Hplc seminar

hplc seminar

1. HPLC CHROMATOGRAPHY Submitted to.- Dr . Bijender Singh Associate professor, department of biotechnology,cuh Presented by-Mahesh Kumar Roll.no.-191506 Deparment of biotechnology,cuh.

2. INTRODUCTION The term hplc is given by Csabe Horvath. This is the type of liquid chromatography. This is known as high performance liquid chromatography. Also known as high pressure liquid chromatography. It’s is used for identification, quantification and separation. It has two types- A.Normal phase HPLC B.Reverse phase HPLC

3. MODES OF HPLC Normal phase chromatography -This method separates analytes on the basis of polarity. Stationary phase –silica(Hydrophilic) Mobile phase(Non polar) –Hexane, chloroform. Reverse phase chromatography - Stationary phase –hydrophobic Mobile phase-polar Mixture 10%+water 90%

4. OBJECTIVE To understand the mechanism of purification, function and importance in analytical field. To validate the developed method as par ICH guidelines.

5. PRINCIPLE HPLC is based on adsorption as well as partition. When mixture of components are introduced into a Hplc column, they travel according to their relative affinities towards the stationary phase. The components which has more affinities towards the Adsorbent, travel slower. The components which has less affinities towards the stationary phase,travels faster.

6. COMPONENTS 1.Stationery phase-Here , column is used as stationary phase. This is a tube like structure. This is made up with stainless steel and plastic. The column is properly packed in which a solid Adsorbent material is contain. Example-Silica. 2.Mobile phase –This is a liquid solution where water is 90%and organic solvent is 10%. 3.Sample-it may be blood, bacteria or fungi and any plant material which we want to analyze.

7. INSTRUMENTATION Solvent reservoir. Degasser. Injector. Column. Oven. Detector. Computer system. Data processor. Recorder. Collecting tube. Pump. Gradient device.

8. FUNCTION OF INSTRUMENTS 1. Solvent reservoir-Here contains solvent and mobile phase. This is bottle like structure. 2.Degassor – this will remove dissolved gas ,and avoiding bubble formation. 3.Gradient device-this allows automatically generation of column before the separation. 4.Pump-it should be capable of delivery the mobile phase , pressure under 3000Psi to 5000Psi. 5.Sample injector-used to inject the sample. Range-0.1-100ml., pressure-400Psi. 6.Detector-used for detection of analyte. It may be –Ir detector,UV detector , electrochemical detector. 7.Data processor-this is send the data from detector to recorder. 8.Recorder-keep the record of exact value of separated molecules. Column-used as a stationary phase. This should be 300mm long. Diameter-2-5mm.

9. EXPERIMENTAL PROCEDURES 1.Firstly ,we pass the buffer through a column. We equilibrate the column to ready for purification. Here we don’t pass the sample,only pass buffer,after that, detector detects the signal to a computer system,and there will be developed a straight line . 2.After that,we load the sample with mobile phase,after that, detector detects the hydrophilic molecules first and on the system, there will generate a peak,this peak will be hydrophilic molecules. Detector is attached with a collecting tube. The elute will be hydrophilic molecules first time. Hydrophilic molecules will form a bond with resin . Resin in hydrophilic in nature. By this binding, Hydrophobic molecules move slow in the column. So for this problem,we will apply here concentration gradient. Here concentration gradient means that we will decrease the concentration of water and increase the concentration of organic solvent.

10. CONTINUOUS .... So, slowly-slowly polarity of mobile phase changes. As a result,bonded molecules elutes slowly-slowly and they pass out through column and detector detects the hydrophobic molecules and send the signal to the computer and this will generate the second peak . Eluted Molecules collected in collecting tube. Here , on computer system ,there will be formation of a graph. This is called chromatogram.

11. Here in this graph ,on the x-axis,there will be retention time and y-axis,there will be concentration or absorbence. By this graph ,we can calculate the area of peak. Peak area, generally tell as the concentration of molecules. which molecules have high peak area , concentration will be more those molecules. By this graph ,we can know that how the peak is quantified.what is the concentration of peak ,we can know by this graph. Here we also know about the retention time. Retention time-the amount of time it takes for the compound to pass through the column. By this graph ,we can also analyze the RRF value. RRF value-known amount of a component/size of the peak Also we determine the retention factor(k)- Time sollute spends in stationary phase /time solute spends in mobile phase

12. ADVANTAGES AND DISADVANTAGES Advantages- 1.High performance. 2.simple and fast. 3.Good accuracy and precision rate. 4.Automated procedure and data handling. 5.Repetative analysis using same column. Disadvantages- 1.High cost. 2.Co-elution is difficult. 3.Requring large quantities of expensive organics. 4.low sensitivity for certain compounds. 5.Not universal.

13. APPLICATION Used in Drug discovery. Used in water purification. To study environment issues. Drug metabolism study. Proteomics analysis. Used in food industry. Used in forensic purpose. Used in clinical industry. Used for cosmetic analysis. Used to determine structure. Determination of retention time.

14. CONCLUSION HPLC play a critical role in analysis of pharmaceutical products. The new technology in chemistry and instrumentation provides more and more information per unit of works as HPLC begins to fulfill the promise of increased speed, resolution and sensitivity predicted for liquid chromatography.

15. REFERENCE Alkesh Pradhan, chochin university of science and technology. School of industrial fisheries. www.water.com www.chemguide.co.uk www.science direct.com. hiq.linde-gas.com www.knaeur.net chem.libretexts.org

16. Thank you