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High Performance Liquid Chromatography:

High Performance Liquid Chromatography Presented by: Surya Narayana Parichha Dept. of Pharmaceutical Chemistry Govt. college of Pharmacy, Aurangabad. 3/12/2011 1


Outline Introduction Principle Theory Types of HPLC Instrumentation Application Advantages References 3/12/2011 2

What is HPLC?:

What is HPLC? The most widely used analytical separation technique Utilizes a liquid mobile phase to separate components of mixture uses high pressure to push solvent through the column Popularity : sensitivity ready adaptability for accurate quantitative determinations suitability for separating non-volatile species or thermally fragile ones 3/12/2011 3


Contd… Widespread industrial applicability. Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids and a variety of other inorganic substances. 3/12/2011 4

History lesson:

History lesson 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! Eureka(1960s)! Decrease particle size of packing causes increase in column efficiency! diameters 3-10 m This technology required sophisticated instruments new method called HPLC 3/12/2011 5


Principle: HPLC separates mixture of compounds on the basis of polarity. "Like attracts Like" i.e. if the column is non-polar the compound to elute first will be the most polar one. 3/12/2011 6


Theory: Plate Theory -series of discrete yet contineous horizental layers (theoretical plate). N=L/H Rate Theory -explains the effect of variables like mobile phase velocity and adsorbabilities. 3/12/2011 7

Classification of HPLC:

Classification of HPLC Normal phase chromatography Reverse phase chromatography Displacement chromatography Liquid-liquid(partition)chromatography Ion-exchange chromatography Size exclusion chromatography Bioaffinity chromatography 3/12/2011 8


Instrumentation Components required: Mobile phase reservoir Pump Injector Column Detector Data system 3/12/2011 9

Instrumentation contd…:

Instrumentation contd… 3/12/2011 10

Mobile phase reservoir:

Mobile phase reservoir Glass/stainless steel reservoir Removal of dissolved gases by degassers vacuum pumping system heating/stirring of solvents sparging vacuum filtration 3/12/2011 11

Pumping System:

Pumping System Provides a continuous constant flow of the solvent through the injector Requirements pressure outputs up to 6000 psi pulse-free output flow rates ranging from .1-10 mL/min flow control and flow reproducibility of .5% or better corrosion-resistant components 3/12/2011 12


Contd… There are three types of pumps used Reciprocating pump Usually consists of a small chamber in which the solvent is pumped by the back and forth motion of a morter driven piston Displacement pump These are the large syringe like chambers equipped with a plunger i.e activated by screw driven mechanism Pneumatic pump It is the simplest pumps mobile phase is contained in a collapsible container housed in a vessel that can be pressurized by compressed gas 3/12/2011 13

Sample Injection Systems:

Sample Injection Systems For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 L 3/12/2011 14

Liquid Chromatographic Column:

Liquid Chromatographic Column Smooth-bore stainless steel or heavy-walled glass tubing Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500) Add columns together to increase length 3/12/2011 15


Contd… Two types of column used Analytical column Guard column Column thermostats maintaining column temperatures constant column heaters control column temperatures z150 o C) columns fitted with water jackets fed from a constant temperature bath 3/12/2011 16

Detectors :

Detectors Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of analytes Non-destructive 3/12/2011 17

Types of Detector:

Types of Detector Refractive index UV/Visible Fluorescence Conductivity Electrochemical 3/12/2011 18

Refractive Index :

Refractive Index Measure displacement of beam with respect to photosensitive surface of dectector 3/12/2011 19


Contd… Advantages universal respond to nearly all solutes reliable unaffected by flow rate low sensitive to dirt and air bubbles in the flow cell Disadvantages expensive highly temperature sensitive moderate sensitivity cannot be used with gradient elution 3/12/2011 20


UV/Visible 3/12/2011 21


Contd… Advantages high sensitivity small sample volume required linearity over wide concentration ranges can be used with gradient elution Disadvantages does not work with compounds that do not absorb light at this wavelength region 3/12/2011 22


Fluorescence For compounds having natural fluorescing capability Fluorescence observed by photoelectric detector Mercury or Xenon source with grating monochromator to isolate fluorescent radiation 3/12/2011 23


Contd… Advantages extremely high sensitivity high selectivity Disadvantages may not yield linear response over wide range of concentrations 3/12/2011 24


Conductivity Measure conductivity of column effluent Sample indicated by change in conductivity Best in ion-exchange chromatography Cell instability 3/12/2011 25

Electrochemical :

Electrochemical Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current 3/12/2011 26


Contd… Advantages high sensitivity ease of use Disadvantages mobile phase must be made conductive mobile phase must be purified from oxygen, metal contamination, halides 3/12/2011 27

Data System:

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 3/12/2011 28

Elution methods:

Elution methods Isocratic elution single solvent of constant composition Gradient elution 2 or more solvents of differing polarity used 3/12/2011 29

Advantages of HPLC:

Advantages of 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 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 3/12/2011 30


Precautions…. Reversed Phase to Normal phase Reversed Phase : Buffered Aqueous methanol Flush : 50: 50 Methanol/Water, Methanol then IPA Normal Phase : Hexane/Ethyl Acetate. * Normal to reversed Phase : Normal Phase : Hexane/Ethyl Acetate Flush : IPA then MethanolFinally 50:50 Methanol/Water ReversedPhase:Buffered Aqueous Methanol. 3/12/2011 31

Applications :

Applications To analyze and identify the compounds For determination of purity of compounds. To quantify the individual components from a mixture of compounds. 3/12/2011 32

References :

References Chatwal G.R.,[Instrumental Methods Of Chemical Analysis],Himalaya Publishing House, Fifth edition 2007. Skoog -Holler- NieMan [Principle of Instrumental Analysis], Harcourt College Publisher, Fifth edition M.W. Dong, Modern HPLC for practicing scientists. Wiley, 2006. L. R. Snyder, J.J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, John Wiley & Sons, New York, 1997. S. Ahuja and H. T. Rasmussen (ed), HPLC Method Development for Pharmaceuticals, Academic Press, 2007


THANK YOU 3/12/2011 34

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