Prep hplc 1

• 1.Presented By : Miss. Bharti G. Jadhav. (First Year M. Pharm.) (Quality Assurance.) Under Guidance of : Mr. Abhay R. Shirode. (Assistant Professor.) Bharati Vidyapeeths College of Pharmacy, C.B.D., Belapur, Navi Mumbai- 400614. PREPARATIVE HIGH PRESSURE LIQUID CHROMATOGRAPHY.

2. 1. Introduction to chromatography. 2. Classification of column chromatographic methods. 3. Preparative chromatography. 4. Preparative HPLC. 5. Objectives. 6. Instrumentation. 7. Method development and optimisation. 8. Applications. 9. Hyphenation with other analytical chromatographic techniques.(Review of reported studies) 10. Commercially available instruments for preparative high pressure liquid chromatography. HIGHLIGHTS: 3. Chromatography is a powerful separation method. The term chromatography( Greek word Chroma= Color and Graphein= To write) meaning color writing. INTRODUCTION TO CHROMATOGRAPHY: What is mean By Chromatography 4. General Classification Specific Method Stationary Phase Type Of Equilibrium Liquid chromatography ( Mobile Phase: Liquid) Liquid-Liquid Liquid adsorbs on solid Partition between immiscible liquids Liquid bonded phase Organic species bonded to a solid surface Partition between liquid and bonded surface Solid-Liquid Solid Adsorption Ion exchange Ion exchange resin Ion exchange Size exclusion Liquid in interstices of a polymeric solid Partition Gas chromatography (Mobile Phase: Gas) Gas-Liquid Liquid adsorbs on solid Partition between gas and liquid Gas bonded phase Organic species bonded to solid surface Partition between liquid and bonded surface Gas-Solid Solid Adsorption Supercritical Fluid chromatography (Mobile Phase: Supercritical Fluid) Organic species bonded to solid surface Partition between supercritical fluid and solid surface. Table 1: Types of Column Chromatographic Methods. 5. Powerful technique for the isolation and purification of variety of chemicals, pharmaceutical compounds, natural products and biological molecules. To increase throughput and separation power, the first preparative HPLC system was developed in the 1970s. Types of HPLC: Based on the scale of operation. PREPARATIVE CHROMATOGRAPHY: 6. Analytical HPLC. Preparative HPLC. 1. Sample goes from detector into waste. 1. Sample goes from detector into fraction collector. 2. Use quantification and/or identification of compounds. 2. Use for isolation and/purification of compounds. 3. Column has internal diameter-1-5mm 3. Column has internal diameter-1-10cm 4. Column particles are 5um or smaller. 4. Column particles are 7um or larger. 5. HPLC pump provide up to 10mL/min. 5. HPLC pump provide >>10mL/min. 6. Solubility of sample in mobile phase usually not important. 6. Solubility of sample usually very important. 7. Mobile phase is not recover. 7. Mobile phase recovery is possible. Table 2: Difference Between Analytical And Preparative HPLC. 7. PRINCIPLE: Adsorption. Similar to HPLC. The only difference is sample goes from detector into fraction collector. PREPARATIVE HPLC: 8. OBJECTIVES OF PREPARATIVE HPLC Purity Yield Throughput 9. 1. Solvent reservoir 2. Pump 3. Preparative injector 4. Preparative columns 5. Detectors 6. Programmer 7. Recycle valve 8. Fraction collector. Figure 2 : Instrumentation Of Preparative HPLC. INSTRUMENTATION: 10. 1. SOLVENT RESERVOIR: Material of construction: Glass or stainless steel For biologically sensitive, or labile substances: Coating of biocompatible material. 11. 2. PREPARATIVE PUMP: Figure 3: Dual Preparative Pump. Figure 4: Industrial Preparative System. Requires high eluent flow rate 10 and 100 ml/min and large internal diameter of columns. A larger piston head is required to work at flows of 10- 100ml/min. 12. Should inject sample within the range of 0.1 to 100 ml. Rheodyne injector is used. 3. PREPARATIVE INJECTOR: Figure 6: Positions Of Rheodyne Injector. Figure 5: Rheodyne Injector. 