CHROMATOGRAPHY: CHROMATOGRAPHY Presented by: Mr. DHRUV K. MANGUKIA Guided by: Mr. SANDEEP KATARIA MAHARISHI ARVIND INSTITUTE OF PHARMACY, JAIPUR 1 INTRODUCTION:: INTRODUCTION: Technique for separation and analysis of components of the mixture. As per IUPAC 1993 International commission ; “Chromatography is a physical method of separation in which components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction.” Various types and its usefulness in evaluation of crude drugs. 2 What is Chromatography?: What is Chromatography? Separation of components of a mixture. Stationary phase. Solid. Liquid ( coated on solid ). Mobile phase (flow over stationary phase). Liquid. Gas. Components distribute over phases based on affinity for either phase. 3 Types of Chromatography…: Types of Chromatography… Paper Thin layer HPLC Gas Column 4 PRINCIPLE:: PRINCIPLE: 5 1 2 TLC [ Thin layer chromatography ]:: TLC [ Thin layer chromatography ]: Principle : Adsorption. Adsorbents : Silica gel, Aluminum oxide, Calcium carbonate, Magnesium carbonate, Zinc carbonate, magnesium oxide, cellulose. Mobile phase : both organic and inorganic solvents as well as water are also used. TECHNIQUE: Preparation of plates: Pouring, Dipping, Spraying, Spreading, pre-coated plates. Activation of adsorbents: Air dry for 30 min + in an oven for 30 min at 110 ⁰C. purification of silica gel G layers : Methanol-conc. HCl (9:1); to remove Iron . Sample application : Agla microsyringe / capillary tube. Development tank : “edge effect”. Evaluation : Direct and indirect methods. 6 PowerPoint Presentation: APPLICATIONS: Separation and identification of components of mixture. Purification. As a check on separation and purification process. Examination of reaction. Identification of organic compounds like Acids, Alcohols, Glycols, Alkaloids, Amines, Amino acids, Proteins and peptides, Antibiotics. EXAMPLES: AMLA ( Emblica officinalis ) Comparison of Rf value of Gallic acid obtained from amla with Gallic acid standard ( Rf value = 0.68 ). 7 HPTLC : HPTLC PRINCIPLE: Involves high speed capillary flow range of the mobile phase. Development tank Linomate type applicator scanner 8 DIFFERENCE: DIFFERENCE PROPERTY HPTLC TLC Particle size of adsorbent 4 – 8 µm 5 – 20 µm Efficiency High due to smaller particle size generated Less Separations 3 - 5 cm 10 - 15 cm Analysis Time Less More 9 PowerPoint Presentation: PROPERTY HPTLC TLC Solid support Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes Silica gel , Alumina & Kiesulguhr Development chamber New type that require less amount of mobile phase More amount Sample spotting Auto sampler Manual spotting Sample volume 0.5 – 5 μ l 1 - 10 μ l 10 PowerPoint Presentation: PROPERTY HPTLC TLC Theoretical plate 4000 IN 3 cm 2000 IN 12 cm Development time 10 min 25 min Size of spot 0.5-1 mm 2-4 mm Capacity Limited sample capacity. More no. Of samples can be handled at the same time. 11 PowerPoint Presentation: APPLICATIONS OF HPTLC: Therapeutic drug monitoring to determine the concentration of drug and it’s metabolites in blood , urine , etc . Characterization of hazards in industrial waste. Determination of mercury in water. Analysis of environmental pollutions levels. Quantitative determination of prostaglandin’s & thomboxanes in plasma. Analysis of nitrosoamine in food and and body fluid. HPTLC is used for determination of β-blockers like atenolol, metoprolol, Propranolol, oxypronolol, alprenolol, timolol. Salicylic acid, salicylamide, paracetamol have been determined in saliva. 12 PowerPoint Presentation: EXAMPLE: Sample = Caesalpinia pulcherrima, f. Caesalpiniacea. Isolated flavonoids were estimated by hptlc. Adsorbents - silica gel F254 aluminium plate. Mobile phase - Benzene : Methanol. Spraying reagent- Vanillin- H2SO4. Color of spots – Purple. 13 GAS CHROMATOGRAPHY:: GAS CHROMATOGRAPHY: Principle: GSC - Adsorption. GLC – Partition. Carrier gas: H, He, Ni, Ar. Sample injection system: liquids – micro-syringe; solids – solution / scaled in thin walled vials; gas – special sampling valves. Columns: a) Packed column – GSC : packed with size graded adsorbent. GLC : liquid is coated with size graded solid adsorbent. 14 PowerPoint Presentation: b) Open column – inside wall coated with a thin film of liquid phase. Detectors: Katharometer. Flame ionisation detector. Thermionic detector. Electron capture detector. KATHAROMETER FLAME IONISATION DETECTOR 15 PowerPoint Presentation: APPLICATIOS OF GAS CHROMATOGRAPHY : Assay of drugs like; Atropine sulphate ointment and injection. Methadione HCl injection. Naloxone injection. Nitrous oxide gas. Scopolamine hydrochloride injection. Separation and purificationof crude drugs from rare natural sources. Evaluation of herbal formulations. EXAMPLE: GLC – Separation of mixture of sterol acetate present in oat seeds, i.e., cholesterol, brassicasterol , campesterol , stigmasterol , sitosterol . 