High Performance Liquid Chromatography .pptx

High-performance liquid chromatography (HPLC) is a sophisticated analytical technique used to separate, identify, and quantify the components of a mixture. It involves passing a sample dissolved in a mobile phase through a column packed with a stationary phase under high pressure, allowing components to separate based on their interaction with the stationary phase.

Separation:
HPLC separates components based on their differing affinities for the stationary phase. The components that interact more strongly with the stationary phase will move more slowly through the column, while those that interact less strongly will move faster.
Identification:
The separated components are detected as they exit the column, and the time at which each component exits the column can be used to identify it.
Quantification:
The area of the peak on the chromatogram (the graph of detector response versus time) is proportional to the amount of each component in the sample.
Principle:
HPLC relies on a high-pressure pump to force the mobile phase through the column. The high pressure allows for faster separations and greater resolution compared to traditional liquid chromatography methods.
Mobile Phase:
The mobile phase is a solvent or a mixture of solvents that carries the sample through the column. The composition of the mobile phase can be adjusted to optimize the separation of different components.
Stationary Phase:
The stationary phase is a solid material packed inside the column that interacts with the sample components. The type of stationary phase is chosen based on the properties of the components being separated.
Applications of HPLC:
Analysis of pharmaceutical compounds: HPLC is widely used for the analysis of drugs and their metabolites.
Environmental monitoring: HPLC can be used to analyze pollutants in water and soil.
Food chemistry: HPLC is used to analyze the composition of food products.
Biochemistry: HPLC is used to analyze proteins, peptides, and nucleic acids.

1. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Department of Molecular Biology and Biotechnology Acharya Narendra Deva University of Agriculture & Technology, Kumarganj, Ayodhya The word was first derived from the Greek word chroma meaning “colour” and graphy means “writing”. Chromatography is the science which studies the separation of molecules based on differences in their structure and/or composition. Definition:“It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.” The Russian botanist Mikhail Semenovitch Tswett coined the term chromatography in 1906. The first analytical use of chromatography was described by James and Martin in 1952,. A wide range of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties to separate materials.  HPLC: A better form of column chromatography. Instead of draining down through the stationary phase, the solvent is forced through under high pressure. 1967 : beginning of HPLC after the works of HUBER and HUZSMAN, this technique was first named « High Speed Liquid Chromatography » then « High Pressure Liquid Chromatography » and finally « High Performance Liquid Chromatography » HPLC is considered to be the major chromatographic technique available today for non-volatile or heat-sensitive substances. Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. According to form of stationary phase a) Planar chromatography i- Paper chromatography ii- Thin-layer chromatography b) Column chromatography i- Gas chromatography ii- Liquid chromatography (LC/HPLC) Douglas A. Skoog, Donald M. West, F. James Holler, & Stanley R. Crouch, Fundamentals of Analytical Chemistry, 9ᵗʰ edition, Brooks/Cole, 20 Davis Drive, Belmont, CA, 2014. Chapter 33 High-Performance Liquid Chromatography 912-932 INSTRUMENTATION HPLC CLASSIFICATION OF CHROMATOGRAPHY INTRODUCTION REFERENCE HPLC profiles of the extracted palants Submitted to: Dr. N.A. Khan Professor (Molecular Biology & Biotechnology) Dr. A.K. Pandey Assistant Professor (Department of Soil Science & Agriculture Chemistry) Submitted by: Ayush Srivastava - A-14690/23 (Molecular Biology & Biotechnology) Method a sample is injected solvent and sample are pushed through under pressure different compounds have different retention times output can be detected by compounds absorbing UV can be connected to a mass spectrometer 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, and a detector. Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.