1. Chromatography High Performance Liquid Chromatography HPLC Chapter 4-5 1 Dr Gihan Gawish

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3.  (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to:  separate,  identify,  and quantify compounds. 3 HPLC

4. HPLC Instrumentation (Schematic diagram) Dr Gihan Gawish 4 Liquid Mobile Phase Pump Injection Valve Separation Column Detector

5. Dr Gihan Gawish 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 5 HPLC- Mobil phase

6. Dr Gihan Gawish  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 6 HPLC- Pumps

7. Dr Gihan Gawish  Valve consists of a rotor and stator (stationary back-plane). 7 HPLC- Injection Valve Animation

8. Dr Gihan Gawish  Generally stainless steel and teflon components.  The stationary phase packings are microporous silica 2-10 μ m in diameter.  Unmodified silica is very polar.  Some systems use Precolumns to remove impurities from solvent or sample 8 HPLC- Analytical Columns

9. Dr Gihan Gawish  2 types porous stainless frit 0.5 to 2 μ m 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. 9 HPLC- Precolumns Filters

10. Dr Gihan Gawish  Ideal Characteristics  Universal  Small volume, prevents remixing & band  broadening  Fast response to flowing system 10 HPLC- Detectors

11.  UV  Single wavelength (filter)  Variable wavelength (monochromator)  Multiple wavelengths (PDA)  Fluorescence  Electrochemical  Mass Spectrometric HPLC- Detectors

12. Dr Gihan Gawish  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. 12 HPLC- Analysis Animation

13. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent (s) used. Retention time Dr Gihan Gawish 13 Time at which a specific analyte elutes (comes out of the end of the column) is called the retention time Retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte.

14. Dr Gihan Gawish  organic molecules organic molecules  Biomolecules Biomolecules  Ions Ions  polymers polymers 14 HPLC- Analyte

15. Dr Gihan Gawish  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. 15 HPLC- Advantage

16. Types of HPLC 1. Normal phase chromatography Dr Gihan Gawish  (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. 16

17. Types of HPLC 2. Reverse Phase Chromatography Dr Gihan Gawish  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 17

18. Practical considerations Dr Gihan Gawish  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. 18

19. Dr Gihan Gawish 19 HPLC- Chromatograph