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  3. 3. INTRODUCTION (HISTORY)  In early 1900`S Mikhail .Tswett a botanist has invented the chromatographic technique  He termed this system of colored bands as the chromatogram and method as chromatography (i.e; chroma -color and graphos -writing )  In 1941 martin and synge introduced partition and paper chromatography and also laid the foundation for gas liquid chromatography and high performance liquid chromatography in the year 1952  Classic chromatography was slow rate for analysis which lead to delay and loss of resolution by diffusion this problem was largely overcome by hplc  In the year 1969 a very marketed revival of interest in the technique of liquid chromatography because of development of HPLC by Kirkland and Huber (waters corporation and named ALC 100 hplc)
  4. 4. CHROMATOGRAPHY: It is defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases. HIGH PRESSURE LIQUID CHROMATOGRAPHY: high performance liquid chromatography is chromatographic instrumental technique of analytical chemistry.(to separate ,identify and quantify the each components)
  5. 5. TYPES OF HPLC TECHNIQUES  Based on modes of chromatography a. b. Normal phase mode Reverse phase mode  Based on principle of separation a. f. Adsorption chromatography Ion exchange chromatography Ion pair chromatography Size exclusion or gel permeation chromatography Affinity chromatography Chiral phase chromatography  Based on scale of operation a. Analytical hplc Preparative hplc b. c. d. e. b.
  6. 6. High pressure liquid chromatography
  7. 7. PRINCIPLE The principle of separation in normal phase mode and reverse phase mode is absorption. When a mixture of components are introduced in the hplc column they travel according to their relative affinities towards the stationary phase. the components which has more affinity towards the adsorption travel s slower. The components which has less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase ,the components are separated.
  8. 8. INSTRUMENTATION  A solvent reservoir and mixing system  A high pressure pump  A sample inlet pump  A column  A detector and recording unit
  9. 9. A SOLVENT RESERVIOR 1. 2. 3. 4. The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with the special caps The choice of mobile phase to be used in any separation will depend on the type of separation to be achieved Isocratic separation may be made with a single solvent or two or more solvents, where as gradient elution system may be used where developing solvent is continuously changed . All solvents for use in hplc system must be specially purified by the sparing process for degassing of aqueous solvents
  10. 10. HIGH PRESSURE PUMPS 1. Pumps performance directly affects the retention time reproducibility and detector sensitivity 2. The main feature of a good pumping system is that it is capable of output of at least 3.4*107 pa(5000 p.s.i) 3. Flow delivery must be at least 10 Cm3 / min for normal analysis Four types of pumps a. Pneumatic pumps b. Syringe type pumps c. Reciprocating pumps d. Hydraulic amplifier pumps
  11. 11. SYRINGE TYPE PUMPS    It works on the principle of positive solvent displacements by a piston mechanically driven at a constant rate of about 250-500ml capacity . They flush solvents simply by a push button action of a purge valve. Double syringe pumps also can be used in which one piston delivering solvent to column while the other is refilling from the reservoir Advantage–potentially quit stable floe delivery because no check valve has to be actuated during an analysis.
  12. 12.  RECIPROCAL TYPE PUMP It employs small volume chamber with reciprocating pistons to work directly on the solvent or diaphragm,pressurises the liquid from solvent reservoir.
  13. 13. SAMPLE INJECTING SYSTEM  A good injecting system should have the smallest possible contribution to peak broadening .The injection system should be I. Convenient to use II. Able to operate at high pressures III. Chemically inert with the eluent and the sample IV. Reproducible  There are 3 important ways of introducing the sample into the injection port
  14. 14. 1. FIXED VOLUME INJECTION :In this a fixed volume is introduced by making use of a fixed volume loop injector. The sample loaded by means of load syringe 2. VARIABLE INJECTION VALVE INJECTION :A variable volume is introduced by making use of an injection valve .Valve injectors contain a needle port which can be closed or sealed at high pressure to insert the syringe then the sample moves to column 3. COLUMN INJECTION : In this ,a syringe is used to inject the sample through an inert septum directly into the mobile phase. Commercially high pressure syringe can inject upto10-20mpa
  15. 15. THE COLOUMN a. The columns are generally made of stainless steel. b. Withstand pressure of 5.5*107pa 8000p.s.i c. Straight columns of 20to50cm in length and 1-4 mm in diameter d. Porous plug of stainless steel or Teflon are used in the ends of the columns to retain the packing material.
  16. 16. COLUMN PACKING MATERIALS  There are three forms a. Microporous- they ramify through particles which are 5 to 10micromt eg :silica ,alumina, lichrosorb-si ,micropak-NH2. b. Pellicular –particle are coated onto an inert solid core (glass bead )of 40 mm. eg: inactive silica , zipax,active silica corasil, polymer coated , permaphase ODS c. Bonded phases -stationary phase is chemically bonded onto an inert support
  17. 17. DETECTOR SYSTEMS  As the quantity of material applied is very small its is imperative that sensitivity is high and stable.  It response is usually presented as a record trace displaying the components as peak on time scale.  Mainly two type Bulk property detectors: Refractive index and conductivity detector Solute property detectors: UV , visible absorption ,fluorescence and electrochemical detector.
  18. 18. RECORDERS AND INTEGRATORS  Recorders are used to record the responses from detectors after amplification .They record the base line and all the peaks obtained with respect to time  Integrators are improved version of recorders with some data processing capabilities  Now a days computers and printers are used for recording process.
  20. 20. ADVANTAGES 1. Separation fast and efficient 2. Can be applied to the separation and analysis of very complex mixtures 3. Accurate quantitative measurements 4. Repetitive And reproducible analysis using the same column 5. Both aqueous and non aqueous samples can be analysed with little or no sample pretreatments 6. A variety of solvents and column are available providing a high degree of selectivity for specific analysis 7. separated components can be easily collected and isolated from the mobile phase for further analysis or characterization.
  21. 21. APPLICATIONS 1. Purification of biological molecules 2. Separation of compound such as drugs and their metabolites ,peptides , vitamins 3. Separation of high polar compounds such as amino acids ,organic ,acids and catecholamine's. 4. Dissolution of pharmaceutical dosages. 5. Shelf life determinations of pharmaceutical products. 6. Identification of counterfeit drug products. 7. Pharmaceutical quality control.
  22. 22. REFERENCE  Instrumentation methods of chemical analysis by Gurdeep r. Chatwal and Sham K. Anand. -Himalayapublishinghouse(revised fifth edition)  AInstrumental methods of chemical Analysis by B.K.Sharma. -GOEL Publishing house 27th edition  Text book of pharmaceutical analysisi by Dr.S.Ravi Sankar -Rx publications third edition  ms&tbm=isch&sa=X&ei=YThlUviSPITWrQfiz4HgCg&ved=0CAcQ_AUoAQ& biw=1024&bih=677#q=sample+injector+in+hplc  