Hplc

A brief introduction and instrumentation on high-performance liquid chromatography which is widely used in the analytical analysis of different samples in the pharmacy field. In this presentation, there is a small case study in finding out the different mobile and stationary phases used in the HPLC analysis.

1. High Performance Liquid Chromatography Concept to Conclusion Mr.AVINASH.db - B.Pharmacy 1

2. HPLC 2

3. HISTORY OF HPLC Liquid chromatography was initially discovered as an analytical technique in the early twentieth century and was first used as a method of separating colored compounds. This is where the name chromatography { chroma means color, graphy means writing, was derived.} A Russian botanist named Mikhail S. Tswett used to separate & purify mixtures of plant pigments into the pure constituents. He separated the pigments based on their interaction with a stationary phase , which is essential to any chromatographic separation. 3

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5. PRINCIPLE followed in HPLC 5

6. COLOUMN DETECTOR SCHEMATIC DIAGRAM OF HPLC 6

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8. DIFFERERNT TYPES TECHNIQUES 8

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10. Components elute in increasing order of polarity Components elute decreasing order of polarity. 10

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12. ISOCRATIC ELUTION 12

13. GRADIENT ELUSION 13

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15. CATIONIC EXCHANGE ANIONIC EXCHANGE 15

16. Types of pumps used in HPLC DIS P L A CEMENT PUMPS R E CI P R O C A T I NG PUMPS PNEUM A T I C PUMPS 16

17. The role of this pump is to force mobile phase through the column at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1to 2-mL/min range. Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). RECIPROCATING PUMPS 17

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19. It consists of large, syringe like chambers equipped with a plunger activated by a screw driven mechanism powered by a motor. DISPLACEMENT PUMPS 19

20. In this pumps, the mobile phase is driven through the column with the use of pressure produced from a gas cylinder. PNEUMATIC PUMPS 20

21. The injector serves to introduce the liquid sample into the flow stream of the mobile phase. Typical sample volumes are 5 to 20 ( μL ). INJECTORS 21

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23. It is Considered as the “heart of the chromatograph” the column’s stationary phase separates the sample components based on affinity. It is usually made of stainless steel to withstand high pressure caused by the pump to move the mobile phase through the column packing. The small particles inside the column are called the “packing material”. Guard column is used to remove particular matter and contamination, it protect the analytical column . COLUMN 23

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25. Ultraviolet (UV) This type of detector responds to substances that absorb light. The UV detector is mainly to separate and identify the active components of a mixture. UV detectors are the most versatile, having the best sensitivity . UV detectors cannot be used for testing substances that are low in chromophores (colorless or virtually colorless) as they cannot absorb light at low range. DETECTORS 25

26. Fluorescence This is a specific detector that senses only those substances that emit light. This detector is popular for trace analysis in environmental science. I t is very sensitive Mass Spectrometer The mass spectrometer detector coupled with HPLC is called HPLC- MS. HPLC-MS is the most powerful detector, widely used in pharmaceutical laboratories and research and development. The principal benefit of HPLC-MS is that it is capable of analyzing and providing molecular identity at minute level . 26

27. Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis). The concentration of each detected component is calculated from the area or height of the corresponding peak and reported. Data processing unit (Computer) 27

28. PARAMETERS DISTRIBUTION CONSTANT RETENTION TIME VOID TIME RETENTION FACTOR 28

29. K c = c S / c M K , the distribution constant , is the ratio of the affinity of compound A in the stationary phase and activity of compound A in the mobile phase The amount of time between the injection of a sample and its elution from the column is known as the retention time. DISTRIBUTION CONSTANT RETENTION TIME 29

30. VOID TIME RETENTION FACTOR The retention factor (k) is a means of measuring the retention of an analyte in the chromatographic column. The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound. The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0, which is known as void time. 30

31. The plate height is related to the flow rate of the mobile phase And is given by VAN DEEMTER EQUATION H = A + B/ υ + C υ A is a constant which represents the different possible paths that can be taken by the analyte through the stationary phase. B is a constant that describes the longitudinal diffusion that occurs in the system. C is a constant that describes the rate of adsorption and desorption of the analyte to the stationary phase. VAN DEEMTER EQUATION υ is the velocity of the mobile phase. 31

32. ADVANTAGES OF HPLC: Separations are fast and efficient (high resolution power). It can be applied to the separation and analysis of very complex mixtures. Accurate quantitative measurements. Repetitive and reproducible analysis using the same column. Adsorption, partition, ion exchange and exclusion column separations are excellently made. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC I t provides a m eans for deter m ina t i o n of m ulti p le components in a single analysis. 32

33. Applications 33

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35. DRUGS DOSAGE FORM MOBILE PHASE STATIONARY PHASE FLOW RATE DETECTOR RETENTION TIME REASON TENOFORVIR-df, LAMIVUDINE,EFAVIRENZ TABLET Methanol:10mM phosphate buffer of pH-5 in 70:30. C 18 COLUMN(150*4.6mm) with inter diameter-5µm 1ml/min PDA detector with UV-245nm L-2.76 T-3.96 E-10.5 Can easily determine the drug-drug interactions. SALBUTAMOL,AMBROXOL,GUAIFENESIN TABLET Aecetonitrile:potassium dihydrogen phosphate of pH-4 in 30:70 C18 column(250*4.6mm)with internal diameter- 5µm 1ml/min PDA detector with UV-215nm S-2.57 A-7.1 G-5.85 Easy for quantification n and estimation. CASE STUDY -1 35

36. DRUGSTHEOPHYLLINE DOSAGE FORM MOBILE PHASE STATIONARY PHASE FLOW RATE DETECTOR RETENTION TIME REASON L-SALBUTAMOL SULPHATE, SYRUP Methanol:10mM tetrabutylammonium hydrogen sulphate in 50:50 C18(250*4.6mm) with internal diameter-5 µm. 1.0mL/min UV-214nm S-2.575 T-4.967 Easy for quantification and estimation. CASE STUDY-2 36

37. Gurdeep R. Chatwal, Sham K. Anand, Instrumental Method Of Chemical Analysis, Himalaya Publishing House. Douglas A. Skoog, Instrumental Analysis. R EFERENCE 37

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