Mysterious White Powder- Jon Benskin

TITLE: Mysterious, White Powders

KEY QUESTION(S): What are the active ingredients in popular pain relieving drugs and how can we identify unknown substances based on known facts and mass spectrometry?

SCIENCE SUBJECT: Chemistry and Biology

GRADE AND ABILITY LEVEL: Honors and Advanced Placement classes

SCIENCE CONCEPTS: Mass spectrometry, atomic structure, data analysis, the use of scientific tools

OVERALL TIME ESTIMATE: Two, 55 minute periods

LEARNING STYLES: Visual, kinesthetic, problem solving.

VOCABULARY: Acetaminophen- A common molecule found in many pain relieving drugs, such as Tylenol®and Excedrin ES®. Aspirin- A common molecule found in many pain relieving drugs, such as Bayer®. Electrospray Ionization- A process that ionizes a molecule in order to be detected via mass spectrometry. Mass spectrometry- A process used to identify the molecular weights of a molecule/solution. Methanol- A common solvent used in electrospray ionization.

LESSON SUMMARY: Students will prepare dilute solutions of caffeine, aspirin, and acetaminophen and then create individual mass spectrometry fingerprints of those pure solutions. Students will then conduct and analyze mass spectrometry results of common pain relievers (purchased over the counter) in order to determine the active ingredients that they contain and the identity of each.

STUDENT LEARNING OBJECTIVES WITH FLORIDA SCIENCE STANDARDS: The student will be able to: SC.912.P.8.4 Explore the scientific theory of atoms (also known as atomic theory) by describing the structure of atoms in terms of protons, neutrons and electrons, and differentiate among these particles in terms of their mass, electrical charges and locations within the atom. SC.912.N.1.1 Define a problem based on a specific body of knowledge, for example: biology, chemistry, physics, and earth/space science, and do the following: 1. pose questions about the natural world, 2. conduct systematic observations, 3. examine books and other sources of information to see what is already known, 4. review what is known in light of empirical evidence, 6. use tools to gather, analyze, and interpret data, 10. communicate results of scientific investigations, and 11. evaluate the merits of the explanations produced by others. SC.912.N.1.3 Recognize that the strength or usefulness of a scientific claim is evaluated through scientific argumentation, which depends on critical and logical thinking, and the active consideration of alternative scientific explanations to explain the data presented. SC.912.N.1.4 Identify sources of information and assess their reliability according to the strict standards of scientific investigation.

MATERIALS: ESSENTIAL: 

1 ml caffeine, aspirin, or acetaminophen pure stock solutions (10-3 M). All three solutions must be used in each class.



Pain relievers (3 different pain relievers would be ideal. They need to contain caffeine, aspirin, and/or acetaminophen. Popular choices would be Tylenol®- acetaminophen, Bayer®- aspirin, Excedrin ES®- caffeine, aspirin, and acetaminophen).



Methanol



Distilled/HPLC water



Formic/Acetic acid



Mortar and pestle



Microcentrifuge tubes



1L beakers



Micropipettes (P1000 and P100)



Micropipette tips



Electronic balance



250ml beakers

SUPPLEMENTAL: 

Additional, or different, pain relievers



Acetone (for cleaning)

BACKGROUND INFORMATION: As technology continues to progress, the use of advanced tools has become common-place in scientific laboratories. Mass spectrometry is a technology that has been used in science since the 1950s1; but just recently, with the incorporation of modern computing, has this technology progressed to a place where it is astoundingly accurate and sensitive. The basic principle of mass spectrometry is to identify the masses of molecules present in a sample. Today, there are many types of mass spectrometers, each with their own benefits and operational differences. Table-top mass spectrometry has become more common and less expensive, allowing undergraduate and high school students a chance to become familiar with this important technology. As previously mentioned, there are many types of mass spectrometers. It is not possible or feasible (at this point) to create portable versions of some of these variants. However, electrospray ionization mass spectrometry (ESI-MS) is variant that can be contained in a relatively small (portable) device. This variant works on the principle of ionizing a molecular sample. This is completed by moving a dissolved molecule through an electrospray needle, through an empty space, and having it enter a small hole in a sampling cone. These ions that move through this opening then travel to the sensors which detect the mass of the ions coming in contact with it (see figure 1). There are basic limitations to tabletop versions of this mass spectrometer (specifically the Advion Compact Mass Spectrometer). Only relatively small molecules (up to about 1,200 AMU) can be ionized and detected and relatively large amounts of solution must be run through the device.

