In modern high performance liquid chromatography, the separation effect depends largely on the choice of chromatographic packing. However, the choice of chromatographic packing is very wide. To make a suitable choice, we must first have a certain understanding and understanding of this. The liquid chromatograph packing may be a ceramic substrate of an inorganic nature or an organic polymer matrix. The organic matrix in the liquid chromatograph packing is primarily silica gel and alumina. The inorganic matrix is ​​rigid and does not easily swell in a solvent. The organic polymer matrix in the liquid chromatograph filler mainly has crosslinked styrene-divinylbenzene and polymethacrylate. The organic polymer matrix is ​​small in rigidity and easy to be compressed, and the solvent or solute easily penetrates into the organic matrix, causing the filler particles to expand, thereby reducing mass transfer and ultimately reducing column efficiency. The following Lutron Analytical Instruments engineers briefly describe the properties of the three most widely used substrates in liquid chromatography.
1) Silica gel matrix Silica gel matrix is ​​the most common matrix in HPLC packing. In addition to high strength, it also provides a surface that can be bonded to various ligands by mature silanization techniques to form a packing for reverse phase, ion exchange, hydrophobic interaction, hydrophilic action or size exclusion chromatography. Silica matrix fillers are suitable for a wide range of polar and non-polar solvents. The disadvantage is that it is unstable in the alkaline water-soluble mobile phase. Typically, silica gel matrix fillers are recommended for routine analytical pH ranges from 2 to 8.
2) Alumina matrix The alumina matrix has the same good physical properties as silica gel and is also resistant to a large pH range. It is also rigid and does not shrink or swell in the solvent. However, unlike silica gel, the alumina bonded phase is unstable in the aqueous mobile phase. However, an alumina bonded phase which is stable in the aqueous phase has now appeared and shows excellent pH stability.
3) Polymer matrix Polymer matrix The high-crosslinking styrene-divinylbenzene or polymethacrylate-based filler is used for HPLC under normal pressure, and its pressure limit is lower than inorganic filler. The styrene-divinyl phenyl group is highly hydrophobic. Use any mobile phase to stabilize over the entire pH range and wash the column with NaOH or a strong base. The methacrylate matrix is ​​inherently more hydrophobic than styrene-divinylbenzene, but it can be rendered hydrophilic by appropriate functional group modifications. This matrix is ​​not as acid and alkali resistant as styrene-divinylbenzene, but can also withstand repeated rinsing at pH 13.
All polymer matrices expand or contract as the mobile phase changes. High cross-linking polymer fillers for HPLC have limited expansion and contraction. Solvents or small molecules readily penetrate into the polymer matrix because the mass transfer of small molecules in the polymer matrix is ​​slower than in ceramic matrices, resulting in low column efficiency in such matrices. For macromolecules like proteins or synthetic polymers, the performance of the polymer matrix is ​​comparable to that of a ceramic matrix. Therefore, polymer matrices are widely used to separate macromolecular substances.
The choice of liquid chromatograph matrix generally follows the following rules. Silica gel matrix fillers are used for most HPLC analyses, especially for small molecular weight analytes. Polymer fillers are used for large molecular weight analytes. Molecular exclusion and ion exchange columns.