Hplc high performance liquid chromatography

1. MEHRAN UNIVERSITY Of ENGINEEING AND TECHNOLOGY JAMSHORO . PRESENTATION TOPIC: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Group Leader: Hameer Khan Khuhro

2. INTRODUCTION 1 CONTENTS PRINCIPLE 3 INSTRUMENTATION 4 ADVANTAGES 5 FIELDS OF APPLICATION 6 DEFINITION 2

3. INTRODUCTION In 1964 J.Calvin Giddings predicted improved liquid chromatography. Few years later C.Horvath and S.R Lipsky built the first HPLC and called it High pressure liquid chromatography. Today HPLC has become known as High performance liquid chromatography .

4. WHAT IS HPLC   HPLC, is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component.

5. PRINCIPLE: The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte.

6.   A flow scheme for HPLC  

7. Instrumentation Solvent Reservoirs Pump Sample Injector Column(s) Detector Data System  

8. 1.  Solvent  Reservoir It stores Mobile Phase. The liquid used as Mobile Phase is called Eluent . Mobile Phase may be a single solvent or solvent mixture of constant composition and is called Isocratic elution. Mobile Phase Reservoir can be filled with a wide range of solvents with different polarities and is called Gradient elution. Example Acetonitrile or Methanol with water. The solvents must be pure and degassed to avoid formation of gas bubbles.

9. 2.Solvent Delivery System (Pump) The pump is the most critical piece of equipment for a successfully operating HPLC. Functions : HPLC Pump has three basic functions: Provide accurate and constant flow. Provide accurate mobile phase compositions. Provide the force necessary to push the mobile phase through the tightly packed column.

10. HPLC Pump Criteria Constructed of materials inert toward solvents to be used Deliver high volumes (flow rates) of solvent (to 10 mL /min) Deliver precise and accurate flow (<0.5% variation) Deliver high pressure (to 6000 psi) Deliver pulse free flow Have low pump-head volume Be reliable HPLC Pumps: Types Reciprocating pumps Syringe pumps Constant pressure pumps

11. 3. Sample Injector The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible so that the sample enters the column as a homogeneous To minimize spreading of the injected volume during transport to the column, the shortest possible length of tubing should be used from the injector to the column.

12. Performance Requirements No sample remaining in unit Minimal broadening of sample band Free adjustment of injection volume Minimal loss Superior durability and pressure resistance

13. Manual Injectors   Front View Inject

14. Automatic Injectors

15. 4. HPLC columns The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from 10 to 30cm. Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Its chromatographic behavior is generally predictable and reproducible.

16. PARAMETERS Length (5-15 cm); much shorter than GC column Diameter (4 mm down to 50 m m) Particle size (3, 5, or 10 m m) Typically detection limit is decreased by decreasing the column diameter

17. 5. Detector There are several ways of detecting when a substance has passed through the column .

18. 6.Data System Data is processed using specific softwares that are connected to HPLC machine . Receive the information and present it as a graph. It automatically compare the graph with standard graph and gives results.

19. Advantages of High Performance Liquid Chromatography High separation capacity, enabling the batch analysis of multiple components Superior quantitative capability and reproducibility Moderate analytical conditions Unlike GC, the sample does not need to be vaporized. Generally high sensitivity Low sample consumption Easy preparative separation and purification of samples

20. Fields in Which High Performance Liquid Chromatography Is Used Biogenic substances Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc. Medical products Drugs, antibiotics, etc. Food products Vitamins, food additives, sugars, organic acids, amino acids, etc. Environmental samples Inorganic ions Hazardous organic substances, etc. Organic industrial products Synthetic polymers, additives, surfactants, etc.