Experiment Nine: HPLC and LC Introduction: High Performance Liquid Chromatography (HPLC) is a type of instrument that separates mixtures of compounds with a water base. Liquid Chromatography Mass Spectrometry (LC-MS) which is very similar to the HPLC, it allows to determine the mass-to-charge ratio of specific elements. Purpose: Within this experiment, the purpose was to test with various standards of different concentrations of caffeine and test the decaffeinated and caffeinated tea. The purpose of that was to determine the concentration of caffeine in the tea samples with aide in the HPLC. Hence, the same experiment was conducted with the LC-MS and to compare the results. SOP for HPLC: 1. Make sure there is enough degassed methanol and water. 2. Turn computer on. 3. Check pump lines for air bubbles. 4. Turn detector on and oven. 5. From the desktop, select LabSolutions->Instruments->Type:LC->CH210SYSTEM1-Instrument1 6. Go to File->Open method file->Project 1->Taylor Test->Test Method 1 7. Select parameters a. Organic/Aqueous ratio can be altered here. b. A 10 minute run is standard. c. Hit Download. 8. Hit Single Run a. Give a Sample Name. b. Give individual ID name. c. Use first yellow folder button to make your own data folder. d. Data File is what it will be saved as. e. Hit Ok. f. Hit Start. 9. Manually set to Load using the black dial next to column. 10. Syringe should be prepped by flushing at least once with deionized water and once with sample. 11. Once pressures stabilize on screen, inject. 12. To start, turn knob quickly, leaving syringe in. 13. Hit start. 14. After run, turn back to Load and remove syringe. 15. Copy chromatogram in paint. Troubleshooting 1. Problem: Where Ready should appear, Not Connected appears in gray box. a. Go to Main->System Configuration->Reset->Auto Configuration->OK Shut Down 1. Purge line by running closed line with 100% solvent from Pump B for 5 min (Acquisition>Instrument Parameters>set parameters>Download). *Note: You will have to time this because the system is closed. Repeat using 0% solvent from Pump B for 5 min. 2. File-> Open method file->Low Flow Method->open->Download 3. Close program. 4. Computer and instrument stay on. 5. Ensure mobile flasks have adequate solvents. 6. Turn off detector and oven. 7. Check waste container. Data: HPLC: Concentration (ppm) Area (Caffeine Peak) 10 344338 20 571396 30 829879 40 986278 1200000 y = 21843x + 136897 R² = 0.9911 1000000 800000 Series1 600000 Linear (Series1) 400000 200000 0 0 10 20 Refer to spectra in notebook. Results LC-MS: 30 40 50 25 20 y = 2.69x + 9.97 R² = 0.7555 15 Series1 Linear (Series1) 10 5 0 1 2 3 4 Conclusion: Overall, the HPLC and LC-MS were not complicated instruments. However, the R squared value for the LC-MS was terrible, having a value of 0.755. Hence, the concentrations of the caffeinated and decaffeinated had skewed results.