1. Gihan Gawish.Dr High Performance Liquid Chromatography

2.  (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to:  separate,  identify,  and quantify compounds.  Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent (s) used. Dr Gihan Gawish

3. LC vs. HPLC Liquid chromatography High-performance liquid chromatography (HPLC)  Use large, non-rigid support material  Particles size dp: > 150 μm, column  size dc: 10 ~ 50 mm, column length L:  50 ~ 500 cm, flow rate F: < 1mL/min  Gravity. Large H, small N  Poor system efficiencies and large  plate heights  Use small, uniform, rigid support material  Particle size dp < 40 μm, usually 3-10 μm in practice  Good system efficiencies and small plate heights, narrow peaks, shorter separation times

4. HPLC components: Liquid Mobile Phase Pump Injection Valve Separation Column Detector Dr Gihan Gawish

5. HPLC Instrumentation (Schematic diagram) Dr Gihan Gawish

6. HPLC Instrumentations High pressure: Several hundred atm Packing: 3 ~ 10 μm Elaborate and expensive 1. Solvent treatment system 2. Pumping system 3. Sample injection system 4. Column 5. detector Dr Gihan Gawish

7. Instruments – Solvent System Mobile phase reservoirs: – Several reservoirs (> 500mL) – Degassing: remove of dissolved gas band spreading and interfering detection Sparging: fine bubble of gas vacuum pumping, distillation, heating – Dust removal: interference with detection, column clogging, damage pumping system Millipore filter under vacuum – Isocratic elution: constant composition – Gradient elution: different solvent systems during elution, continuous change or step wise, solvent proportion valve

8. Mobile phase Solvent Reservoirs can be single solvent or multi-solvents The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte Dr Gihan Gawish

9. HPLC pumps  Requirements for HPLC apply high pressure to force liquid through the beads faster pressures to 6000 psi control flow rate from 0.1 to 10 mL/min Types of HPLC pumps  Reciprocating pumps: most commercial systems are based on this design.  Syringe pumps Dr Gihan Gawish

10. Instruments – Sample Injection System Sample Injection system: – Limit of precision of HPLC – Sample size: 0.5 ~ 500 μL – No interference with the pressure – Based on a sample loop, 1 ~ 100 μL, Reproducibility: 0.1%, P < 7000 psi – Auto sampler: inject continuously variable volume 1 μL – 1 mL Controlled temperature environment for derivatization reaction

11. Injection valve  Valve consists of a rotor and stator (stationary back-plane). Dr Gihan Gawish

12. Analytical Columns  Generally stainless steel and teflon components.  The stationary phase packings are microporous silica 2-10 μm in diameter.  Unmodified silica is very polar.  Some systems use Precolumns to remove impurities from solvent or sample Dr Gihan Gawish

13. Precolumn filters  2 types porous stainless frit 0.5 to 2 μm or a little piece of sacrificial column.  Injection valve => Precolumn => Column => Detector  Prevents the contamination of the expensive analytical columns with fine particles that can eventually clog the mobile phase flow. Dr Gihan Gawish

14. Detectors in HPLC  Ideal Characteristics  Universal  Small volume, prevents remixing & band  broadening  Fast response to flowing system Dr Gihan Gawish

15. Instruments – Detectors 1 Absorption detectors: – UV-Vis: Most widely used Z-shape, flow-through cell (V, 1 ~ 10 μL and b, 2 ~ 10 mm) Photometer: Hg 254 nm and 280 nm line for organic, D2 or W filament + interference filter Spectrophotometer: more versatile – IR: filter instrument or FTIR Similar cell (V, 1.5 ~ 10 μL and b, 0.2 ~ 1.0 mm) Limit: no suitable solvent, special optics – Fluorescence: Hg or Xe lamp Fluorometer and spectrofluorometer Fluorescing species or fluorescent derivatives A UV-Vis detector for HPLC

16. Instruments – Detectors 2 Electrochemical detectors: – Amperometry, voltammetry, coulormetry and conductormetry – A: simplicity, high sensitivity, convenience and wide-spreading application – Thin-layer flow cell of Teflon : 50 μm thick, 1 ~ 5 μL volume – Indictor E: Pt, Au, C – RE and CE: down stream – Multi-electrode: simultaneous detection or sample purity indication Amperometric thin-layer cell for HPLC

17. Analyze  The sample to be analyzed is introduced in small volume to the stream of mobile phase.  The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses the length of the column.  The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition.  The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. Dr Gihan Gawish

18. Analytes  organic molecules organic molecules  Biomolecules Biomolecules  Ions Ions  polymers polymers Dr Gihan Gawish

19. Advantage of HPLC  HPLC results in high resolution (sharp peaks), and rapid separation (minutes to 1 hour).  HPLC can be analytical or preparative.  HPLC can be used for all types of chromatography: size exclusion, ion exchange, reversed phase, and affinity chromatography. Dr Gihan Gawish

20. Types of HPLC 1. Normal phase chromatography  (NP-HPLC), this method separates analytes based on polarity  NP-HPLC uses a polar stationary phase and a non-polar mobile phase  Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. Dr Gihan Gawish

21. 2. Reverse Phase chromatography  Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase.  One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group  With these stationary phases, retention time is longer for molecules which are more non-polar Dr Gihan Gawish

22. Practical considerations:  Not all proteins can withstand the pressure of HPLC  All materials must be of the highest quality.  Solvents must be degassed to eliminate formation of bubbles. Dr Gihan Gawish

23. HPLC example Dr Gihan Gawish