high performance liquid chromatography instrumentation
TRANSCRIPT
High Performance Liquid Chromatography
Instrumentation
solventsolvent
pumppump
injectorinjector
columncolumn
detectordetector
HPLC Instrument
Schematic of HPLC Instrument
Mobile phase Quality
High purity
Reasonable cost (and disposal)
Boiling point 20-50 °C above column temperature
Low viscosity
Low reactivity
Immiscible with stationary phase
Compatible with detector
Safety – limited flammability and toxicity
Mobile phase selection
k’ of 2-5 for two or three component mixturek’ of 0.5-20 for multicomponent mixtureMatch analyte polarity to stationary phase polarityMobile phase of different polarity
Normal Phase:
nonpolar solvent, polar stationary phase least polar component elutes first increasing mobile phase polarity decreases elution time
Reversed Phase:
polar solvent (water, MeOH, ACN), nonpolar stationary phase most polar component elutes first increasing mobile phase polarity increases elution time most widely used
Solvent Treatment Systems
For Dissolved gases (Irreproducible flow rates and band broadening)
Degassers
i. Vacuum system and distillating system.ii. Sparging system
(inert gas insoluble in mobile phase)
For Dust and particulate matter(Damage the pumping or injection system and clog the column)
Filtering the mobile phase through millipore filters.
Solvent Reservoir and Elution
Elution :
Isocratic : Elution with a single or mixed solvents of constant composition.
Gradient Elution: Elution of mixed solvents, with different polarities with composition varied with time.
HPLC instruments equipped with proportioning valves to introduce solvents from different reservoirs.
The ratio is preprogrammed before elution.
Pumping Systems
Why pressure?
The typical particle sizes in HPLC is 3-10 μm. In order to achieve flow rates of 0.5 to 5 mL/min, for a 10-30 cm column, pressures of 1000 to 6000 psi are required.
Requirements for HPLC pumping system
pressures to 6000 psipulse free outputcontrol flow rate from 0.1 to 10 mL/minresistance to corrosion by solvents
Different types of pumping Systems
Displacement syringe pump Pulse free Small capacity (250ml)
Only for isocratic elution.
Reciprocating pumps Small internal volume High output pressure Adaptable for gradient elution Large capacity Constant flow rate
Pump used in most commercial design.
Reciprocating pumps
www.lcresources.com/resources/getstart/2b01.htm
Disadvantage: Causes pressure pulses which leads to
i. poor quatitative analysis
ii. detection problems.
Solution
i. Dual pump system
ii. Pulse dampers ( act as shock absorbers)
Injection ( Sampling Valves)
Six-port sample injection system is used. It takes
i. Small amount of sample (≤ 500µl of sample)
ii. In a pressurized system
Injection ( Sampling Valves)
http://www.restek.com/info_sixport.asp
Guard Column ( Pre-column)
Prevents the contamination of the expensive analytical columns with fine particles that can eventually clog the mobile phase flow.
Porous stainless steel column(0.5 -2µm)
Composition same as analytical column.
Particle size is large to minimize pressure drops.
When contaminated, discarded and replaced by new one.
Analytical Column
Generally made of stainless steel or teflon components
10-30 cm long x 4-10 mm internal diameter
Packing usually 5 or 10 µm diameter
Analytical Column Packaging
Pellicular
Spherical, nonporous, glass or polymer beads 30-40- µm diameter Thin porous layer of silica, alumina, or ion-exchange resin deposited on surface
Porous
Most common 3-10- µm diameter Silica (most common), alumina, or ion-exchange resin Thin organic film bonded to surface
solventsolvent((mobile phasemobile phase))
andandsamplesample
wax coated beadswax coated beads
HPLC ColumnHPLC Column
to detectorto detector
Normal vs Reverse phase
nonpolarnonpolarstationary phasestationary phase
3 mmicroscopic view of beadmicroscopic view of bead
Reverse phase(C4, C8 or C18): Non-polar stationary phase
Normal phase: Polar stationary phase
Detectors
Ideal Characteristic of a detector
Adequate sensitivity
Good stability and reproducibility
Linear response to solutes
Short response time
Response to all solutes in a mixture
Non-destructive
Temperature stability
Types of Detectors
Bulk property Detectors (Mobile
phase property)Refractive index
Density
Dielectric constant
Electrochemical
Solute property DetectorsUV-Visible
Fluorescence
Diode array
Mass spectrometry
Diode array 3D plot