HPLC

High Performance Liquid Chromatography FEROSEKHAN S FNB-41 CIFE MUMBAI

Liquid Chromatography

Elution chromatography
Increasing polarity of pure solvents
hexane
ether
acetone
methanol
water
acetic acid
Solvents mixed
%hexane and % methanol
miscible
can be mixed continuously (solvent programming)

Outline
What is HPLC?
An Overview
Types of HPLC
Partition Chromatography
Adsorption Chromatography
Ion Chromatography
Size-Exclusion Chromatography

What is HPLC?
The most widely used analytical separations technique
Utilizes a liquid mobile phase to separate components of mixture
uses high pressure to push solvent through the column
Popularity :
sensitivity
ready adaptability to accurate quantitative determination
suitability for separating nonvolatile species or thermally fragile ones

HPLC is….
Popularity:
widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public
Ideally suited for separation and identification of,
proteins,
amino acids
nucleic acids,
hydrocarbons,
carbohydrates,
pharmaceuticals,
pesticides,
pigments,
antibiotics,
steroids, and a variety of other inorganic substances

History
Early LC carried out in glass columns
diameters: 1-5 cm
lengths: 50-500 cm
Size of solid stationary phase
diameters: 150-200  m
Flow rates still low! Separation times long!
Decrease particle size of packing causes increase in column efficiency!
diameters 3-10  m
This technology required sophisticated instruments
new method called HPLC

Advantages to HPLC
Higher resolution and speed of analysis
HPLC columns can be reused without repacking or regeneration
Greater reproducibility due to close control of the parameters affecting the efficiency of separation
Easy automation of instrument operation and data analysis
Adaptability to large-scale, preparative procedures

Advantages to HPLC
Advantages of HPLC are result of 2 major advances:
stationary supports with very small particle sizes and large surface areas
appliance of high pressure to solvent flow

Instrumentation
Instruments required:
Mobile phase reservoir
Pump
Injector
Column
Detector
Data system

Schematic of liquid chromatograph

LC column LC injector

Most widely used
Bonded-phase Chromatography
Silica Stationary Phase:
OH OH OH OH
O O O
Si Si Si Si
Siloxanes: O CH 3
Si O Si R R= C 8 , C 18
O CH 3

Reverse Phase Chromatography
Nonpolar Stationary Phase
Polar Mobile Phase
Normal Phase Chromatography
Polar Stationary Phase
Nonpolar Mobile Phase
Column Selection
Mobile-Phase Selection

Applications
Parathion in Insecticides:
O
CH 3 CH 2 O P O NO 2
CH 3 CH 2 O

Adsorption Chromatography
Classic
Solvent Selection
Non-polar Isomeric Mixtures
Advantages/ Disadvantages
Applications

What is Ion Chromatography?
Modern methods of separating and determining ions based on ion-exchange resins
Mid 1970s
Anion or cation mixtures readily resolved on HPLC column
Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations

Size Exclusion Chromatography(SEC)
Gel permeation(GPC), gel filtration(GFC) chromatography
Technique applicable to separation of high-molecular weight species
Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products
Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase

Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules
larger molecules
smaller molecules
intermediate size molecules
SEC(continued)

SEC Column Packing
Small (~10 µm) silica or polymer particles containing a network of uniform pores
Two types (diameters of 5 ~ 10 µm)
Polymer beads
silica-based particles

The Mobile Phases are...
Aqueous solutions
containing methanol, water-miscible organic solvents
also contain ionic species, in the form of a buffer
solvent strength & selectivity are determined by kind and concentration of added ingredients
ions in this phase compete with analyte ions for the active site in the packing

Properties of the Mobile Phase
Must
dissolve the sample
have a strong solvent strength leads to reasonable retention times
interact with solutes in such a way as to lead to selectivity

Mobile phase reservoir
Glass/stainless steel reservoir
Removal of dissolved gases by degassers
vacuum pumping system
heating/stirring of solvents

Elution methods
Isocratic elution
single solvent of constant composition
Gradient elution
2 or more solvents of differing polarity used

Sample Injection Systems
For injecting the solvent through the column
Minimize possible flow disturbances
Volumes must be small
.1-500  L
Sampling loops
interchangeable loops (5-500  L at pressures up to 7000 psi)

Types of Detector
Refractive index
UV/Visible
Fluorescence
Conductivity
Evaporative light scattering
Electrochemical

Detectors
Refractive Index detector
All solutes
Measures change in RI of the mobile phase due to solutes
Carbohydrates and lipids

Refractive Index
Measure displacement of beam with respect to photosensitive surface of dectector

Refractive Index
Advantages
universal respond to nearly all solutes
reliable
unaffected by flow rate
low sensitive to dirt and air bubbles in the flow cell

Refractive Index
Disadvantages
expensive
highly temperature sensitive
moderate sensitivity
cannot be used with gradient elution

Detectors
UV-VIS absorption detector (190-800 nm)
Aromatics
Conjugated molecules

UV/Visible
Mercury lamp
Photocell measures absorbance

UV/Visible
Advantages
high sensitivity
small sample volume required
linearity over wide concentration ranges
can be used with gradient elution
Disadvantage
does not work with compounds that do not absorb light at this wavelength region

Fluorescence
For compounds having natural fluorescing capability
Fluorescence observed by photoelectric detector
Mercury or Xenon source with grating monochromator to isolate fluorescent radiation

Fluorescence
Advantages
extremely high sensitivity
high selectivity
Disadvantage
may not yield linear response over wide range of concentrations

Conductivity
Measure conductivity of column effluent
Sample indicated by change in conductivity
Best in ion-exchange chromatography
Cell instability

Data System
For better accuracy and precision
Routine analysis
pre-programmed computing integrator
Data station/computer needed for higher control levels
add automation options
complex data becomes more feasible
software safeguard prevents misuse of data system

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HPLC

by ferosecife on Mar 27, 2009

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HPLC — Presentation Transcript