HPLC process and it's working ejisiejejdidididdid

HPLC process and it's working

1. HPLC HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Produced By :- Anshul N. Wakode Directed By :- Kanchan Mawande Mam

2. Separate : Resolve mixtures into individual components Identify : Determine the presence and characteristics of each component Quantify : Measure the amount of each component present Define -

3. History - - 1960s: The concept of HPLC emerged, building on earlier liquid chromatography techniques. Researchers like Csaba Horvath and Jack Kirkland pioneered the development of modern HPLC . Csaba Horvath, a Hungarian-American chemist Jack Kirkland, an American chemist.

4. High resolution: The combination of high pressure and small particle sizes enables HPLC to achieve high resolution, allowing for the separation of complex mixtures and closely related compounds . In the context of High-Performance Liquid Chromatography (HPLC), resolution refers to the ability of the chromatographic system to separate and distinguish between two or more closely related compounds or peaks in a mixture.

5. Principle ( hplc ) - Partitioning : Molecules distribute between the mobile and stationary phases based on their affinity Adsorption : Molecules interact with the stationary phase through weak intermolecular forces . Size exclusion: Molecules are separated based on their size and shape . Ion exchange: Molecules are separated based on their ionic properties.

6. Types of hplc 1. Reverse-Phase HPLC (RP-HPLC): Most common type, uses a hydrophobic stationary phase and a hydrophilic mobile phase . 2. Normal-Phase HPLC (NP-HPLC): Uses a hydrophilic stationary phase and a hydrophobic mobile phase . 3. Ion-Exchange HPLC (IE-HPLC): Separates molecules based on their ionic properties . 4. Size-Exclusion HPLC (SE-HPLC): Separates molecules based on their size and shape.

7. Instrumentation and working

8. Instrumentation and working Solvent reservoirs Solvent storage: Reservoirs hold the mobile phase solvents, typically in large volumes (e.g., 1-5 liters ). Solvent filtration: Some reservoirs have filters to remove impurities and contaminants from the solvents . Solvent delivery: Reservoirs supply the solvents to the pump, which pressurizes and delivers the mobile phase to the column . Solvent recycling: Some HPLC systems have reservoirs that collect and recycle solvents for reuse . Buffer preparation: Reservoirs can be used to prepare buffers or other mobile phase components . Reduced risk of contamination Improved HPLC performance and reproducibility

9. Solvent degasser The degasser removes these gases by : 1 . Heating: Warming the solvent to increase gas solubility 2 . Vacuum: Applying a vacuum to reduce gas solubility 3 . Agitation: Gently stirring the solvent to release dissolved gases Types of degassers: 1. In-line degasser: Integrated into the HPLC system, degassing occurs in-line with the solvent flow 2 . Off-line degasser: A separate unit that degasses solvents before they enter the HPLC system 3 . Membrane degasser: Uses a semipermeable membrane to remove dissolved gases A solvent degasser is a crucial component that removes dissolved gases from the mobile phase solvents.

10. A gradient valve mixes two or more solvents in varying proportions to create a continuous gradient of mobile phase composition. Types of gradient valves: 1. Low-pressure gradient valve: Operates at low pressures (<100 psi) and is used for isocratic and gradient elution. 2. High-pressure gradient valve: Operates at high pressures (up to 400 bar) and is used for high-pressure gradient elution. Gradient valve

11. pump The high-pressure pump uses a reciprocating piston design to generate high pressure. In High-Performance Liquid Chromatography (HPLC), a high-pressure pump is responsible for delivering the mobile phase to the column at extremely high pressures (up to 400 bar or 5800 psi).

12. In High-Performance Liquid Chromatography (HPLC), the sample injection loop plays a crucial role in introducing the sample into the column . - The sample injection loop is a valve with a loop-shaped channel that holds a precise volume of sample -Types of injection loops : 1 . Fixed loop: Has a fixed sample volume. 2 . Variable loop: Allows adjustment of sample volume. Sample injector

13. Guard column The guard column removes impurities and contaminants from the mobile phase and sample, preventing them from reaching the analytical column . - Types of guard columns : 1 . Pre-column: Placed before the analytical column . 2 . In-line filter: Integrated into the HPLC system . 3 . Replaceable cartridge: Can be replaced when contaminated. -Characteristics: 1 . Short length: Typically 1-5 cm . 2 . Large particle size: 5-20 μm . 3 . Same stationary phase: As the analytical column.

14. Analytical column The analytical column uses a stationary phase and a mobile phase to separate molecules based on their interactions and affinities . Benefits:- 1 . High resolution: Separates complex mixtures into individual components . 2. Sensitive detection: Enables detection of trace amounts of molecules . 3. Quantitative analysis: Allows for accurate quantification of molecules.

15. Detector The detector responds to changes in the mobile phase composition, detecting the presence of molecules based on their properties . - Working process: 1 . Molecules exit the column: Separated molecules enter the detector flow cell . 2. Detection occurs: The sensor detects changes in the mobile phase or molecules . 3. Signal generation: The detector generates an electrical signal proportional to the molecule's presence . 4. Signal processing: The electronic circuitry amplifies and processes the signal . 5. Chromatogram generation: The processed signal is plotted against time, creating a chromatogram.

16. waste The waste management system collects and disposes of the waste mobile phase, solvent, and sample components that are not detected or are excess . Data acquisition Data acquisition involves converting the detector signal into digital data, processing, and storing it for analysis.

17. Define Stationary phase :- the stationary phase is the material that is fixed in placed within the chromatoghraphic column . It is the phase that does not move . It is typically solid or liquid . Common types of stationary phase are : - silica gel - alumina - c18 , c8 - ion-exchange resins

18. Mobile phase :- The mobile phase is the substance that flows through the chromatographic column and carries the analyte through the column. It is the phase that move. Common types of mobile phase includes : - solvent : (e.g. water , acetonitrile , methanol ) - buffer solution – (e.g. phosphate buffer, ammonium acetate,) - gases – (e.g. helium, nitrogen,)

19. Advantages Precision: HPLC is a precise and quick quantitative analysis method. Separation : HPLC can separate complex mixtures and resolve ions with similar mass but different electronic configurations. Reproducibility : HPLC is highly reproducible because the cycle is mechanized. Sensitivity : HPLC is sensitive and selective, especially for vitamin analysis . Dis-advantages Cost: HPLC can be expensive because it requires a power supply, regular maintenance, and a large number of expensive organics. Troubleshooting: It can be complicated to develop new methods or troubleshoot problems.

20. Applications:- - Forensics: HPLC can be used to detect drugs in crime labs.  - Food and beverages: HPLC can detect adulteration of ingredients and pesticides in food and beverages.  - Quality control: HPLC can be used to monitor the quality of raw materials and finished goods in manufacturing.  - Pharmaceuticals: HPLC is used to detect impurities in pharmaceutical products, monitor drug levels in plasma, and develop new drugs. 

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