13. Column is the heart of the liquid chromatography. Sample distribution plate is used to distribute the sample across the column. It consists of a disc with series of radial slots. 4. PREPARATIVE COLUMNS: Figure 7: A typical Preparative Column 14. Scale Column I.D. (mm) Quality of Product Typical Column Length (mm) Purpose Analytical 4.6 1 - 40mg 250 Biological materials for activity testing Semi-Prep 10 - 30 100mg - 3g 250 Reference compounds Preparative 50 - 70 5 - 10g 250 - 1000 Intermediates for lab synthesis Pilot 100 - 300 20g - 5kg 300 - 1000 Pharmaceutical development Process >300 kg - tons 500 - 1000 Large scale production 15. Fig: Largest column in preparative HPLC with 4000 mm x 1600 mm i.d. 16. It depends on the particle size, scale of the separation and on the nature of the material to be separated. There are two type of Column Packings. Particle size more than 20mm- Dry Packing. Particle size less than 20mm- Slurry Packing. Packing of Preparative Columns: Packing of preparative columns 17. Once column diameter approaches 5 cm, additional difficulties arises. Formation of channels/bridges in the column bed. Chances of friction between particles and wall of the column. Figure 9: Difference between Analytical and Preparative Column. Difficulties Aries During Packing of Analytical Column 18. Techniques For Preservation of Column Radial compression Packing Technique. Longitudinal Compression Packing Technique. For preservation of column bed, two techniques are used: 19. Figure 10: Radial Compression Packing Technique. Figure 11: Longitudinal Compression Packing Technique. 20. COMMERCIALLY AVAILABLE PREPARATIVE COLUMNS: 21. In preparative HPLC eluent should be diluted with more mobile phase and then passed through the detector. Detectors are same as that of HPLC. 5. PREPARATIVE DETECTOR: 22. In preparative HPLC sample goes from detector to fraction collector. The fraction collector diverts the flow either to waste or, to a fraction container via the fraction collection needle which can achieve by using diverter valve. 6. FRACTION COLLECTOR: Figure 14: Fraction Collector System. 23. The preparative scale fraction collector is designed for flow rates up to 100 mL/min. Figure 15: Eluent Flow Rate. Designing of Fraction Collector: 24. Based on a signal plot. Highest flexibility. Manual fraction collection Based upon detector response. Peak-based fraction collection Compound with the desired mass is selectively collected Mass-based fraction collection Time-based fraction collection Fraction Collection Methods: Based on time of interval 25. Scale Up Optimization of Throughput ( Column Overloading) Optimization of Separation (Mobile Phase, Stationary Phase, Temperature, Retention, Selectivity.) Selection of Appropriate Mode of Separation Definition of Separation Problem METHOD DEVLOPMENT AND OPTIMIZATION OF PREPARATIVE HIGH PRESSURE LIQUID CHROMATOGRAPHIC METHOD: 26. The first step in preparative method development is to identify the problem and challenges associated. Sample information. Analyte(s) of interest. (type, number, concentration, required level of purity) Other separation strategies suitable for your sample. Detection. Amount of material to be isolated. Required degree of accuracy, precision etc. Method verification. DEFINITION OF SEPATION PROBLEM: 27. The following factors should be considered when selecting the appropriate HPLC mode for your separation. Solubility. Molecular weight. Sample matrix. Detectability. Other separation alternatives. SELECTION OF APPROPRIATE MODE OF SEPARATION: 28. 1. Mobile Phase: Viscosity of mobile phase. sample solubility in mobile phase. pH volatility of solvents/buffers. solvent cost. 2. Stationary Phase: The chemistry of the stationary phase controls, Selectivity. Production rate. 3. Temperature: Increase in temperature, Improves resolution and solubility. Decreases the viscosity of the mobile phase. Increase in production rate. Optimisation of Separation: 29. 