What does HPLC stand for?

1. What does HPLC stand for? • High Performance Liquid Chromatography

2. Theory behind it • Based on partition chromatography • Components that are like the stationary phase stay on the column longer than those that are like the mobile phase

3. “Normal” vs “Reverse” PhaseA History lesson Normal Phase Column is polar Mobile phase is non polar (Chloroform) Never use water Reverse Phase Column is non-polar Mobile phase is more polar (water, methanol ect.)

4. Columns • Bonded phase columns • Solid support (usually 5 micron Silica) is chemically attached to the stationary phase (C-18) via covalent bonds • Stationary phase can not be easily washed off • Most common are C-8 and C-18 • Have 8 or 18 carbon long straight alkanes attached to the silica

5. Normal phase HPLC Columns • Cyano Rugged, moderate polarity, general use • -OH (Diol) More polar and retentive • Amino Highly polar, less Stable • Silica Very rugged, low cost, (Unbonded) adsorbent

6. Reverse Phase HPLC Columns • C-18, C-8 Rugged, general purpose, highly retentive for non polar compounds • C-3, C-4 Less retentive, used mostly for peptides and proteins not as retentive because have less carbons • Phenyl Bonded Greater selectivity for aryl compounds • Cyano phase Moderate retention, normal and reverse useful for moderately polar analytes • Amino Weak retention, good for carbohydrates

7. Column Performancedepends on…… • Tubing -Length • Packing -Particle size, Geometry • Chemistry - Surface Chemistry, Adsorption, Selectivity • Mechanics - Efficiency

8. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

9. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

10. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

11. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

12. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

13. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

14. Methanol pump Injector Column Mixer become mobile phase Data Station Detector pump Water

15. HPLC Instrumentation:Detection • Fixed wavelength UV • Variable Single wavelength UV-vis • Diode Array (multi wavelength) • Refractive index • Fluorescence • And others based on electrochemical techniques • Mass Spectrometry

16. HPLC Instrumentation • Solvent Delivery Systems • Syringe pump • Diaphragm pump • Single piston reciprocating pump • Dual-piston reciprocating pump

17. Solvent Delivery SystemsDegassing • Outgassing is the number #1 problem in HPLC • If mobile phase contains air it must be degassed before placing on the system. • 1 of two methods • Sonicate with vacuum • Sparge with Helium for about 15 minutes

18. Solvent Delivery SystemsGeneral Guidelines • ALWAYS have mobile phase reservoir higher than the pumps • If system is dry physically prime BEFORE attempting to auto purge • Best to prime and purge with methanol or isopropanol first to ensure the system is properly wetted

19. Solvent Delivery SystemsGradient mixing • Low pressure gradient elution (LPGE) • Solvents are mixed before the pump • High pressure gradient elution (HPGE) • Solvents are mixed after the pump

20. Resolution • The completeness of separation of one analyte from other analytes in the mixture • Separations are optimized by exploiting differences between chemical and physical properties of the sample molecules. • Could also be called “differential adsorption”

21. Retention Time Variation • Four Main Causes 1. True Flow Rate- Check to see if void time has changed 2. Temperature- A 1degree (C) temperature shift can result in a 1 – 2 % shift in retention time 3. Mobile Phase pH - A shift in the mobile phase pH may effect some peaks and not others (primarily those with ionizable functionalities) 4. Percent Organic mobile phase- Since k’ is strongly affected by changes in strong solvent concentration, be careful of how mobile phases components are mixed - Improper degassing technique can change mobile phase concentration

22. Retention Time Variation • System leaks • Poor gradient mixing • Mobile phase composition varying • Contaminated guard column • Old column • System not yet equilibrated • Must run 10 min to equilibrate

23. Causes of Tailing peaks • Incorrect solvent for the sample • Extra-column effects • Build up of garbage on the column inlet • Secondary retention (Silanol sites) effects • Contamination of column packing material • Inadequate or inappropriate buffering

24. Indicators of Impending Column Failure • Pressure increase • Tailing increase • Plate loss • Selectivity (co elution) • Retention( usually less)

25. Trouble Shooting Detector • Mobile phase pulsing pump stroke • Drift - if stops when pumped turned off then impure mobile phase • Swell - if stops when pumped turned off then impure mobile phase

26. Trouble Shooting Detector • Bubble IN cell At each pump stroke No equilibrium of base line • Bubbles passing Through the cell • Transient spiking -Continuous • Saw tooth