HPLC 4
HPLC 4
HPLC 4
General components of a high performance liquid chromatograph. HPLC solvent delivery systems. How automatic injectors work. Common HPLC detectors.
Solvent Filters
Analytical Column
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.
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Vacuum Degassing
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.
Determines mobile phase composition. Largest solvent plug fills first. Agilent 1100 and 1200 quaternary pump.
Piston B Retracting
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.
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1 2 3 4 5
1. 2. 3. 4. 5.
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Purge valve
PTFE Frit
Frits, Filters, and Sieves are used to protect other parts of the LC from pump and seal material.
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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.
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Gradient Formation
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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
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Summary Video
Sample Injectors
Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Two major designs: Automatic Injectors or Manual Injectors
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Manual Injectors
Sample Loop
Load - Inject
Front View
Rear View
Inject
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Manual Injectors
Sample Load
From Pump To column
Solvent in Solvent out Sample in
Sample in
Sample Inject
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Automatic Injectors
Step 1
Step 2
Step 3
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Rotor Seals
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Column Oven
Constant temperature for solvent and column is required to perform reproducible results.
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WL 241/394
Py re ne C hr ys en e
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
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UV signal FL signal
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Wavelength (nm)
Mass/Charge 26
Detector performance characteristics: Sensitivity (LoD, LoQ) Selectivity Linearity Qualitative information Reliability Ease of use Universality
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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
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Slope = sensitivity
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.
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UV-Vis Detectors
Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beers Law.
Detector Flow Cell
I0
c b
Log I0 = A = abc
I
Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).
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UV Lamp
Mirror 1 Grating
Single wavelength detection of multi wavelength detection possible. Wavelength calibration is done automatically using a holmium filter.
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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.
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Spectra
Wavelength
Time
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Fluorescence Detection
Trigger pack
Mirror
Diffuser
Electrochemical Detectors
Thin-layer design
Wall-jet design
Gold for carbohydrates. Platinum for chlorite, sulfate, hydrazine, etc. Carbon for phenols, amines. Silver for chloride, bromide, cyanide.
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Conductivity Detectors
Schematics
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Applications
water
cell C
A ref.capacitor
variable resistances
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