Knowledge About Normal Phase Solid Phase Extraction

In the lab, we often use SPE cartridges, but do we understand the principle of normal phase solid phase extraction?

Normal phase solid phase extraction is the dissolution of polar substances in non-polar solvents (diethyl ether, n-butane, etc.), which are adsorbed by a strong polar stationary phase. The interaction between them includes dipole pairs, hydrogen bonds, and electron pairs. This material needs to be eluted with a strong polar substance, and the solvent strength factor ε>0.6 of the eluent is required, such as methanol (ε=0.73). The commonly used adsorbents are alumina, magnesium silicate, diatomaceous earth, and silica gel.

Normal phase solid phase extraction has been used to separate and purify vitamin D and its metabolites, fatty acids, carbohydrates, and phenolics in biological samples. Generally used as a strong polar adsorbent such as diatomaceous earth, silica gel, alumina, magnesium silicate, etc., the target analyte is a non-polar or weakly polar compound, such as fat-soluble vitamins, pesticides, and the like. The filler is most widely used in silica gel.

A large amount of silanol-based polar sites present on the surface of silica gel can adsorb moderately polar compounds such as alcohols, aldehydes, and organic halides dissolved in non-polar solvents, and then washed with a solvent larger than 0.6. Take off. In recent years, molecular sieves, glass beads, graphite carbon, and chemically bonded phases with amino groups and cyano groups, which are slightly weaker than silica gel, have been used as fillers.

The latter applies to the separation of moderately polar compounds such as aldehydes, ketones, and nitro compounds. Hawach’s normal phase solid phase extraction columns include Normal Phase Alumina SPE Cartridges, Normal Phase CN SPE Cartridges, Normal Phase Diol SPE Cartridges, Normal Phase PR Grade Florisil SPE Cartridges, Normal Phase Silica SPE Cartridges.

Cleaning HPLC (High-Performance Liquid Chromatography) columns is an essential maintenance task to ensure the accuracy and reproducibility of your chromatographic analyses. When selecting a cleaning solvent for HPLC columns, you need to consider the type of column, the nature of the contaminants, and the compatibility of the solvent with your specific application. Here are some common cleaning solvents used for HPLC columns:

1. Reversed-Phase Columns (C18, C8, etc.):

  • Acetonitrile: Acetonitrile is a widely used cleaning solvent for reversed-phase columns. It effectively removes nonpolar contaminants and can be mixed with water in varying proportions to create a gradient for more efficient cleaning.
  • Methanol: Methanol is another popular choice for cleaning reversed-phase columns. Like acetonitrile, it’s effective at removing nonpolar residues. Methanol can also be mixed with water to create a cleaning gradient.

2. Normal-Phase Columns:

  • Hexane: Hexane is often used to clean normal-phase columns, which are typically used for separating nonpolar compounds. It helps remove nonpolar residues and contaminants.
  • Isopropanol (IPA): Isopropanol is another option for cleaning normal-phase columns. It’s a polar solvent and can be used to remove polar contaminants.

3. Ion-Exchange Columns:

  • Buffer Solutions: For ion-exchange columns, cleaning with buffer solutions at different pH levels is common. The choice of buffer depends on the specific type of ion exchange (e.g., anion or cation) and the nature of the contaminants.

4. Size-Exclusion Columns:

  • Buffer Solutions: Size-exclusion columns are best cleaned with buffer solutions that are compatible with the column and the sample.

5. Specialty Columns (Chiral, Affinity, etc.):

  • Consult Manufacturer’s Recommendations: Specialty columns may require specific cleaning solvents or procedures recommended by the manufacturer. Always refer to the manufacturer’s guidelines for cleaning such columns.

General Cleaning Guidelines:

  • Always check the manufacturer’s recommendations for your specific HPLC column. They may provide specific cleaning procedures and solvents.
  • When changing between different mobile phase solvents, perform equilibration runs to ensure that the new solvent does not interfere with the previous solvent used in the column.
  • It’s essential to ensure that the cleaning solvent is compatible with the column’s stationary phase material.
  • For stubborn contaminants, consider using a stronger solvent or a solvent with a higher organic content, but be cautious not to damage the column.
  • Perform a system suitability test or a blank run after cleaning to ensure that the column is functioning correctly.
  • Dispose of waste solvents and rinse solutions properly, following laboratory waste disposal protocols.

Proper cleaning and maintenance of HPLC columns are crucial for achieving accurate and reproducible chromatographic results. Always follow the manufacturer’s guidelines and best practices to extend the lifespan of your columns and maintain the integrity of your analyse