Liquid chromatography plays a crucial role in the biomedical industry due to its high precision in analyzing low molecular weight polymer compounds. However, over time, polymeric columns used for separating biomolecules can become contaminated, leading to reduced efficiency and impacting detection sensitivity and speed. Regular cleaning is therefore essential to maintain column performance.
The chemical stability of polymeric materials is typically evaluated based on their resistance to various solvents and pH conditions. 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 monoliths, and Swift columns, are capable of withstanding a broad pH range—usually from pH 11 to 13, and sometimes even from pH 0 to 14. However, when using strong organic solvents, it's important to consider the degree of cross-linking, as this affects how the polymer expands or contracts during washing.
Polymers with a cross-linking level above 8–10% tend to have better mechanical stability, shrink minimally in aqueous solutions, and remain relatively stable in organic solvents.
For regenerating a PS-DVB monolithic column, the recommended procedure involves flushing 10 column volumes at half the working flow rate with 0.1% tribasic 2-propanol. Then, wash 5 or more column volumes with 100% mobile phase B at half the working flow rate, followed by equilibration with at least 10 column volumes of 100% mobile phase A at the working flow rate.
For mono- and butyl methacrylate monolithic columns, a common method to remove protein residues from silica gel-bonded reverse-phase columns is back-flushing with 10 column volumes of sodium hydroxide solution, followed by rinsing with water, 20% ethanol, and the working buffer. If there are many hydrophobic proteins, adding a step with 30% isopropanol or 70% ethanol after water can help improve the cleaning process.
To eliminate microbial contamination, PS-DVB columns can be fully washed with 0.5 to 1.0 M sodium hydroxide, and the column should be left in contact with the solution for at least one hour at room temperature.
When dealing with less soluble proteins like membrane proteins, structural proteins, or viral coat proteins, stronger cleaning conditions may be required. For example, a 50% isopropanol solution containing 3 M guanidine hydrochloride can effectively remove these proteins at 60°C.
In solid-phase synthesis, the removal of synthetic peptides can lead to the formation of positive ions that react with anisole and thioanisole, producing large aromatic molecules that contaminate the column during purification. These contaminants are highly retained on C18 columns and cannot be removed by 100% methanol or acetonitrile. To clean such columns, flush with 5 volumes of 100% isopropanol, then 3–5 volumes of dichloromethane, followed by another 3–5 volumes of isopropanol, and finally return to the initial solvent system. The presence of aromatic impurities can be detected using UV absorbance at 260 nm.
Optical Fibre To Rj45 Converter,Sc To Sc Coupler,Sc Duplex Adapter,Sc To St Adapter
Ningbo Fengwei Communication Technology Co., Ltd , https://www.fengweicommunication.com