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					High Performance Liquid Chromatography
K.J.C.Bose M.Sc.,M.Tech.,

HPLC
originally referred to: High Pressure Liquid Chromatography

HPLC
originally referred to: High Pressure Liquid Chromatography


high pressure to be able to use small particle size to allow proper separation at reasonable flow rates

HPLC
originally referred to: High Pressure Liquid Chromatography


high pressure to be able to use small particle size to allow proper separation at reasonable flow rates

currently refers to: High Precision Liquid Chromatography

Types of Liquid Chromatography

Types of Liquid Chromatography


Partition (liquid-liquid)

Types of Liquid Chromatography


Partition (liquid-liquid)  reverse-phase

Types of Liquid Chromatography


Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase

Types of Liquid Chromatography


Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase

Types of Liquid Chromatography


Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase

Types of Liquid Chromatography




Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase adsorption (liquid-solid)


used to separate relatively nonpolar species with MW < 5000

Types of Liquid Chromatography




Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase adsorption (liquid-solid)


used to separate relatively nonpolar species with MW < 5000



a particular strength of this method is that it resolve isomeric mixtures such as meta and para benzene derivatives much easier than other techniques

Types of Liquid Chromatography




Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase adsorption (liquid-solid)


used to separate relatively nonpolar species with MW < 5000



a particular strength of this method is that it resolve isomeric mixtures such as meta and para benzene derivatives much easier than other techniques



ion-exchange

Types of Liquid Chromatography




Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase adsorption (liquid-solid)


used to separate relatively nonpolar species with MW < 5000



a particular strength of this method is that it resolve isomeric mixtures such as meta and para benzene derivatives much easier than other techniques





ion-exchange gel-permeation


packing is hydrophobic-used to separate nonpolar species

Types of Liquid Chromatography




Partition (liquid-liquid)  reverse-phase  nonpolar stationary phase  normal-phase  polar stationary phase adsorption (liquid-solid)


used to separate relatively nonpolar species with MW < 5000



a particular strength of this method is that it resolve isomeric mixtures such as meta and para benzene derivatives much easier than other techniques





ion-exchange gel-permeation


packing is hydrophobic-used to separate nonpolar species packing is hydrophilic -used to separate polar species



gel-filtration


Applications of Liquid Chromatography

Effect of Particle Size and Flow Rate on Efficiency

Components
Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System Columns Temperature Control Detectors

Schematic of an apparatus for HPLC

Components


Solvent Reservoir


usually one or more glass or stainless steel reservoirs each of which contains 200-1000 ml of solvent

Components


Solvent Reservoir


usually one or more glass or stainless steel reservoirs each of which contains 200-1000 ml of solvent


isocratic elution - single solvent separation technique

Components


Solvent Reservoir


usually one or more glass or stainless steel reservoirs each of which contains 200-1000 ml of solvent
isocratic elution - single solvent separation technique  gradient elution - 2 or more solvents, varied during separation


Improvement in separation efficiency by gradient elution..

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector


vacuum pumping systems

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector
 

vacuum pumping systems distillation system

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector
  

vacuum pumping systems distillation system a system for heating and stirring the solvents

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector
   

vacuum pumping systems distillation system a system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility through the solvent

Components
Degassing System - its function is to remove
dissolved oxygen or nitrogen which interfere by forming bubbles the bubbles can cause band broadening and can interfere with the performance of the detector
    

vacuum pumping systems distillation system a system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility through the solvent *** can be done before solvent is added to the reservoirs

Components
Some “Requirements” for LC Pumping Systems are:

Components
Some “Requirements” for LC Pumping Systems are:


ability to mix solvents and vary polarity of mobile phase during run

Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir


Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir  generation of pressures up to 6000 psi


Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir  generation of pressures up to 6000 psi  flow rates ranging from 0.1 to 10 mL/min


Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir  generation of pressures up to 6000 psi  flow rates ranging from 0.1 to 10 mL/min  flow reproducibility’s of 0.5 % or better


Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir  generation of pressures up to 6000 psi  flow rates ranging from 0.1 to 10 mL/min  flow reproducibility’s of 0.5 % or better  resistance to corrosion by a variety of solvents


Components
Some “Requirements” for LC Pumping Systems are:
ability to mix solvents and vary polarity of mobile phase during run  “unlimited” solvent reservoir  generation of pressures up to 6000 psi  flow rates ranging from 0.1 to 10 mL/min  flow reproducibility’s of 0.5 % or better  resistance to corrosion by a variety of solvents  pulse-free output


Components
Three basic types of LC Pumps are:


pneumatic pumps motor driven syringe type pumps reciprocating pumps





Components
 pneumatic


pumps

 advantages

simple  inexpensive  pulse free
 disadvantages

limited capacity  limited pressure output (< 2000 psi)  not amenable to gradient elution


Components
Three basic types of LC Pumps are:


pneumatic pumps motor driven syringe type pumps reciprocating pumps





Motor driven syringe type pumps
 Advantages:
 simple  inexpensive

 pulse

free

 Disadvantages

capacity  not amenable to gradient elution

 limited

Components
Three basic types of LC Pumps are:


pneumatic pumps motor driven syringe type pumps reciprocating pumps





Reciprocating Pumps


Advantages
small internal volume  high output pressures (up to 10,000 psi)  readily adaptable to gradient elution  “unlimited” solvent reservoir




Disadvantages
produces a pulsed flow  expensive


A reciprocating pump for HPLC
The piston expels liquid through a one-way valve (check valve). The pumping rate is usually adjusted by controlling the distance the piston retracts, thus limiting the amount of liquid pushed out by each stroke, or by the cam rotating speed.

The main disadvantage of this type of pump is sinusoidal pressure pulsations which lead to the necessity of using pulse dampeners.

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A reciprocating pump for HPLC

A dual piston reciprocating pump for HPLC
Both pump chambers are driven by the same motor through a common eccentric cam; this common drive allows one piston to pump while the other is refilling.

A dual piston reciprocating pump for HPLC
Another type of dual piston pump uses only two check valves, but piston volumes are different. While the smaller piston dispenses an eluent in the HPLC system, the bigger piston is sucking an eluent. When pistons change their direction, the bigger piston simultaneously refill the smaller chamber and dispenses eluent into the system.

Gradient Controller


The gradient controller is the device that allows you to create a gradient program (vary the composition of the solvent during the run)
Gradients are produced differently for different types of pumping systems



Components
Sample Injection System
sample valve  syringe


Components
Sample Injection System
sample valve  syringe


A sampling loop for liquid chromatography

Figure 30.5 in textbook

Sampling Loop Systems for Liquid Chromatography


Both manual and automated valve systems are available A major limitation that the sample size is fixed by the volume of the sample loop. The loop must be changes in order to alter sample size.



Columns


straight, 15 to 150 cm in length; 2 to 3 mm i.d.


packing - silica gel, alumina, Celite

Components
Precolumns
remove impurities from solvent  saturates mobile phase with liquid of stationary phase before the analytical column


Components
Detectors
single wavelength UV  tuneable UV  diode-array  refractive index  MS


Performances of LC Detectors

Ultraviolet detector cell for HPLC

Diode-Array Absorption Spectra

Refractive-Index Detector
“Schematic of a differential refractiveindex detector.”


				
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posted:5/20/2009
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Description: HPLC.ppt