High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) utilizes a high-pressure chromatographic column to separate components of liquids or solids dissolved in liquids, followed by detection and analysis. The molecular species of interest (termed analyte) together with the solvent and other components (termed matrix) form the ‘mobile phase’ that flows through the column. The Stationary phase in the column can be a porous solid, stagnant liquid, a liquid layer on the solid phase, or an interfacial layer between liquid and solid.

In HPLC, the analyte molecules are partitioned between mobile and stationary phases, depending on their relative affinities for the stationary phase. The extent of separation is influenced by the molecular structure of the analyte and properties of stationary and mobile phases. Analyte molecules with greater affinity to the stationary phase are held back for longer durations within the column, while the other molecules are eluted earlier. All eluted molecules are detected when they exit the column, with the signal intensity (peak) characterizing the nature and amount of each molecule. Considerations for mobile phase selection include composition, solubility, UV transparency, viscosity, and miscibility with other solvents. Stationary phases are typically porous particles made of inorganic silica compounds, Aluminum oxide or polymeric beads packed into a column.

The HPLC equipment includes injector arrangement, high pressure pump, column, detector and computer based data acquisition, control and analysis. The major types of liquid chromatography include normal phase chromatography, reverse phase chromatography, ion exchange, size exclusion chromatography, and affinity chromatography. Various types of detectors can be used, such as differential refractive index (DRI), UV, fluorescence, radiochemical and electrochemical. HPLC can also be coupled to Mass spectrometer or NMR for further analysis.

The HPLC technique is widely used for separation, purification and analytical applications in academic and industrial research, especially for mixtures of complex synthetic and natural organic molecules.

Common Uses of High Performance Liquid Chromatography (HPLC)

• Molecular weight determination of complex organics
• Molecular weight distribution determination
• Separation and purification of organic compound
• Forensics
• Pathology testing
• Drug evaluation
• Proteins fractionation
• Environmental testing

Advantages of High Performance Liquid Chromatography (HPLC)

• High sensitivity
• Identifies complex molecules
• Well defined separation.
• Wide range of solvents and column packings available
• Small quantity of sample.

Limitations of High Performance Liquid Chromatography (HPLC)

• Structural information on substances separated by HPLC is limited by the detector system employed.
• Compounds with similar structure and polarity can elute simultaneously making identification difficult
• In normal phase HPLC, silica packing can be affected by water or protic organic solvents which change the hydration state of the silica.

 Industrial Applications of Gel Permeation Chromatography (GPC)

• Pharmaceuticals,
• Foods and beverages,
• Polymers,
• Environmental monitoring
• Forensics