Presentation of HPLC

HPLC Presentation at IICT Hyderabad

1. CRCL Group A Officer’s Training at IICT Welcome to All Distinguished Faculty Members of IICT & Officers of Revenue Laboratories. 05/04/2019 1 IICT Training Presentation-2 Group-III

2. CRCL Group A Officer’s Training at IICT 05/04/2019 2 IICT Training Presentation-2 Group-III Don’t worry Singh Sir, I have some knowledge about HPLC, we will discuss and our team work together to get a better solution Mr. Suresh, I have one sample and I need a quantitative separation of it, have you any idea about that?

3. CRCL Group A Officer’s Training at IICT 05/04/2019 3 IICT Training Presentation-2 Group-III Topic: H igh P erformance L iquid C hromatography( HPLC ) Group-III Team Members: T.R Suresh A.J Aleyamma Ajay Kumar Singh H S Bhandarkar K Thambidurai 05/04/2019 3 IICT Training Presentation-2 Group-III

4. CRCL Group A Officer’s Training at IICT 05/04/2019 4 IICT Training Presentation-2 Group-III Topic to be Covered 1. Introduction 2. Basics of HPLC 3. Types of Liquid Chromatography 4. Instrumentation 5. Application 05/04/2019 4 IICT Training Presentation-2 Group-III

5. CRCL Group A Officer’s Training at IICT 05/04/2019 5 IICT Training Presentation-2 Group-III Introduction HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, detector and a recorder or output device. 05/04/2019 5 IICT Training Presentation-2 Group-III

6. CRCL Group A Officer’s Training at IICT 05/04/2019 6 IICT Training Presentation-2 Group-III Basic Principles of HPLC 05/04/2019 6 IICT Training Presentation-2 Group-III Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the solvent along with the sample through the stationary phase as it moves through the column. Compounds are separated based upon their differential migration / partition behaviour between the stationary and mobile phases .

7. CRCL Group A Officer’s Training at IICT 05/04/2019 7 IICT Training Presentation-2 Group-III Types of Liquid Chromatography 05/04/2019 7 IICT Training Presentation-2 Group-III Partition chromatography Adsorption, or liquid-solid chromatography Ion exchange chromatography Size exclusion, or gel, chromatography

8. CRCL Group A Officer’s Training at IICT 05/04/2019 8 IICT Training Presentation-2 Group-III Types of Liquid Chromatography 05/04/2019 8 IICT Training Presentation-2 Group-III In Partition chromatography, the molecules are separated in between two phases based ion the partition coefficient of the analyte . The partition chromatography is the basic principle involved in many separation techniques like high performance liquid chromatography and gas chromatography.

9. CRCL Group A Officer’s Training at IICT 05/04/2019 9 IICT Training Presentation-2 Group-III Types of Liquid Chromatography 05/04/2019 9 IICT Training Presentation-2 Group-III Adsorption, or liquid-solid Here separation is based on the adsorption/desorption of the analyte onto a polar surface(silica). The solid surface is called adsorbent which is generally porous in nature with a high surface area to adsorb substances on its surface by  intermolecular forces  . Some commonly used adsorbents are Silica gel H, silica gel G, silica gel N, silica gel S, hydrated gel silica, cellulose microcrystalline, alumina, modified silica gel, etc.

10. CRCL Group A Officer’s Training at IICT 05/04/2019 10 IICT Training Presentation-2 Group-III Types of Liquid Chromatography 05/04/2019 10 IICT Training Presentation-2 Group-III Ion Exchange chromatography In this type of Chromatography the sample components are separated based upon ionic interaction and exchanging of ions between the charged groups of molecules of analyte and charged groups in the stationery phase opposite opposite to that of analyte molecules. Polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

11. CRCL Group A Officer’s Training at IICT 05/04/2019 11 IICT Training Presentation-2 Group-III Types of Liquid Chromatography 05/04/2019 11 IICT Training Presentation-2 Group-III Size Exclusion (OR) Gel chromatography (Gel Permeable ) Seperation based on analyte’s molecular size and sieving action of the column packing. In size exclusion the column is consisted of substances which have controlled pore sizes and is able to be filtered according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute first before the smaller molecules.

12. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Schematic Diagram of working principle: 05/04/2019 12 IICT Training Presentation-2 Group-III

13. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation 05/04/2019 13 IICT Training Presentation-2 Group-III Mobile Phases Pump / Mixing Chamber Injector port Column Chamber Recorder / Control System

14. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Solvent Solvent Delivery System (Pump) Injector Sampler Column Detectors (Diode Array) Fraction Collector Recorder (Data Collection) 05/04/2019 14 IICT Training Presentation-2 Group-III

15. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Solvent (Mobile Phase): Mobile phase is usually a mixture of one or more solvents and it should be High, Purity, Low Viscosity and Low Boiling Points. The Common Solvents used are N-Hexane, Toluene, Methylene Chloride, THF, ACN, Methanol and Water Binary system contains Two Solvents, and Quaternary System Contains Four Solvents 05/04/2019 15 IICT Training Presentation-2 Group-III

16. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Pump: Pump is a important device used to mix the solvents, to maintain the required flow rate of Mobile phase in the system. 05/04/2019 16 IICT Training Presentation-2 Group-III

17. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Injector and Sampler: [ Autosampler , Sample Manager] Injector – It is a simple device to introduce the sample volume of a sample solution into the flowing mobile phase stream. The injector can be a simple manual device, or a sophisticated autosampler that can be programmed for unattended injections of many samples from an array of individual vials. 05/04/2019 17 IICT Training Presentation-2 Group-III

18. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Column: Types of Columns – Microbore •Diameter 1-2 mm; Length 7-30 cm; sample size 0.01 mg; flow rate 0.1 mL /min –Std. analytical •Diameter 3-5 mm; Length 7-30 cm; sample size 0.1 mg; flow rate 1 mL /min; Particle Size 3-10 μ m –Preparative •Diameter 5-20 mm; Length 25-50 cm; sample size 10 mg; flow rate 10 mL /min ; Particle size >10 μ m 05/04/2019 18 IICT Training Presentation-2 Group-III

19. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Column: Column Packing Material • Support Type –Silica (or alumina) or polymer (cross-linked polystyrene ) •Bonded groups –C18, C8, C4, amino, cyano, phenyl – Diethylaminoethyl (DEAE), sulfonate , quaternary ammonium •Particle size ( dp ): 3-, 5-, 7,-10-or 20 μ m –Efficiency is inversely proportional to dp –Column pressure is inversely proportional to ( dp )2 • Pore size ( dpore ): 60-300A –Wide pore materials (300 A) are used for biomolecules or polymers •Surface area: 90-400 m2/g –High surface area maximizes solute interaction with bonded groups 05/04/2019 19 IICT Training Presentation-2 Group-III

20. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Column Length (L) •Column efficiency (n) is proportional to Length •Common column length is 10-25 cm •Column can be connected together to produce a longer column with higher efficiency •Column back pressure is proportional to L •Fast LC column are short column (3-10 cm) packed with small-particles ( dp = 3 μm ) 05/04/2019 20 IICT Training Presentation-2 Group-III

21. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Column Diameter (d) Sample capacity is proportional to (d) 2 Flow rate is proportional to (d) 2 Detection limits is inversely proportional (d) 2 Commonly used column with following diametrs : Dia Sample Wt. Flow Rate For Semi-preparative 8-20 mm 10-50mg 5-30 ml/min Standard Analytical 4.6 mm 1 mg 1 ml/min Narrow bore 2.0 mm 0.2 mg 0.2 ml/min Mirco bore 1.0 mm 0.05 mg 0.05 ml/min 05/04/2019 21 IICT Training Presentation-2 Group-III

22. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Detector A device that indicates the composition of the eluent by measuring physical or chemical properties [ e.g.,  UV/visible light absorbance, differential refractive index, fluorescence, or conductivity]. 05/04/2019 22 IICT Training Presentation-2 Group-III

23. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Types of Detector UV-Visible Detector Fluorescence Detector Photo-Diode Array (PDA) Evaporative Light Scattering Detector (ELSD) Refractive-Index (RI) Mass-Spectrometric (MS) 05/04/2019 23 IICT Training Presentation-2 Group-III

24. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Components of HPLC: Types of Detector UV-Visible Detector - For Unsaturated Organic & Carbonyl Compounds Fluorescence Detector – It is one of the most sensitive detector and often used for trace analysis. Photo-Diode Array (PDA) - The diode arrays work in parallel simultaneously monitoring all the wavelengths Evaporative Light Scattering Detector (ELSD) - Universal detection of non-volatile compounds Refractive-Index (RI) - this detector is extremely useful for detecting those compounds that are nonionic , do not adsorb in the UV, and do not fluoresce. Mass-Spectrometric (MS) 05/04/2019 24 IICT Training Presentation-2 Group-III

25. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Instrumentation Fraction Collector : 05/04/2019 25 IICT Training Presentation-2 Group-III Fraction collector is used to collect the different fraction of the compounds based on their retention time in semi preparative scale

26. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III Application of HPLC 05/04/2019 26 IICT Training Presentation-2 Group-III

27. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III 05/04/2019 27 IICT Training Presentation-2 Group-III Analysis of Morphine Content in Indian Opium Samples by HPLC Mobile phase: Dissolve 1.0 g of sodium heptane sulfonate monohydrate R in 420 mL of water, adjust to pH 3.2 with phosphoric acid (4.9 g/L H3PO4) (about 5 mL ) and add 180mLof acetonitrile . Stationary phase: O ctylsilyl silica gel for chromatography R(5 μ m). C-18 Column Flow Rate: 1.5 ml / Minute Detection: Spectrophotometer at 280nm Reference solution : Dissolve 25.0 mg (M1) of Morphine Standard in the mobile phase and dilute to 25.0mL (V1) with the mobile phase. Dilute 10.0 mL (V2) of this solution to 100.0 mL (V3) with the mobile phase.

28. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III 05/04/2019 28 IICT Training Presentation-2 Group-III Analysis of Morphine Content in Indian Opium Samples by HPLC Sample Solution Preparation : Suspend 1.00 g (M2) of the substance to be examined, cut into thin slices, in 50 mL of ethanol (50 per cent V/V) ,mix with the aid of ultrasonic bath for 1 hours, allow to cool and dilute to 100.0 mL (V4) with the same solvent. Allow to stand.   To 10.0 mL (V5) of the supernatant liquid add 5 mL of ammonium chloride buffer solution pH 9.5 R, dilute to 25.0 mL (V6) with water and mix.   Transfer 20.0 mL (V7) of this solution to a chromatography column about 0.15 m long and about 30 mm in internal diameter containing 15 g of kieselguhr for chromatography. Allow to stand for 15 min. Elute with 2 quantities, each of 40 mL , of a mixture of 15 volumes of 2-propanol R and 85 volumes of methylene chloride R. Evaporate the elute to dryness in vacuum at 40 °C using rotary evoporator . Transfer the residue to a volumetric flask with the aid of the mobile phase and dilute to 25.0 mL (V8) with the mobile phase. Inject this sample in HPLC.

29. CRCL Group A Officer’s Training at IICT 05/04/2019 IICT Training Presentation-2 Group-III 29 Analysis of Morphine Content in Indian Opium Samples by HPLC HPLC Chromatogram for Standard Morphine Morphine

30. CRCL Group A Officer’s Training at IICT IICT Training Presentation-2 Group-III 05/04/2019 30 Analysis of Morphine Content in Indian Opium Samples by HPLC HPLC Chromatogram for Raw Opium Sample Morphine

31. CRCL Group A Officer’s Training at IICT IICT Training Presentation-2 Group-III 05/04/2019 31 Analysis of Morphine Content in Indian Opium Samples by HPLC Calculation

32. CRCL Group A Officer’s Training at IICT IICT Training Presentation-2 Group-III 05/04/2019 32 Analysis of Morphine Content in Indian Opium Samples by HPLC Typical Analysis of Different Raw Opium Samples

33. CRCL Group A Officer’s Training at IICT IICT Training Presentation-2 Group-III 05/04/2019 33