HPLC Instrumentation In This Section We Will Discuss

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. 2

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

Solvent Filters Guard column Injector Precolumn Filter Analytical Column Solvent Inlet Filter Solvent Inlet Filer · Stainless Steel or glass with 10 micron porosity. · Removes particulates from solvent. Precolumn Filter · Used between the injector and guard column. · 2 to 0. 5 micron · Removes particulates from sample and autosampler wear debris. · Must be well designed to prevent dispersion. 4

Vacuum Degassing 5

Functions of the Solvent Delivery System The solvent delivery system has three basic functions: 1. Provide accurate and constant flow. 2. Provide accurate mobile phase compositions. 3. Provide the force necessary to push the mobile phase through the tightly packed column. 6

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

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

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. 9

Ballvalves for Reciprocating Piston Pumps 10

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

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

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. 13

Gradient Formation Low Pressure Gradient High Pressure Gradient 14

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 15

Summary Video Click picture to activate video. 16

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

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

Manual Injectors Sample Load 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

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

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

Necessity for More Than One Detector Sensitivity 24

Necessity for More Than One Detector Selectivity 25

Necessity for More Than One Detector Qualitative Information Chlortoluron ? Take peak spectrum (UV) Atrazine ? Take peak spectrum (MS) 200 58 215 44 172 68 96 104 60 Wavelength (nm) 80 100 120 132 138158 140 160 180 200 220 Mass/Charge 26

HPLC Detector Characteristics Detector performance characteristics: · Sensitivity (Lo. D, Lo. Q) · Selectivity · Linearity · Qualitative information · Reliability · Ease of use · Universality 27

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. Peak Noise 28

Response Limit of Detection - Limit of Quantitation Linear range Slope = sensitivity MQL MDL Intercept e. g. , RSD<10%, S/N > 20 e. g. , S/N > 3 Amount · 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. 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. Detector Flow Cell c I 0 I b Log I 0 = A = abc I Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ). 30

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

UV-Vis Detector with Spectral Capability Vis Lamp Achromatic Lens UV Lamp Detector Flow Cell Diode Array Homium Filter Optical Slit · · Grating 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. 32

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

Fluorescence Detection 34

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

HPLC-MSD API- Electrospray 36

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

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