Best Broken into four categories

1. Best Broken into four categories Theoretical Background Gas Chromatography HPLC Columns and Interactions

2. HPLC High Performance Liquid Chromatograpy Use for: • Small molecules • Charged, Neutral, Hydrophillic, Hydrophobic • Large molecules • Synthetic polymers, proteins, carbohydrates • Thermolabile molecules Must be soluble in mobile phase Not used for: • Gasses • Very small simple molecules • Alkanes

3. HPLC Setup

4. Mobile Phase Reservoir Mixture of Different Solvents Examples: Organic, Buffers, Water Methanol, Ethanol, Acetonitrile, Hexane, DCM Buffers: Phosphate, Acetate, Formate All solvents must be HPLC quality grade (Extremely pure) Before use the mobile phase must be • Filtered through fine filters to remove any particles from solvents • Degassed • Remove dissolved gas from the solution • Vacuum Degasser • Helium as a degassing gas

5. Pumps Pumps for HPLC need to be: • Able to pump at high pressure • Very precise control of flow rate Isocratic Elution Use of single solvent or mixed solvent of constant composition Gradient Elution Two pumps, or one pump and proportioning valve, used to deliver changing mixture of two or more solvents Useful to achieve better separation and shorter elution times by creating mobile phase gradients

6. Injectors Introduces sample to system Difficult as under high pressure Direct injection is difficult Two types of injectors: • Manual • Auto Sampler Sample loop fixes the exact quantity of sample to be injected each time

7. Column Contains stationary phase particles Usually made from stainless steel or plastic Pre-Columns, Guard Cartridge, used either to: • Protect column • Same stationary phase • Pre-separate sample • Different stationary phase Once activated: Washed after use, and kept in solvent

8. Column Stationary Phases Normal Phases NP • Polar Stationary Phases • Silica, Alumina • Adsorption Chromatography Reversed Phases RP • Non-polar Stationary Phases • Polar Stationary Phases • Bonded silica, C2, C4, C8, C18 • Absorption / Partition Chromatography pH Sensitive (Between pH 2 to 8) Silica is slightly soluble at alkaline pH Requirements • Column must be equilibrated before separation process • Shorter Narrower tubing reduces dead volume (extra column volume) Ovens Retention time temperature dependant Two injections of same sample can be significant in temperature changes Oven maintains temperature

9. Detectors Examples Ultraviolet and Visible absorption Differential refractive index Electrochemical: Amperometric Electrochemical: Conductometric Fluorescence Mass Spectrometry Solution Light Scattering Evaporative Light Scattering

10. Detectors UV/VIS Detectors Diode Array Detector (DAD)

11. Detectors UV/VIS Detectors Photo Diode Array Detector Peak Purity • Comparison of spectra at three different points of peak elution time (start, middle, end) • If identical three spectra identical

12. Detectors Refractive Index Detectors Universal Bulk Property Good for polymers Good for analytes with no chromophores 1000 times less sensitive than UV Useless in gradient elution Sensitive to temperature and pressure Not good for trace analysis

13. Sample Preparation Sample must be soluble in mobile phase Overloadof sample may damage column Concentration of key analytesshould be in linear range of detector Quantity of sample introduced to system depends on sample loop size Separated compounds can be collected and isolated after detection point (if not destroyed)

14. Separation Process - Parameters Column Length Longer Column, Better Separation, Longer Retention Time Particle Size of Stationary Phase Smaller Particles, Narrower Peaks, Higher Pressures Temperature Higher Temperature, Faster Diffusion, Better Separation, Shorter Retention Times, Column Degrades Combination of Mobile Phases Gradient Elution, Best Separation, Shortest Time pH of Mobile Phase Ionised compounds not retained well on RP-Columns

15. Best Broken into four categories Theoretical Background Gas Chromatography HPLC Columns and Interactions

16. Effect of Particle Size Smaller Particles increases Efficiency due to Sharper Peaks! More theoretical plates N, smaller HETP h. Smaller Particles need Higher Pressure to force mobile phase through column Maximum flow rates are limited by high pressures

17. Silica Surface Dominated by Polar Functional Groups

18. Reversed – Phase Silica Acid condensation of silica forms RP silica Hydrophobic molecules bonded to silica surface Switches to a non-polar environment

19. Analyte Interactions

20. Silica Surface NonPolar RP Silica Surface Polar NP RP vs NP Like Attract Like Opposite Phases Repel What do you expect to happen when red line breaks? Blue solvent: Water

21. NP vs RP Like Attracts Like Solvents with similar functionalities to the analytes will adsorb in the same way and compete for the same interaction sites. This will effect the binding equilibrium Reversed Phase Silica is more common Why? Better Peak Shape Less Tailing due to strong interactions Aqueous content of mobile phase allows buffering Control analyte ionisation with pH Better Control of mobile phase gradients Composition varies more widely

22. Predicting the Order of Elution Identify key points of difference Polarity More Polar = Stronger Interaction in Normal Phase More Polar = Weaker Interaction in Reversed Phase What is eluted first in a Normal Phase Column? Opposite applies for Reversed Phase First Last Morphine Codine Heroin

23. Effect of Stationary Phase For reverse phase silica Longer chain lengths result in longer elution times and better resolution

24. Effect of Mobile Phase For reverse phase silica Increasing the proportion of ‘weak ‘ solvent reduces competition for adsorption – analytes are retained longer and resolved better

25. Optimising Mobile Phase If some analytes are insufficiently retained while others are excessively retained, vary strength of mobile phase! Use of gradients allow peaks at certain intervals to spread or narrow giving good resolution and improve overall run time 

26. Effect of Mobile Phase pH General Rule Analytes should be uncharged Low pH for acids High pH for bases BUT! Remember silica has limited stability at high and low pH.