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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
• Must be well designed to prevent dispersion. Precolumn Filter • Used between the injector and guard column. 4 .5 micron • Removes particulates from sample and autosampler wear debris. • 2 to 0.Solvent Filters Guard column Injector Precolumn Filter Solvent Inlet Filter Analytical Column Solvent Inlet Filer • Stainless Steel or glass with 10 micron porosity. • Removes particulates from solvent.
Vacuum Degassing 5 .
Provide the force necessary to push the mobile phase through the tightly packed column.Functions of the Solvent Delivery System The solvent delivery system has three basic functions: 1. Provide accurate and constant flow. 3. 2. 6 . Provide accurate mobile phase compositions.
Multichannel Gradient Valve • • • Determines mobile phase composition. 7 . Largest solvent plug fills first. Agilent 1100 and 1200 quaternary pump.
Dual Piston Parallel Pump Check Valves Pumphead Piston A B Rotary Switching Valve Single Combined Piston Delivery Delivery Piston 'A' Advancing Piston B Retracting 8 .
9 . Provides constant flow and the pressure necessary to get through column.Dual Piston in Series Pump First piston displaces solvent at twice the speed and stroke volume of the second piston.
Ballvalves for Reciprocating Piston Pumps Gold Seal Sapphire Insert Ruby Ball Spring Insert 10 .
Piston Support Rings Seal Keepers Seals Wear Retainers 11 .Pump Seals and Pistons 1 2 3 4 5 1. 2. 5. 4. 3.
12 . and Sieves are used to protect other parts of the LC from pump and seal material.Frits and Filters Purge valve PTFE Frit Frits. Filters.
13 .Damping Units Damping Unit Pump Ripple 2% P/P Pressure • 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 14 .
•Performance parameters for HPLC pumps: Flow Precision Flow Range Delay Volume Pressure Pulse Composition Precision 15 .Summary •The pump is the most critical piece of equipment for a successfully operating HPLC.
16 .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 17 .
Inject Front View Rear View Inject 18 .Manual Injectors Sample Loop Load .
Manual Injectors Sample Load From Pump To column Solvent in Solvent out Sample in From Pump To column Solvent in Solvent out Sample in Sample Inject 19 .
Automatic Injectors Step 1 Step 2 Step 3 20 .
Rotor Seals Rotor Seal found within valve 21 .
22 .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 23 .
WL 241/394 Py re ne C hr ys en e Necessity for More Than One Detector Sensitivity PAH's extracted from soil.LC-PAH 150x4.: H2O/CH3OH= 10:90 WL 270/388 WL 248/411 Be nz o( Pe e)p yr ry e le ne ne Be nz o( k) f lu or Be an nz th o( en a) e py re ne WL 302/420 WL 247/504 Be nz o( gh i)p er In de no ( yle ne 12 3c d) py re ne Fluorescence UV-signal 24 .6mm. Sup. Solv.
8mg/l. 20ul injected UV and fluorescence signal 25 .Necessity for More Than One Detector Selectivity Flecainide in Serum UV signal FL signal Therapeutic concentration: 1.
Necessity for More Than One Detector Qualitative Information Qualitative Information Chlortoluron ? Atrazine ? Take peak spectrum (UV) Take peak spectrum (MS) 200 58 215 44 68 96 104 60 80 100 120 172 132 138158 140 160 180 200 220 Wavelength (nm) Mass/Charge 26 .
HPLC Detector Characteristics Detector performance characteristics: • Sensitivity (LoD. LoQ) • Selectivity • Linearity • Qualitative information • Reliability • Ease of use • Universality 27 .
Peak Noise 28 .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.
Limit of Detection .Limit of Quantitation Response Linear range Slope = sensitivity MQL MDL Intercept e. not only the detector performance • Limit of quantification (LOQ) is a defined limit for a method used for a specific purpose. S/N > 20 e.g. S/N > 3 Amount • Limit of detection (LOD) is a result of the whole chromatography system.RSD<10%..g. 29 ..
UV-Vis Detectors Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. good stability. Concentration Sensitive. Special: UV-Vis Spectral capability (Diode Array Technology ). gradient capability. Detector Flow Cell I0 c b I Log I0 = A = abc I Characteristics: Specific. 30 .
UV-Vis Detectors . • Wavelength calibration is done automatically using a holmium filter. Flow cell Mirror 2 Reference diode 31 .Design Principles UV Lamp Cut-off filter Holmium oxide filter Slit Variable Wavelength Detector Sample diode Mirror 1 Grating • Single wavelength detection of multi wavelength detection possible.
Wavelength Resolution: Up to 1 nm. Wavelength calibration with Holmium oxide filter. 32 . Wavelength range 190 .950 nm.UV-Vis Detector with Spectral Capability Vis Lamp Achromatic Lens UV Lamp Homium Filter Optical Slit Grating Diode Array Detector Flow Cell • • • • Diode Array UV-Vis Detector allows online measurement of spectra.
Online Spectra .UV-Vis Detector Absorbance Spectra Wavelength Time 33 .
auto-recognition 34 .Fluorescence Detection Trigger pack Xenon flash Lamp. signal & spectra mode Lens (condensor EX) Slit EM Slit EX Emission Monochromator signal & spectra mode Slit PMT PMT detector Lens (condensor EM) Mirror Diffuser Reference Diode 8 µl Flow Cell. 15 W Exitation Monochromator.
Electrochemical Detectors Thin-layer design • • • • Wall-jet design Porous flowthrough design Gold for carbohydrates. cyanide. hydrazine. etc. 35 . bromide. Silver for chloride. amines. Platinum for chlorite. Carbon for phenols. sulfate.
Electrospray 36 .HPLC-MSD API.
Refractive Index Detector Design The Refractive Index Detection is strongly influenced by: Pressure changes Temperature changes Flow pulse Gradient elution is not possible! 37 .
Light Scattering Detector 38 .
capacitor D E } soft drinks blood plating baths nuclear fuel reprocessing streams variable resistances ~ B 39 .Conductivity Detectors Schematics F Applications water cell C r fixed resistor Balance control Ions Acids in Bases Salts soap products detergents A ref.