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 Controller Solvent Cabinet Vacuum Degasser Binary Pump Autosampler Thermostatted Column Compartment Detector
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
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.
Multichannel Gradient Valve • Determines mobile phase composition. • Largest solvent plug fills first. • Agilent 1100 and 1200 quaternary pump.
Dual Piston Parallel Pump Check Valves Rotary Switching Valve Pumphead Piston A B Combined Single Piston Delivery Delivery Piston 'A' Advancing Piston B Retracting
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.
Pump Seals and Pistons • Piston • Support Rings • Seal Keepers • Seals • Wear Retainers 1 2 3 4 5
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.
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.
Gradient Formation Low Pressure Gradient High Pressure Gradient
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
Summary Video Click picture to activate video.
Sample Injectors Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Two major designs: Automatic Injectors or Manual Injectors
Manual Injectors Sample Loop Load - Inject Front View Rear View Inject
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
Step 1 Step 2 Step 3 Automatic Injectors
Rotor Seal found within valve Rotor Seals
Column Oven Constant temperature for solvent and column is required to perform reproducible results.
Common HPLC Detectors • UV-VIS • Diode Array • Multiple Wavelength • Variable Wavelength • Mass Spectrometers • Refractive Index • Fluorescence • Light Scattering • Electrochemical • Radioactivity • Conductivity
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
HPLC Detector Characteristics Detector performance characteristics: • Sensitivity (LoD, LoQ) • Selectivity • Linearity • Qualitative information • Reliability • Ease of use • Universality
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.
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.
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 ).
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
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.
Absorbance Spectra Wavelength Time Online Spectra - UV-Vis Detector
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.
Refractive Index Detector Design The Refractive Index Detection is strongly influenced by: • Pressure changes • Temperature changes • Flow pulse Gradient elution is not possible!
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