1. Tools for Identifying Differences Among Samples

2. Are You Certain? Question Every Result
The More You Test the More You Know
However… Everything You Do Costs Money

3. Identifying Differences in Samples
You will analyze 4 samples
Sample 1: Pre epizootic
Sample 2: Early epizootic
Sample 3: Peak epizootic
Sample 4: Post epizootic
*Epizootic?
a disease or condition that occurs at about the same time in many individuals of the same species in a geographic area

4. Identifying Differences in Samples
4 groups of 2 students
Each group will choose an envelope that has the identifier for sample they will analyze.
Each group will use their sample through all phases.
After the tests are run, all teams will need to work together to identify the differences and any potential “toxin.”

5. Side note on Sample Information
Blinded Study = Increased Integrity
Teams will not know the date or time of collection for their sample during the analysis.
Four water samples will be analyzed (based on time points).
The experiments are designed to test the hypothesis that the spike in dolphin deaths was caused by something in the water.

6. Tools for Identifying Differences in Samples
HPLC (LCMS)
Colorimetric (Ninhydrin and Ellams’s) pH
The above represent three analysis stations.
Each tool has its own inherent strengths and weaknesses.
False positives are possible.
A combination of results will lead a more accurate prediction.

7. Station 1: HPLC High Performance Liquid Chromatography
Goals
Prepare serial dilutions
Run samples on HPLC machine
Analyze results from HPLC
Use results to identify which samples if any should be used for LCMS (liquid chromatography mass spectroscopy)

8. HPLC Prepare Serial Dilutions -clean water sample
-non-diluted lagoon water sample
-10 fold dilution of lagoon water sample
-100 fold dilution of lagoon water sample
You will need to calculate how to make the dilutions and record them in your notebooks (what you did and which sample is which).
Prepare and perform 4 runs on the HPLC at this station.

9. HPLC Run samples on HPLC
Working with a technician from TPIMS (Torrey Pines Institute for Molecular Studies), you will run each of the samples on the HPLC. Each of the sub-samples will take approximately 15 minutes to run on the HPLC.
The technician will show you how to use the HPLC with the first 2 samples. One student will run the 3rd sub-sample and the other student the 4th sub-sample.
It is your responsibility to inform the TPI technician the name identification of each sample.

10. An HPLC Chromatogram
Retention time varies depending on interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.

11. LCMS Liquid Chromatography with Mass Spectrometry
Analysis will be done by TPIMS technicians at TPIMS.
You will prepare, label, and package vials and ship them to TPIMS for LCMS analysis.
It will be the your responsibility
to properly label the samples.
TPIMS will mail (or FedEx) the results to the schools.

12. Station 2: Colorimetric Tests
Goals
Convert chemical formulas to structures
Identify functional groups on compounds and predict results of tests based on the presence or absence of certain functional groups
Perform colorimetric tests on lagoon water samples as well as other known samples
Analyze and record results

13. Colorimetric Tests Two different tests:
Ninhydrin (identifies free primary amines)
Same reaction used to look for fingerprints
Ellman’s (identifies free sulfur groups)

14. Colorimetric Tests Convert Chemical Formulas to Structures
A TPIMS technician will be at the station to help students.
You will consider a number of different chemical structures and their related formulas.
You will consider that the same chemical formula can produce multiple chemical structures.
You will consider the role of chemical bonds.
You will see what compounds with free primary amines look like compared to other nitrogen containing compounds.
You will be given chemical formulas and be asked to draw as many feasible structures as possible.

15. A TPIMS technician will be at the station to help the students.
Colorimetric Tests
Predict results of tests based on the presence or absence of certain functional groups (Part 1)
A TPIMS technician will be at the station to help the students.
You will be given samples and chemical structures of a number of compounds.
You will predict the results of the tests based on the chemical structures.
You will then perform the tests on each compound, record their results and compare to their predictions.

16. A TPIMS technician will be at the station to help the students.
Colorimetric Tests
Predict results of tests based on the presence or absence of certain functional groups (Part 2)
A TPIMS technician will be at the station to help the students.
You will be given unknown samples as well as the chemical formula of each sample.
You will test each sample and, based on the results, draw a plausible chemical structure.

17. Colorimetric Tests Perform test on lagoon water samples
A TPIMS technician will be at the station to help the students.
You will test the lagoon water samples and record the results.
You may or may not need this information to help identify a toxin.

18. A TPIMS technician will be at the station to help the students.
Station 3: pH
Goals
Test standards using a pH meter and/or strips
Test 5 unknowns and predict which compound is which based on results
Analyze lagoon water sample
A TPIMS technician will be at the station to help the students.

19. A laboratory notebook is a legal document.
Recording Results
Students should use a table that lists all the compounds they tested and all the results.
A laboratory notebook is a legal document.