4.Retention: Minimum retention factor(k) necessary for isolating the product and providing the desired purity, cycle time is decreased and the production rate increased. Concentration of the product in collected fractions decreases when retention increases, Column efficiency increases, but the cycle time and solvent consumption are increased as well. k= 1.2-2.0 for isocratic separation, k = 3-4 for gradient separations. 5. Selectivity: Increasing the selectivity value up to 2 or 3 significantly improves the throughput of separation. Selectivity can be optimised by changing the solvent composition as well as pH and nature of buffer added to the mobile phase. 30. Two ways of performing column overloading: 1. volume overloading. 2. concentration overloading. Table 4: Difference between Volume and Column Overloading. OPTIMIZATION OF THE THROUGHPUT: Volume Overloading. Concentration Overloading. 1. Determined by injection volume. 1. Determined by solubility of the compound in mobile phase. 2. Appropriate when sample has poor solubility. 2. Appropriate when sample has good solubility 3. Throughput determined by column diameter. 3. Throughput determined by selectivity. 4. Analytical area of adsorption isotherm. 4. Preparative area of adsorption isotherm. 5. Small particle size improves 5. Particle size has very little influence 31. 1. Purification in medicinal or high-throughput chemistry. 2. Purification in natural product chemistry. 3. Purification of by-products for impurity analysis. 4. Recovery collection. 5. Automated fraction re-analysis. APPLICATIONS OF PREPARATIVE HPLC: 32. 1. Preparative High-Performance Liquid ChromatographyMass Spectrometry for the High- Throughput Purification of Combinatorial Libraries. Marcus Bauser* Medicinal Chemistry VII, Business Group Pharma, BAYER AG, 42096 Wuppertal, Germany Preparative HPLC when coupled/hyphenated with other analytical techniques: (review of reported studies) 33. 2. Excellent combination of counter-current chromatography and Preparative high-performance liquid chromatography to separate galactolipids from pumpkin. A. Berthod, G.G. Leitao, I.A. Sutherland and W.D. Conway 3. Preparative high pressure liquid chromatography-flash chromatography.(Puriflash) 34. Commercially available instruments for preparative high pressure liquid chromatography: 1. Japan analytical Industry: Features: Reduces expenses of purchasing columns. Achieves the same separation as if using a longer column. 35. 2. Waters: Features: Flexible solvent delivery options allowing binary or quaternary based pumps providing low- pressure multi-solvent blending or high pressure gradient mixing of flow rates up to 150 ml/min. Easy to use. highly sensitive UV/Visible or Photodiode Array detectors are use. 36. CONCLUSION: Preparative chromatography is powerful technique for the isolation and purification of variety of chemicals, including pharmaceutical compounds, natural products and biological molecules. If we optimise all the parameter like column loadability, selectivity, flow rate, particle size, we can scale up the technique from analytical to preparative scale. 37. REFERENCES: 1. Taylor T., White C.A. ,The CHROMacademy Essential Guide - Basics of Preparative HPLC;105-115. 2. Kazakevich Y., LoBruto R., John Wiley & Sons. HPLC for Pharmaceutical Scientists ISBN-13:2007; 937 980. 3. Truei Y., Tingyue Gu, Tsai G., Large-Scale Gradient Elution Chromatography Advances in Biochemical Engineering/Biotechnology.1992,( 47);1-44. 4. Breslav M., Leshchinskaya V., Preparative High performance Liquid Chromatography Optimisation;1903-0909. 5. Ganetsos G., Barker P.E.,Preparative and Production scale Chromatography, Chromatographic Science,1993(61);786. 6. Berthod A., Leitao G.G., Sutherland I.A., Excellent combination of counter-current chromatography and preparative high-performance liquid chromatography to separate galactolipids from pumpkin,8 May 2009, 19 (1216); 4176418.