Figure 1. Basic diagram of ESI-MS (from http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html )

Once a sample has been ionized and detected, the mass spectra is represented via software on an attached computer. An example of what this spectra could look like can be seen in figure 2. The tall peak indicates that a molecule with an approximate weight of 92 AMU (toluene) was detected more than any other molecule. A very important limitation to make note of is that the device is not capable of specific molecular identification. The student/scientist would need to have an idea of what molecule they were looking for in the sample, and could then identify if that molecule was present or not.

Figure 2- Example of mass spectra produced by toluene.

An important class of molecules in everyday life is pain relievers. Although many different pain relievers are now produced, some of the most common are acetaminophen and aspirin. These molecules, each with their own specific molecular weights, can be identified in samples via ESI-MS. This activity allows students to explore the constituents of different pain relievers in order to determine the identity of an unknown substance.

ADVANCE PREPARATION: Very little advance preparation is required once the mass spectrometer is setup and operational.* Students should have the following materials at each station: 

100ml distilled/HPLC water



100ml methanol



1ml formic/acetic acid



Wash bottle with acetone (optional, but recommended if multiple classes will be using the glassware)



Pain reliever stock solutions (can be teacher or student choice of which letter/pain reliever to use) 



Label each pain reliever as follows (only include the letter, not the pain reliever name) and put in 1.5ml microcentrifuge tubes: 

“A” - Tylenol®. Add .15g crushed powder to beaker, fill to 1L with water for a 10-3 M stock solution (assuming tablet contains 500mg acetaminophen).



“B” - Bayer® Aspirin. Add .18g crushed powder to beaker, fill to 1L with water for a 10-3 M stock solution (assuming tablet contains 300mg aspirin).



“C” - Excedrin ES®. Add .30g crushed powder to beaker, fill to 1L with water for a 10-3 M stock solution (assuming the tablet is 250mg acetaminophen, 250mg aspirin, and 65mg caffeine. Note- caffeine concentration will be lower than 10-3, but will still be evident in the spectral data).

Pure chemical stock solutions 

Preparation instructions (put in 1.5ml microcentrifuge tubes): 

.19g of caffeine powder in a beaker, fill to the 1L mark



.18g of asprin powder in a beaker, fill to the 1L mark



.15g of acetaminophen powder in a beaker, fill to 1L mark



Micropipettes (P1000, P100)



Micropipette tips (for P1000 and P100)



Five 1.5 ml microcentrifuge tube, or equivalent (for making solution)

*Note: The portable mass spectrometer kit should come with methanol, water, formic/acetic acid.

PROCEDURE AND DISCUSSION QUESTIONS WITH TIME ESTIMATES: 1. Introduction to quadrupole mass spectrometry (3 Minute video) http://www.youtube.com/watch?v=IowMQnI6Rxc 2. Mass spectrometer analysis of pure solutions (50 minutes) - Students will need to dilute their pure solutions to a concentration of 10-5 M in preparation for mass spectrometry. This procedure is found below and additionally in the student handout. In the microcentrifuge tube, students should: - Add 700ul methanol - Add 300ul water - Dispose of 20ul of this solution - Add 10ul of pure stock solution (10-3 M caffeine, asprin, or acetaminophen) - Add 10ul of acetic/formic acid - This 10-5 M solution is now ready for analysis in the mass spectrometer



Mass spectrometer instructions (see teacher instructional manual for further information)

-

The teacher should call over a group of students to the mass spectrometer. 

