HPLC (High pressure liquid chromatography)

HPLC , pumps used in hplc , column used in hplc, detector used in hplc.

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PRESENTED BY: Khaja Anees Ahmed M Pharm 1 st semester Dept. of Pharmaceutics HKE’S MTRIPS Gulbarga. 1

2. CONTENTS Introduction Principle Instrumentation 2

3. INTRODUCTION HPLC stands for High performance liquid chromatography and it is also known as high pressure liquid chromatography. It is extension of conventional liquid chromatography. HPLC is a analytical technique used to separate, identify & quantify the mixture of compounds. It gives high performance due to the small particle size of stationary phase (30µm). High pressure is applied for rapid separation of compound that is 1000-4000 psi. It is used in biochemistry and analytical chemistry to identify, quantity and purify the individual components of a mixture. 3

4. PRINCIPLE The principle of separation in normal phase mode and reverse phase mode is adsorption . When a mixture of components are introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase and the components are separated. 4

5. INSTRUMENTATION 1. Mobile phase reservoir 2. Pumps 3. Mixing unit and solvent degassing 4. Injector 5. Column 6. Detectors 5

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8. MOBILE PHASE RESERVOIR :- In HPLC, the reservoir are usually made up of stainless steel or borosilicate glass with the capacity of 500 ml to 1 L of solvent ( HPLC grade ). Beside, they are duly equipped with adequate mean for the removal of dissolved gaseous product viz O 2 and N 2 that may produce undesired small bubbles in the column as well as detector system engaged. Used to store the mobile phase. Two types of solvent reservoirs are i . Binary system ii. Quarternary system. HPLC grade solvents are used, these are highly pure. 8

9. PUMPS The role of the pump is to force a liquid called the mobile phase through the liquid chromatography at a specific flow rate, expressed in milliliter per min (1ml/min). Normal flow rates in HPLC are in the 1-2ml/min range. Typical pump can reach pressure in the range 6000-9000psi. During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition(gradient). Types of pumps Reciprocating pump. Displacement pump. Pneumatic or constant pressure pump. 9

10. RECIPROCATING PUMP :- The reciprocating pump it consist of a small chamber where the solvent is moved in and by an eccentric system or gear. The forward stroke shut down the inlet check valve, whereas the outlet check valve open the flood gate and allow the respective intended mobile phase to pumped in to the Colum efficiency. In the particular instance, the mobile phase is in direct contact with the piston. Reciprocating pump critically give rise to pulsed flow. 10

11. DISPLACEMENT PUMP :- It consists of large, syringe like chamber equipped with a plunger activated by a screw driven mechanism powdered by a stepping motor. A positive-displacement pump makes a fluid move by trapping a fixed amount and forcing (displacing) that trapped volume into the discharge pipe. Some positive-displacement pumps use an expanding cavity on the suction side and a decreasing cavity on the discharge side. Liquid flows into the pump as the cavity on the suction side expands and the liquid flows out of the discharge as the cavity collapses. The volume is constant through each cycle of operation. 11

12. MIXING UNIT Mixing unit is used to mix solvents in different proportions and pass through the column. There are two types of mixing units. Low pressure mixing chamber- which uses helium for degassing solvents. High pressure mixing chamber- It does not require helium for degassing solvents. Mixing of solvents is done either with a static mixer which is packed with beads or a dynamic mixer which uses magnetic stirrer and operates under high pressure. 12

13. INJECTOR The injector serves to introduce the liquid sample into the flow of the mobile phase. Typical sample volumes are 5-20 microliters . The injector must also be able to withstand the high pressure of the liquid system. Several devices are available either for manual or auto injection of the sample. Types of injector Septum injector Stop flow injector Rheodyne injector 13

14. Septum injector: It is used for injecting the sample through a rubber septum. This is not common, since the septum has to withstand high pressure. 2. Stop flow injector: In which the flow of mobile phase is stopped for a while and the sample is injected through a valve device. 14

15. 3. Rheodyne injector : It is the most popular injector. This has a fixed volume loop like 20µL or 50µL or more. Injector has 2 modes, i.e., load position when the sample is loaded in the loop and inject mode when the sample is injected. 15

16. Column Material Most widely used columns are made up of stainless steel which can withstand high pressure. Column length: Varies from 5cm to 30cm Column diameter: Ranges from 2mm to 50mm Particle size: From 1µ to 20μ Particle Nature: Spherical, uniform sized, porous materials are used. 16

17. Normal Phase Columns: These columns have a polar stationary phase and are used for separating polar compounds. Common stationary phases include silica gel bonded with polar functional groups like amino (NH2) or diol groups. Ion Exchange Columns: These columns contain ion exchange resins as the stationary phase and are used for separating charged compounds based on their ionic properties. Affinity Columns: These columns contain a stationary phase with specific affinity ligands that selectively bind to target molecules based on their biological or chemical properties. 17

18. DETECTORS Detectors used depends upon the property of the compounds to be separated. Different detectors available are UV detector Refractive index detector Flourimetric detector Conductivity detector Amperometric  detector 18

19. Refractive Index Detector Detector is used to measure changes in the refractive index of the mobile phase as analyte molecules pass through the detector. This type of detector is particularly useful for compounds that lack chromophores or fluorophores, making them invisible to UV or fluorescence detectors. The RI detector is sensitive to changes in the concentration of solutes in the mobile phase, allowing for the detection and quantification of analytes based on their refractive index differences from the solvent. 19

20. Conductivity Detector A conductivity detector in HPLC measures changes in the electrical conductivity of the mobile phase as analyte molecules pass through the detector. This type of detector is particularly useful for ionizable compounds, such as acids, bases, and salts. When ions are present in the mobile phase, they affect the conductivity of the solution. The conductivity detector detects these changes and can quantify the concentration of ions in the sample. This detector is especially valuable for analyzing ionic compounds or mixtures containing ions. 20

21. REFERENCES Instrumental methods of chemical analysis by B. K. Sharma, Published by Satyendra Rastogi , Meerut, India. Instrumental methods of chemical analysis by Gurudeep.R . Chatawal , Sham.K . Anand . Text book of Pharmaceutical Analysis, fourth edition by Dr. S Ravi Sankar . 21