High Performance Liquid Chromatography (HPLC)

• The HPLC mobile phase, typically a liquid solvent or a mixture of solvents, carries the analyte through the column.
• It plays a crucial role in the separation process by interacting with the stationary phase and the analytes.
• The choice of mobile phase composition, including solvent type, pH, and buffer concentration, influences the selectivity, resolution, and efficiency of the separation.
• Common mobile phase solvents include water, acetonitrile, methanol, and various buffers, depending on the analyte's properties and separation requirements.
• Optimization of the mobile phase column chromatography is essential for achieving optimal chromatographic performance in HPLC analysis.

• The stationary phase is a solid or liquid material packed inside the HPLC column, where the analytes are separated.
• It interacts with the analytes based on differences in polarity, size, charge, and other chemical properties.
• The choice of stationary phase chemistry, such as reversed-phase, normal-phase, ion exchange, or affinity chromatography, determines the selectivity of the separation.
• Stationary phases can be made of silica-based materials, polymers, or other specialty materials, depending on the application.

Fully porous particles are the traditional particles used in HPLC columns. These particles have pores that allow the mobile phase and analytes to diffuse throughout the entire particle. This leads to a high surface area, enabling good separation efficiency. Fully porous particles are versatile and are often applied in a wide range of analytical applications, particularly when high resolution is required.

Superficially porous particles, also known as core-shell particles, consist of a solid, non-porous core surrounded by a thin, porous layer. This unique design reduces the diffusion path of analytes, leading to faster separations with high efficiency, often comparable to sub-2 micron particles but with the advantage of lower back pressure. These particles are ideal for applications requiring rapid analysis without compromising the resolution.

Monolithic particles are made from a continuous porous rod rather than packed particles. This structure offers high permeability, which allows for faster flow rates with lower back pressure. Monolithic columns are known for their robustness and are particularly effective in handling complex matrices. They are well-suited for high-speed separations and are often used in bioanalytical applications, such as the analysis of proteins and peptides.

Sub-2-micron particles are extremely small, providing a very high surface area that results in high efficiency during separations. These particles are commonly used in Ultra-High Performance Liquid Chromatography (UHPLC), a technique that operates at higher pressures to achieve faster and more efficient separations. Sub-2-micron particles are ideal when the highest possible resolution and speed are required, particularly in complex separations involving closely related compounds.

Silica-based particles are the most commonly used stationary phases in HPLC due to their mechanical strength, chemical stability, and compatibility with various modifications, such as bonded phases. These particles can be fully porous or superficially porous and are available in various particle sizes. Silica-based particles are used in a wide range of HPLC applications where chemical stability and mechanical strength are essential.