Liquid chromatography chromatograph column regeneration method - Database & Sql Blog Articles

Liquid chromatography plays a crucial role in the biomedical field due to its high precision in analyzing low molecular weight polymer compounds. Over time, polymeric columns used for separating biomolecules can become contaminated, leading to reduced efficiency and affecting detection sensitivity and speed. Regular cleaning is essential to maintain column performance. The chemical stability of polymeric materials is typically evaluated based on their structural integrity. Common cleaning agents include nitric acid or sodium hydroxide solutions. Some reverse-phase polymer columns, such as polystyrene-divinylbenzene (PS-DVB) beads, CIM RP-SDVB disks, and Swift columns, are designed to withstand a broad pH range—usually from pH 11 to 13, and sometimes even from pH 0 to 14. However, when using strong organic solvents, users must be cautious, as the degree of cross-linking determines how the polymer responds. Some polymers may expand or contract when exposed to certain solvents. Columns with a cross-linking level above 8–10% generally exhibit better mechanical stability, minimal shrinkage in aqueous environments, and limited expansion in organic solvents. To regenerate a PS-DVB monolithic column, it is recommended to flush 10 column volumes at half the working flow rate with 0.1% tribasic 2-propanol. Then, wash with 100% mobile phase B at half the working flow rate for at least 5 column volumes, followed by equilibration with 100% mobile phase A at the working flow rate for at least 10 volumes. For columns packed with methacrylate-based monoliths, cleaning involves backwashing 10 column volumes of sodium hydroxide solution, followed by washing with water and a 20% ethanol solution, along with the working buffer. If there is a high concentration of hydrophobic proteins, an additional step using 30% isopropanol or 70% ethanol after water is advised. If microbial contamination is present, the PS-DVB column can be fully cleaned using 0.5–1.0 M sodium hydroxide. The column should be washed with sodium hydroxide for at least one hour at room temperature. For columns packed with conventional polymeric matrices used to separate less soluble proteins like membrane proteins, structural proteins, or viral coat proteins, more aggressive cleaning conditions may be required. For example, a 50% isopropanol solution containing 3 M guanidine hydrochloride can effectively remove these proteins at 60°C. When removing synthetic peptides from solid-phase resins, positive ions can form during the cleavage process. These ions can react with anisole and thioanisole, generating large aromatic molecules that may contaminate the column during purification. These contaminants tend to strongly retain on C18 columns and cannot be removed with 100% methanol or acetonitrile. To clean such columns, reverse flushing with 5 volumes of 100% isopropanol, followed by 3–5 volumes of dichloromethane, then another 3–5 volumes of isopropanol, and finally returning to the initial solvent system is recommended. Aromatic impurities can be detected using UV absorption at 260 nm.

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