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Hplc
- 2. •HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High- pressure liquid chromatography). •The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett •By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. INTRODUCTION neethu asokan
- 3. •High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase. •HPLC is a chromatographic technique that can separate a mixture of compounds •It is used in analytical chemistry,Biotechnilogy,Micobiology and Biochemistry to identify, quantify and purify the individual components of a mixture. neethu asokan
- 4. The principle of separation in normal phase mode and reverse phase mode is adsorption. When a mixture of components are introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no 2 components have the same affinity towards the stationary phase, the components are separated PRINCIPLE: neethu asokan
- 5. TYPES OF PHASES Mobile Phase Stationary Phase Solvent Bonded Phase Separation is based on the analyte’s relative solubility between two liquid phases neethu asokan
- 6. Normal Phase. - Polar stationary phase and non-polar solvent. Reverse Phase. - Non-polar stationary phase and a polar solvent. neethu asokan
- 7. Components Of A Liquid Chromatograph System • Mobile Phase / Solvent Reservoir • Degasser • Solvent Delivery System (Pump) • Injector • Pre-column • Column (Stationary phase ) • Detectors • Recorder (Data Collection) neethu asokan
- 8. Instrumentaion 0f HPLC 1. Mobile phase reservoir: • Commonly glass bottles with caps are used. • Teflon tubings and filters are connected to purge gas (helium) for degassing. • Vaccum for 5-10 min is also used for degassing. 2. Pump: • It forces the mobile phase to pass through column. • Flow rate is 1-2 ml/ min. • Trypical pressure is 6000 – 9000psi. neethu asokan
- 9. Instrumentaion 0f HPLC 3. Injector: Can be manually (syringe) or automated. Sample volume 5-20µl. Ideal to stand pressure of mobile phase. Autosampler is used for analysis of many samples automatically. 4. Stationary phase (Column): Heart of HPLC. Separate sample components on basis of physical and chemical parameters. Lenght 10-30cm. Diameter 4-10nm. Packing material 5-10nm thick.neethu asokan
- 10. 5. Detector: Detection of elutes from column. Detectors used depends upon the property of he compounds to be separated. Different detectors available are: 1. Refractive index detectors 2. U.V detectors 3. Fluorescence detectors 4. Electro chemical detectors Computer: Data system that controls modules of HPLC. Signals from detector are interpreted to determine elution time, quantitative and qualitative analysis of sample. neethu asokan
- 11. 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 Sampling loops interchangeable loops (5-500 L at pressures up to 7000 psi) neethu asokan
- 12. Direct injection auto sampler from Pump from Pump to Column Vial Needle Measuring Pump to Column LOAD INJECT neethu asokan
- 13. Smooth-bore stainless steel or heavy-walled glass tubing Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500) India 13,000- 33,000 Add columns together to increase length hpLc Column neethu asokan
- 14. HPLC Basic Instrumentation Mobile phase Pump Solvent Delivery Injector Sample Injection Column Separation Detector Data Processor neethu asokan
- 15. Separations Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste neethu asokan
- 16. SeparationsInjector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph neethu asokan
- 32. The Chromatogram Injection to tR mAU time tR to - elution time of unretained peak tR- retention time - determines sample identity Area or height is proportional to the quantity of analyte. tK = neethu asokan
- 33. neethu asokan
- 34. HPLC system Mobile Phases Flow Rate Composition Injection Volume Column Oven Temperature Detectors neethu asokan
- 35. ADVANTAGES OF HPLC Needs a small sample with a high accuracy and precis Non-destructed sample during operation compared to GC. Separation is fast and efficient Separation &analysis of very complex mixture Separation components can be easily collected Determination of multiple components in a single analysis neethu asokan
- 36. DISADVANTAGES OF HPLC Need a skill to run the instruments High cost neethu asokan
- 37. Chemical Environmental Pharmaceuticals Consumer Products Clinical polystyren es dyes phthalates tetracyclines corticosteroids antidepressants barbiturates amino acids vitamins homocysteine Bioscience proteins peptides nucleotides lipids antioxidants sugars polyaromatic hydrocarbons Inorganic ions herbicides APPLICATIONS neethu asokan
- 38. APPLICATIONS: HPLC is one of the most widely applied analytical separation techniques. Pharmaceutical: Tablet dissolution of pharmaceutical dosages. Shelf life determinations of pharmaceutical products. Identification of counterfeit drug products. Pharmaceutical quality control. neethu asokan
- 39. APPLICATIONS Environmental Phenols in Drinking Water. Identification of diphenhydramine in sediment samples. mountain lakes through the analysis of fish bile. Estrogens in coastal waters - The sewage source. Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria. Assessment of TNT toxicity in sediment.neethu asokan
- 40. Forensics A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy. Identification of anabolic steroids in serum, urine, sweat and hair. Forensic analysis of textile dyes. Determination of cocaine and metabolites in meconium. Simultaneous quantification of psychotherapeutic drugs in human plasma. neethu asokan
- 41. Clinical Quantification of DEET in Human Urine. Analysis of antibiotics. Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis. Detection of endogenous neuropeptides in brain extracellular fluids. neethu asokan
- 42. APPLICATION Food and Flavor Ensuring soft drink consistency and quality. Analysis of vicinal diketones in beer. Sugar analysis in fruit juices. Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits. Trace analysis of military high explosives in agricultural crops. Stability of aspartame in the presence of glucose and vanillin. neethu asokan