Students should fill a syringe with their 10-5M solution (there should be 1ml of the solution).



The syringe should be attached to the mass spectrometer via the brown tube originating from the front of the device.



The syringe should be placed in the syringe pump, and the flow rate should be set at 20ul. Start the pump.



Click the “play” button on the top of the software package



A dialog box will appear allowing you to name the file



Data collection will begin once the file is named



Click on the “manual collection” tab (on the left side) to be shown real-time data from the mass spectrometer





Allow students to record the masses of the main peaks



The run can be stopped at any time by clicking “standby”



The device must be flushed out after each run. Use a clean syringe with methanol.

Analysis of peaks in the data

- Students will have (on their attached worksheet) the molecular formulas for each molecule. Have students determine the molecular mass via each formula to ensure that their mass spectrometry fingerprints match their results (remember, the mass spectrometry peaks will be one more AMU than the molecular masses of each molecule because of the ionization of the sample). - Caffeine- C8H10N4O2 MW= 194.19 - Asprin- C9H8O4 MW= 180.157 - Acetaminophen- C8H9NO2 MW=151.169

3. Mass spectrometer analysis of “unknown” drug solutions (55 minutes) 

Students will need to dilute their “unknown” drugs to a concentration of 10-5 M in preparation for mass spectrometry. This procedure is found below and additionally in the student handout. In the microcentrifuge tube, students should:

- Add 700ul methanol - Add 300ul water - Dispose of 20ul of this solution - Add 10ul of 10-3 M pain relieving solution - Add 10ul of acetic/formic acid - This 10-5 M is now ready for analysis in the mass spectrometer (see above for procedure) 

Analysis of data 

Students need to figure out the main constituents of each drug (“A”, “B”, “C”) via the analysis of the mass spectra. Students will then need to look at the pain relievers packaging to figure out which “unknown” is which drug.

Student Handout- Spectrometer Analysis of Know Compounds and Pain Relievers 1. What is the purpose of mass spectrometry?

2. Common pain relievers usually have a couple common active ingredients. You will need to analyze three pure, common ingredients in order to establish control data for the identification of “unknown” pain relieving compounds. - You will need to dilute the pure compounds (either caffeine, aspirin, or acetaminophen powder) to a concentration of 10-5 M in preparation for mass spectrometry. This can be done as follows. In the microcentrifuge tube: - Add 700ul methanol - Add 300ul water - Dispose of 20ul of this solution - Add 10ul of pure stock solution (10-3 M caffeine, aspirin, or acetaminophen solution) - Add 10ul of acetic/formic acid - This 10-5 M is now ready for analysis in the mass spectrometer



Mass spectrometer instructions 

Students should fill a syringe with their 10-5M solution (there should be 1ml of the solution inside of the syringe).



The syringe should be attached to the mass spectrometer via the brown tube originating from the front of the device.



The syringe should be placed in the syringe pump, and the flow rate should be set at 20ul. Start the pump.



Click the “play” button on the top of the software package



A dialog box will appear allowing you to name the file



Data collection will begin once the file is named



Click on the “manual collection” tab (on the left side) to be shown real-time data from the mass spectrometer



Record the masses of the main peaks (see below)



The run can be stopped at any time by clicking “standby”



Flush the system with methanol when run is complete.

3. Analysis of peaks: What was the mass of the tallest peak? The tallest peak indicates the most prevalent molecule(s) in the sample.

4. Based on your knowledge of molecular structures, how many AMUs are the following molecules (show all work)? Caffeine- C8H10N4O2

Asprin- C9H8O4

Acetaminophen- C8H9NO2

5. When the molecule is ionized, it will gain a proton. Does your mass spectra result match the expected molecular weight + 1 AMU?

