Capitulo III HPLC columnas Video 11

Arquitectura y Propiedades de Columnas Cromatográficas

This section discusses the architecture and physical-chemical properties of chromatographic columns available in the market.

Column Characteristics

• Stainless steel columns come in various diameters and lengths, resistant to typical pressures in HPLC (up to 6000 psi) and UHPLC (up to 15,000 psi). They are corrosion-resistant.

• Columns have internal surfaces without grooves or micro-porous structures. Terminations consist of porous material discs serving as a filtering membrane.

• Column diameters vary based on purpose; preparative columns range from 10mm to 30cm, while analytical columns range from 2mm to 46mm internally.

Configuración de Columnas y Pre-columnas

This section explains the installation of guard or pre-columns between the injector and analytical column for particle retention and prolonging analytical column lifespan.

Guard and Pre-columns

• A guard column is typically two to five centimeters long, containing the same stationary phase as the analytical column. It retains particles that could contaminate or reduce efficiency.

• Pre-columns retain strongly bound compounds with high k values, extending the life of expensive analytical columns by preventing contamination.

Selección de Columnas en HPLC

Factors influencing High-Performance Liquid Chromatography (HPLC) column selection include stationary phase morphology, particle size, pore size, chemical properties, length, and diameter.

Considerations for Column Selection

• Morphology of stationary phase particles can be irregular or spherical; particle size ranges from 2 to 60 microns with pore sizes between 80 and 300 angstroms.

• Chemical properties such as constituent chemical groups in the stationary phase affect stability, permeability, capacity, selectivity, and efficiency.

Evolución de Partículas Cromatográficas

Evolution of chromatographic particles from irregular porous alumina to homogeneously packed particles enhancing efficiency.

Chromatographic Particle Evolution

• Original irregular porous alumina particles evolved into more homogeneous packed particles ranging from 2 to 5 microns. These smaller particles improve interaction with biomolecules like proteins.

Tipos de Partículas Cromatográficas y Usos

Different types of chromatographic particles impact interactions within columns based on their porosity and size.

Chromatographic Particle Types

New Section

This section discusses the efficient separation of proteins and small molecules using columns in chromatography.

Understanding Column Handling

• Use 0.45-micron filters for mobile phase solvents.

• Follow recommended pH ranges, avoid mechanical vibrations, extreme temperatures, and respect pressure limits.

• Silica surface consists of chemically reactive groups that covalently bond to different chemical groups.

• Different types of stationary phases are discussed, including polar and non-polar ones like C18.

New Section

This section delves into the characteristics of various stationary phases used in liquid chromatography.

Types of Stationary Phases

• Polar and non-polar stationary phases like C8 are explained.

• Stationary phases confer selectivity towards similar groups based on their chemical structure.

• Strategies to prevent partition absorption in columns are discussed for efficient chromatography.

New Section

This section focuses on selecting the most suitable column for optimal chromatographic performance.

Choosing the Right Column

• Factors influencing column efficiency include chemical groups in the stationary phase and mobile phase.

• Smaller particle diameter leads to more theoretical plates but also higher pressure due to reduced permeability.