will be the ones moving faster through the plate (carried by the solvent).
Discuss the importance of testing different solvents or solvent mixtures to find the best conditions to separate the pigments.
They should understand that their challenge will require them to identify the best solvent, or mixture of solvents, to EXTRACT the pigments, and then the best solvent, or mixture, to SEPARATE them.
Different combinations of solvents induce different degrees of separation. Which one is best?

13. Chromatography Rf Retention Factor
Discuss the advantages of calculating the Rf of the different substances separated on the plate.
Potential comments: The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions are also constant (solvent system, adsorbent , thickness of the adsorbent , amount of material spotted, temperature). Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. “Relative Rf” means that the values are reported relative to a standard, or it means that you compare the Rf values of compounds run on the same plate at the same time.
The Rf can provide corroborative evidence as to the identity of a compound. If the identity of a compound is suspected but not yet proven, an authentic sample of the compound, or standard, is spotted and run on a TLC plate side by side (or on top of each other) with the compound in question. If two substances have the same Rf value, they are likely (but not necessarily) the same compound. If they have different Rf values, they are definitely different compounds. Note that this identity check must be performed on a single plate, because it is difficult to duplicate all the factors which influence Rf exactly from experiment to experiment.
Different substances are expected to have different Rf values. Thus, the Rf can provide corroborative evidence as to the presence of different components in a sample.

14. Extract and separate the pigments from spinach leaves.
Skill Development
Extract and separate the pigments from spinach leaves.
Available resources:
Solvents: Acetone, hexane.
Pigment sources: Spinach leaves;
TLC strips
Others: Sand, mortar and pestle, glassware.
Students should work in groups trying to generate the best extract (most concentrated) from spinach. The idea is to use the spinach experiment for students to practice the techniques (extraction and TLC) before facing their actual challenge (the separation of pigments in chili pods). Talk about this with the students so that they understand that first they need to become skillful with a technique, and then they are expected to apply their skills and creativity to solve the challenge.
You have 60 minutes

15. Claims, Evidence, Reflections
Share your results for the separation of the pigments in spinach leaves with your classmates:
How successful were you in the separation?
What problems did you encounter?
What suggestions do you have to improve the separation?
Have the students share their results with the rest of the class. Analyze with them the problems they encountered and how to deal with them in the future.

16. Your Real Challenge
Your task is to devise the most efficient method to extract and separate the pigments in a colored natural product (chili see pods).
Discuss in your group what you propose to do.
Have students discuss how they are going to face their challenge. Discuss with them what questions they need to answer (what is the best solvent to extract the pigments? What is the best mobile phase for TLC? How many pigments are there? What are their Rfs? Etc.)
What questions do you need to answer in order to succeed in this task?

17. Your Real Challenge
Design and implement an experimental procedure to extract and separate the pigments in chili pods.
Available resources:
Solvents;
TLC strips;
Glassware.
Once you think they are ready to face their challenge, send them to the lab.
You have 60 minutes

18. Claims and Evidence
Based on the results of your experiments, present your major claims and the evidence that you have to support the answer to your beginning questions.
Did the group answer their beginning questions?
Are their claims clear?
Is the evidence reliable?
Is the evidence appropriate to support the claims?
Ask each of the groups to report their results on the board. Have the rest of the groups question the claims and the evidence using the question on the slide as a guide.

19. Final Reflections What did you learn from doing your experiment?
How would you improve what you did?
How have your ideas changed as a result of this lab?
What do you not completely understand?
What new questions do you have?
Use these questions to motivate a discussion and emphasize major points you want students to understand.

20. Your Report
Beginning questions (2 p): What questions guided your explorations?
Safety Considerations (2 p): What did you do to stay safe in the lab?
Procedures and Tests (2 p): What experiments did you do to answer your questions?
Data, calculations, and representations (6 p): What observations did you make? What data did you collect? What calculations and representations helped you make sense of the data?
You will now go over the basic components of the report. Students should work together in groups, but insist on having an individual report from each student. Talk to your students about your expectation for the report. Have them share ideas on what to write in the different sections.

21. Your Report Claims (2 p): What can you claim to answer your questions?
Evidence and Analysis (6 p): How did you interpret your results to support your claims?
Reflections and additional questions (10 p): What did you learn? What do you not completely understand? How have your ideas changed as a result of this lab? What new questions do you have? How would you improve what you did?
Continue description

22. Your Report Post-Laboratory Questions:
Is it practical to use TLC to separate pigments from natural products? Explain.
What other separation techniques may you use to face a similar challenge to the one posed in this experiment?
How would you use the particulate model of matter to explain a major result from your experiment?
The answer to these questions should be included as part of the lab report (they could be included as part of the reflections)

23. How do we use models to derive properties?
Our Next Lab
Present the next lab
How do we use models to derive properties?

24. The Challenge Butanone Ethanol Hexane
Three volatile solvents have been found in different containers in an illegal drugs lab. Based on prior experiences, you infer the potential identity of these substances:
C4H8O
Butanone
C2H6O
Ethanol
Present the challenge and remind students about what they need to do before coming to lab (complete IBIS quiz, do the pre-lab assignment, complete their report, BRING A LAPTOP WITH EXCEL).
Hexane
C6H14
How can you determine the identity of the solvent in each container?
BRING A LAPTOP WITH EXCEL