3. Why HPLC? Almost universal applicability Remarkable precision Highly commercially available (competition)

4. Publications and $$$$$ Money spent on HPLC exceeds any other analytical technique Source: Introduction to Modern Liquid Chromatography by Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan 3 rd Ed., 2011

5. Hands-on learning SJU Chemistry CHROMATOGRAPHIC THEORY AND METHODS IN THE INSTRUMENTAL ANALYSIS COURSE CHE 3300 AND 3301L Instrumental analysis is a four credit undergraduate course with two one-hour lectures and two 3-hour labs per week with hands-on use of all instruments. The course covers chromatography (30%) the rest of the course covers digital electronics, spectroscopy, thermal analysis, and electrochemistry. Chromatography covers both the equilibrium and kinetic theories of separations. Practical methods to develop methods of analysis are discussed and implemented in lab. Methods of analyzing data and optimizing results are presented in lecture and used in lab. Two instruments used in this course are reversed phase high performance liquid chromatography (RP-HPLC) and gas chromatography with a mass spectrometer detector, (GC-MS). Theory and application of mass spectrometry are also discussed in lecture. INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS CHE 101 AND 101L Principles and practices of instrumentation used to provide quantitative chemical information. Basic electronics, transducers for chemical systems. Applications of instrumental measurements and techniques including; electrochemical methods - voltammetry, potentiometry. Spectrometric methods: emission and absorption, visible, ultraviolet and infrared. Nuclear magnetic resonance. Mass spectrometry. Gas chromatography, liquid chromatography and electrophoresis. Thermal analysis.

6. How Does It Work? Provides separation of compounds in a mixture based on polarity You want a “highly resolved elution pattern” (nice sharp distinct peaks) 80% of all application is reversed-phase (stationary) Hydrophobic compounds will stay on the column longer (“increased retention times”) Mobile phase will carry each compound off the column, to the UV detector, then to waste Diagram source: lab-training.com

8. Let’s see HPLC in action https://youtu.be/kz_egMtdnL4

9. 5 QUICK TIPS: Try ISOCRATIC first  change flow rate, column temp, and inj. volume only If you must GRADIENT then vary % of aqueous buffer to organic modifier (MeOH and ACN) Start with 100% Aqueous Equilibrate for 30 min. using starting conditions Watch for STEADY pressure or you will not have a baseline.. AIR IS THE ENEMY! Prime again until air bubbles are gone!

10. How to make sure the air is OUT! https://youtu.be/_vilKzYfox8

11. How To Set Up an HPLC Run https://youtu.be/qgdGzM6ym7A

12. Review of Key Steps for Set Up Open Project, Go to lamp icon – check settings of PDA for the correct wavelengths Go to pump icon – click flow tab for isocratic/gradient, column heater settings Save this new “Instrument Method” Tie a “Method Set” to it Create “Sample Set” in main window and save

13. 7 More Super Simple Quick Tips Purge injector for 6.5 minutes Make sure you are using appropriate wash Equilibrate column for 30 minutes (watch pressure) Inject the first sample 2x Wash column after run (40 min. water then 50/50 water/organic) For really impure stuff: wash for 10 minutes in 100% organic (and re-equilibrate your column) between injections Use a standard (or structural analog)

14. How to Review Chromatograms in Empower https://www.youtube.com/watch?v=2fXtOQyKJzI

15. Review of Key Steps for Data Review All runs are automatically saved “Review” your Sample Set in “Browse Project” Extract each chromatogram at desired single wavelength Manually integrate peaks for AU (y-axis) and retention times (x-axis) Print chromatogram, spectra, and table

16. Q&A