Gel-Permeation Chromatography
Introduction:
Gel Permeation Chromatography (GPC) is a separation technique used to detect and identify polymeric and biological compounds such as proteins. GPC analysis is also used to determine the molecular weight of different molecules. In GPC analysis, the sample molecules are first completely dissolved in a suitable solvent. These molecules then pass through porous columns containing gel particles, where they are separated based on their size. As the polymer solution passes through the column, smaller molecules penetrate the gel matrix, while larger molecules are excluded and exit with a delay.
Essentially, this analysis separates molecules based on their molecular weight, considering the time they exit the column. It is used to identify and diagnose polymeric and biological compounds, such as proteins. This technique relies on the separation of molecules based on their size and molecular weight. GPC is a powerful tool in chemical analysis, providing precise separation of polymeric and biological compounds. It is used in a variety of applications, making it a key technique in fields such as analytical chemistry, materials science, and biology.
Principle of Operation:
Dissolving the Sample: The GPC process begins by dissolving the sample molecules in a suitable solvent.
Separation through the Column: The solution is passed through a column containing a porous stationary phase, where the molecules are separated based on their size. Larger molecules exit the column first, while smaller molecules are delayed because they penetrate the smaller pores of the column.
Molecular Weight Measurement: The molecular weight of the compounds is calculated relatively by comparing the molecules’ elution time with standard data or a calibration curve.
Advantages of GPC:
1. Accurate Separation: GPC offers high accuracy in separating molecules based on their size and molecular weight.
2. Ease of Use: Sample preparation and analysis are simple, making it a convenient technique for laboratory use.
3. Molecular Weight Determination: GPC is effectively used to determine the molecular weight of polymers and biological compounds.
Applications:
Protein Analysis: GPC is used to study proteins and determine their molecular weight.
Polymer Analysis: It is used to determine the properties of polymers that are soluble in organic solvents.
Environmental Sample Analysis: GPC is used to analyze environmental pollutants with low molecular weights.
Drug Development: It is used in drug development to separate active compounds from impurities.
Reagents Used in GPC:
Refractive Index (RI) Detector: Measures changes in the refractive index of the solution.
UV-Visible Detector: Used to detect compounds that absorb light in the UV-visible range.
Infrared (IR) Detector: Used to analyze chemical composition based on infrared absorption.
Fluorescence Detector: Detects fluorescent compounds in the sample.
Electrochemical Detector: Measures the current produced by chemical reactions.