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HPLC Instrumentation

HPLC Instrumentation. In This Section, We Will Discuss:. General components of a high performance liquid chromatograph. HPLC solvent delivery systems. How automatic injectors work. Common HPLC detectors. HPLC Instrumentation Overview. Principle Pattern. An Example. Solvent Reservoirs.

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HPLC Instrumentation

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  1. HPLC Instrumentation

  2. In This Section, We Will Discuss: • General components of a high performance liquid chromatograph. • HPLC solvent delivery systems. • How automatic injectors work. • Common HPLC detectors.

  3. HPLC Instrumentation Overview Principle Pattern An Example Solvent Reservoirs Controller Solvent Cabinet Vacuum Degasser Binary Pump Autosampler Thermostatted Column Compartment Detector

  4. Solvent Inlet Filer Stainless Steel or glass with 10 micron porosity. Removes particulatesfrom solvent. Precolumn Filter Used between the injector andguard column. 2 to 0.5 micron Removes particulates from sampleand autosampler wear debris. Must be well designed to preventdispersion. Solvent Filters Guard column Injector Analytical Precolumn Column Filter Solvent Inlet Filter

  5. Vacuum Degassing

  6. Functions of the Solvent Delivery System The solvent delivery system has three basic functions: • Provide accurate and constant flow. • Provide accurate mobile phase compositions. • Provide the force necessary to push the mobile phase through the tightly packed column.

  7. Multichannel Gradient Valve • Determines mobile phase composition. • Largest solvent plug fills first. • Agilent 1100 and 1200 quaternary pump.

  8. Dual Piston Parallel Pump Check Valves Rotary Switching Valve Pumphead Piston A B Combined Single Piston Delivery Delivery Piston 'A' Advancing Piston B Retracting

  9. Dual Piston in Series Pump • First piston displaces solvent at twice the speed and stroke volume of the second piston. • Provides constant flow and the pressure necessary to get through column.

  10. Ballvalves for Reciprocating Piston Pumps

  11. Pump Seals and Pistons • Piston • Support Rings • Seal Keepers • Seals • Wear Retainers 1 2 3 4 5

  12. Frits and Filters Purge valve PTFE Frit Frits, Filters, and Sieves are used to protect other parts of the LC from pump and seal material.

  13. Damping Pump Unit Ripple 2% P/P Pressure Damping Units • Filled with compressible liquid separated from the mobile phase by a membrane. • Pressure ripples reduced to < 2% original value.

  14. Gradient Formation Low Pressure Gradient High Pressure Gradient

  15. Summary • The pump is the most critical piece of equipment for a successfully operating HPLC. • Performance parameters for HPLC pumps: • Flow Precision • Flow Range • Delay Volume • Pressure Pulse • Composition Precision

  16. Summary Video Click picture to activate video.

  17. Sample Injectors Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Two major designs: Automatic Injectors or Manual Injectors

  18. Manual Injectors Sample Loop Load - Inject Front View Rear View Inject

  19. Manual Injectors Sample Load From Pump Solvent in Sample in Solvent out To column From Pump Solvent in Solvent out Sample in To column Sample Inject

  20. Step 1 Step 2 Step 3 Automatic Injectors

  21. Rotor Seal found within valve Rotor Seals

  22. Column Oven Constant temperature for solvent and column is required to perform reproducible results.

  23. Common HPLC Detectors • UV-VIS • Diode Array • Multiple Wavelength • Variable Wavelength • Mass Spectrometers • Refractive Index • Fluorescence • Light Scattering • Electrochemical • Radioactivity • Conductivity

  24. Necessity for More Than One Detector - Sensitivity

  25. Necessity for More Than One Detector - Selectivity

  26. Qualitative Information Chlortoluron Atrazine ? ? Take peak spectrum Take peak spectrum (MS) (UV) 200 58 215 44 172 68 158 138 132 96 104 60 80 100 120 140 160 180 200 220 Wavelength (nm) Mass/Charge Necessity for More Than One Detector - Qualitative Information

  27. HPLC Detector Characteristics Detector performance characteristics: • Sensitivity (LoD, LoQ) • Selectivity • Linearity • Qualitative information • Reliability • Ease of use • Universality

  28. Peak Noise LOD The limit of detection for a detector can be characterized by its signal to noise ratio (S/N) for an analyte under a given set of conditions.

  29. Response Linear range Slope = sensitivity e.g.,RSD<10%, S/N > 20 MQL MDL e.g., S/N > 3 Intercept Amount Limit of Detection - Limit of Quantitation • Limit of detection (LOD) is a result of the whole chromatography system, not only the detector performance • Limit of quantification (LOQ) is a defined limit for a method used for a specific purpose.

  30. Detector Flow Cell c I0 I b Log I0 = A = abc I UV-Vis Detectors Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).

  31. UV Lamp Cut-off filter Holmium oxide filter Slit Sample diode Mirror 1 Grating Flow cell Mirror 2 Reference diode UV-Vis Detectors - Design Principles • Single wavelength detection of multi wavelength detection possible. • Wavelength calibration is done automatically using a holmium filter. Variable Wavelength Detector

  32. Vis Lamp Achromatic Lens Diode Array Detector Flow Cell UV Lamp Homium Filter Grating Optical Slit UV-Vis Detector with Spectral Capability • Diode Array UV-Vis Detector allows online measurement of spectra. • Wavelength range 190 - 950 nm. • Wavelength Resolution: Up to 1 nm. • Wavelength calibration with Holmium oxide filter.

  33. Absorbance Spectra Wavelength Time Online Spectra - UV-Vis Detector

  34. Fluorescence Detection

  35. Thin-layer design Porous flow-through design Electrochemical Detectors Wall-jet design • Gold for carbohydrates. • Platinum for chlorite, sulfate, hydrazine, etc. • Carbon for phenols, amines. • Silver for chloride, bromide, cyanide.

  36. HPLC-MSD API- Electrospray

  37. Refractive Index Detector Design The Refractive Index Detection is strongly influenced by: • Pressure changes • Temperature changes • Flow pulse Gradient elution is not possible!

  38. Light Scattering Detector

  39. Conductivity Detectors Applications Schematics F water fixed r soap products cell resistor detergents Ions C } soft drinks Acids in Balance A E Bases ref.capacitor control blood D Salts plating baths nuclear fuel reprocessing streams B ~ variable resistances

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