6. If there were other peaks in your mass spectra, supply a hypothesis for why they appear?

7. Prepare your “unknown” white powder (crushed pain reliever) as follows. In the microcentrifuge tube: - Add 700ul methanol - Add 300ul water - Dispose of 20ul of this solution - Add 10ul of 10-3 M pain relieving solution - Add 10ul of acetic/formic acid - This 10-5 M solution is now ready for analysis in the mass spectrometer

8. Letter of the “unknown” white powder assigned to your group:

9. What is/are the main peak(s) (masses) in your “unknown” sample?

10. Based on the classes previously collected data what is the molecular identity/identities of this/these peak(s)?

11. Based on the packaging of the pain relievers, which pain relieving compound did your group have?

12. If you were a scientist asked to identify this same mysterious white powder, write a short paragraph defending your conclusion (how you arrived at your conclusion and what evidence you have to support it).

Key to Student Handout: Mass Spectrometer Analysis of Know Compounds and Pain Relievers 2. What is the purpose of mass spectrometry? To determine the masses of the molecules present in a sample.

3. Analysis of peaks: What was the mass of the tallest peak? The tallest peak indicates the most prevalent molecule in the sample. Caffeine- 195 Asprin- 181 Acetaminophen- 152

4. Based on your knowledge of molecular structures, how many AMUs are the following molecules (show all work)?

Caffeine- C8H10N4O2 Carbon= 8x12= 96, Hydrogen 10x1= 10, Nitrogen= 4x14= 56, Oxygen= 2x16=32. 96 + 10 + 56 + 32 = 194

Asprin- C9H8O4 Carbon= 9x12= 108, Hydrogen= 8x1= 8, Oxygen= 4x16= 64 108 + 8 + 64 = 180

Acetaminophen- C8H9NO2 Carbon= 8x12= 96, Hydrogen 9x1= 9, Nitrogen= 1x14= 14, Oxygen= 2x16=32 96 + 9 + 14 + 32 = 151

5. When the molecule is ionized, it will gain a proton. Does your mass spectra result match the expected molecular weight + 1 amu? Yes, if the procedure was done correctly and the sample was ionized, the masses should all have an additional proton.

6. If there were other peaks in your mass spectra, supply a hypothesis for why they appear? Contaminants, possible fragmentation of the molecules.

8. Letter of the “unknown” white powder assigned to your group: This will vary per group.

9. What is/are the main peak(s) (masses) in your “unknown” sample? This will vary depending on what sample they were assigned.

10. Based on the classes previously collected data what is the molecular identity/identities of this/these peak(s)? Caffeine- 195 Asprin- 181 Acetaminophen- 152

11. Based on the packaging of the pain relievers, which pain relieving compound did your group have? Groups should match their spectral data to the data collected on the pure stock solutions. They should identify the peaks, and compare the constituents of their white powders to the active ingredients of the pain relievers.

12. If you were a scientist asked to identify this same mysterious white powder, write a short paragraph defending your conclusion (how you arrived at your conclusion and what evidence you have to support it). Students should be able to write a short paragraph connecting the accuracy of the mass spectrometry data to the known molecules and published drug information.

ASSESSMENT SUGGESTIONS: Completing the attached worksheet fulfills the following: 1. Students are able to determine molecular mass based on a molecular formula. SC.912.P.8.4 2. A mysterious white powder is presented to the students. They must use sources of information (periodic table), mass spectra data, and class data to reach a conclusion of the identity of this white powder. SC.912.N.1.1 3. Students must defend the results of their work through the connection of multiple scientific concepts. SC.912.N.1.3 4. Students must rely on the accuracy of mass spectrometry, the information found on the periodic table, and drug information. SC.912.N.1.4

EXTENSIONS: ACTIVITIES: Use other/additional drugs in the analysis. Additionally, the same drugs but different brands could be examined in order to see if one brand is more pure than the other brand. LITERATURE: There are many other types of mass spectrometry. Students can use the Internet to research different version of this technology and write a summary of the differences/similarities.

RESOURCES/REFERENCES: 1. Carl-Ove Andersson, Acta. Chem. Scand. 1958, 12, 1353.