16 COLUMN CHROMATOGRAPHY: COLUMN CHROMATOGRAPHY Principle: Adsorption. Partition. Development techniques: Frontal development. Displacement development. Elution development . Example: Adsorbents : silica gel and sephadex LH-20. Sample: methanolic extract of whole plant of Diodia trees. Asperulosidic acid, asperuloside , geniposidic acid, scopolin and flavonoids . 17 HPLC [ HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ]: HPLC [ HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ] Principle: Application of pressure to increase the flow of solvent through the column packed with fine adsorbents. 18 PowerPoint Presentation: PUMPING SYSTEMS: CONSTANT FLOW RATE PUMP RECIPROCATING PUMP. SYRINGE PUMP. CONSTANT PRESSURE PUMP PNEUMATIC PUMP. Reciprocating pump Syringe pump 19 PowerPoint Presentation: Constant pressure pump 20 PowerPoint Presentation: Precolumn : Mainly used to remove impurities from the solvent; thus prevents contamination of the column. Also prevents clogging of column. Increases column life. Sample injection systems: Syringe injection. Stop flow injection. Sampling loops/ injection valves. 21 PowerPoint Presentation: COLUMNS: Made of stainless steel. Sometimes from glass for low pressure columns. Usually contains 40,000 – 60,000 plates/ metre. Some also contains upto 1,00,000 plates/metre. DETECTORS: UV-VISIBLE DETECTORS: Photometers and spectrophotometers are used. FLUORIMETRIC DETECTORS: For compounds which exhibits fluorescence. 22 PowerPoint Presentation: APPLICATIONS OF HPLC: Used for quality control of drug components like morphine, ergot alkaloids, cardiac glycosides, sennosides, vitamins, rhubarb constituents. Routine assay of drugs and excipients. Study of drug levels in biological fluids. Used to study metabolic pathways. In clinical and toxicological studies. Used for the analysis of amino acids, essential oils & terpenes. Separation of alkaloids and water soluble vitamins. EXAMPLE: Separation of flavonoids from 2 speices ofchondropetalum. 23 FLASH CHROMATOGRAPHY: FLASH CHROMATOGRAPHY A rapid form of preparative column chromatography based on optimised pre-packed columns through which is pumped solvent at a high flow rate. Sample range varies from several mg to over 150 g. Linear flow rates up to 15 cm/min. Pressure 10-100 psi. Basic design of flash chromatography column 24 PowerPoint Presentation: Cartridges are stackable. Different chemistries may be combined in a single run. Specific separated zone may be collected on smaller cartridge and further purified under different conditions. VersaFlash Stand VersaPac Cartridges Spherical silica of various grades is used to pack cartridges. 25 PowerPoint Presentation: IntelliFlash ™ 310 26 APPLICATIONS OF FLASH CHROMATOGRAPHY: APPLICATIONS OF FLASH CHROMATOGRAPHY Sugar impurities from rutin can be isolated and purified. Can isolate and detect acarbose, from it's sugar components. Can isolate and collect the critical components of the plant extract. Isolation and purification of catechins from Green tea extract. Isolation & extraction of flavonoids form Ginkgo biloba leaves extract. Separation & isolation of α -santalol and β santalol from sandalwood extract. Isolation and purification of ginsenosides from red panax ginseng extract. Example: Flash pyrolysis of asphaltite. 27 References:: References: Chatwal GR, Anand SK. Instrumental methods of chemical analysis. 5 th and enlarged ed. Mumbai(INDIA): Himalaya publishing house; 2002. p. 2.566 – 2.654, 2.673 – 2.700. Harborne JB. Phytochemical methods. 3 rd indian ed. UK: Springer; 1998. p. 16 -32. Pawar CR, Landge AD, Surana SJ. Quantitative estimation of phytoconstituents of caesalpinia pulcherrima. Ijprd [0974-9446] 2009 Oct [cited 2011 Aug 21]; 8(003): [5 screens]. Available from : URL: http://www.ijprd.com/QUANTITATIVE%20ESTIMATION%20OF%20PHYTOCONSTITUENTS%20OF%20CAESALPINIA%20PULCHERRIMA.pdf Poole C.F., Dias N.practitioner’s guide to method development in thin layer chromatography.elsevier [0021-9673]2000[cited 2011 Sept 1]; A(892):[19 screens].Available from URL: http://www.sciencedirect.com/science/article/pii/S002196730000162X. H. Jork , W. Funk, W. Fischer, H. Wimmer , Thin-Layer Chromatography Reagents and Detection Methods, Vol. 1, VCH, Weinheim , 1990. 28 PowerPoint Presentation: THANK YOU … … … 29 MAHARISHI ARVIND INSTITUTE OF PHARMACY, JAIPUR.