In chromatographic analysis, how to choose the best chromatographic conditions to achieve the most ideal separation is one of the important aspects of chromatographic work. The HPLC column can be a ceramic inorganic matrix or a mailing polymer matrix. The inorganic mechanism is mainly silica gel and alumina. Inorganic substrates are rigid and not easy to swell in solvents. The organic polymer matrix is mainly cross-linked styrene and polymethacrylate. The organic polymer matrix is small in rigidity and easy to compress. Solvents or solutes can easily penetrate into the organic matrix, causing the filler particles to expand, resulting in reduced mass transfer, and ultimately reduced column efficiency.
1. The matrix applied in HPLC Column is divided into silica gel and alumina. Silica gel is the most common matrix in HPLC packing materials. In addition to high strength, it also provides a variety of ligands on the surface that can be bonded by mature silanization technology to make packing for reverse phase, ion exchange, hydrophobic interaction, and hydrophilic interaction chromatography.
Silica-based 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. Generally, the recommended routine analysis pH range for silica-based fillers is 2-8.
2. Alumina has the same good physical properties as silica gel and can withstand a larger pH range. It is also rigid and will not shrink or swell in solvents. But unlike silica gel, the alumina bonded phase is unstable in the aqueous mobile phase.
Fillers based on highly cross-linked styrene or polymethacrylate are used for HPLC under ordinary pressure, and their pressure limit is lower than that of inorganic fillers. The styrene matrix is highly hydrophobic. The methacrylate matrix is inherently more hydrophobic than styrene, but it can be modified by appropriate functional groups to become hydrophilic. This matrix is not as acid and alkali resistant as styrene, but it can withstand repeated washing at pH 13.
3. All polymer matrices will swell or shrink when the mobile phase changes. Highly cross-linked polymer fillers used in HPLC have restrictions on their expansion and contraction. Solvents or small molecules easily penetrate into the polymer matrix. Because the mass transfer of small molecules in the polymer matrix is slower than that in the ceramic matrix, the column efficiency of small molecules in this matrix is low. For large molecules like proteins or synthetic polymers, the performance of the polymer matrix is comparable to that of the ceramic matrix. Therefore, polymer matrices are widely used to separate macromolecular substances.
4. Reversed-Phase Chromatography:
Matrix: Usually composed of hydrophobic materials like hydrocarbon chains (e.g., C18 or C8), which interact with non-polar or weakly polar analytes.
5. Normal Phase Chromatography:
Matrix: Consists of polar materials like silica gel or alumina. This type of matrix interacts with polar analytes, making it suitable for separating polar compounds.
6. Ion Exchange Chromatography:
Matrix: Contains charged functional groups (e.g., positively charged for cation exchange, or negatively charged for anion exchange) that interact with oppositely charged analytes.
7. Size Exclusion Chromatography (SEC):
Matrix: Made up of porous materials designed to separate analytes based on their size. Larger molecules are excluded from the pores and pass through the column faster.
8. Affinity Chromatography:
Matrix: Contains ligands that specifically bind to certain types of analytes (e.g., antibodies and antigens). This type of chromatography is highly selective for particular interactions.
The choice of matrix is crucial, as it directly influences the separation efficiency and selectivity of the chromatographic process. Factors such as the nature of the analytes, the sample matrix, and the separation goals will dictate which type of matrix is most appropriate for a given application.
It’s important to note that the matrix is packed into the column with precision to ensure a consistent and uniform stationary phase. Additionally, the quality and properties of the matrix significantly affect the performance and longevity of the column, making it a critical consideration in HPLC method development.