How to clean C18 packing? C18 packing is a commonly used reversed-phase chromatography packing and is widely used in liquid chromatography analysis. Since its surface has long-chain alkyl groups, it is easy to adsorb impurities in the sample, so regular cleaning of C18 packing is an important step to ensure its separation performance.
1. Routine cleaning methods 1. Solvent flushing: Use an appropriate pure solvent (such as methanol, acetonitrile, etc.) to flush through the chromatographic column at an appropriate flow rate (usually 1~2 mL/min). The flushing time can be adjusted according to the degree of contamination of the chromatographic column and the required flushing effect, generally 15 to 30 minutes. This method is suitable for situations where there is no obvious contamination on the surface of the chromatographic column and can effectively remove impurities inside the column. 2. Solvent gradient flushing: Use different proportions of organic solvents and water as mobile phases, and gradually change the proportion of solvents to achieve flushing of the chromatographic column. Start with a lower proportion of organic solvent (e.g. 80% organic solvent concentration) and then gradually increase the proportion of organic solvent to 100%. The solvent ratio and flow rate should be kept stable to ensure uniform flushing results. This method is suitable for serious contamination of the chromatographic column and can more thoroughly remove residues from the chromatographic column.
2. Cleaning methods for specific pollution 1. Pollution caused by the precipitation of buffer salts: If the use of buffer salts (such as phosphates, etc.) that are not easily soluble in the organic phase causes precipitation and blockage of the column, you can first use hot water at about 60°C at low temperature to Flush the column at a flow rate that lasts for a long time. If the buffer salt precipitates in the chromatographic column, you can also try to increase the column temperature and use a large proportion of water phase or pure water phase to rinse for a long time. The most common practice is to flush 10 column volumes with 5% methanol (or acetonitrile) aqueous solution to remove residual buffer salts and polar compounds. 2. Conventional organic pollution: First flush 10 column volumes with 5% methanol (acetonitrile) aqueous solution to flush out the buffer salt and some polar compounds in the column. Then flush 10 to 20 column volumes with pure methanol or pure acetonitrile to flush out some hydrophobic impurities. If the pressure is still high, the flow rate can be appropriately reduced and the flushing time extended. If the above method is ineffective, you can choose isopropyl alcohol (or tetrahydrofuran) to flush 10 to 20 column volumes. 3. Protein and peptide clogging: 0.1% trifluoroacetic acid (TFA) can be added to the cleaning mobile phase to enhance the elution ability of adsorbed proteins. If the problem still cannot be solved, you can try injecting 100 μL trifluoroethanol into the column. For old columns, the number of injections can be increased appropriately to clean out strongly retained proteins.
3. Regeneration and reverse connection of chromatographic column 1. Regeneration: Use a series of solvents (such as 10% methanol, acetonitrile, isopropyl alcohol, etc.) to flush the chromatographic column at a low flow rate. Each solvent flushes for a period of time (such as 4 hours), totaling 20 hours. During the regeneration process, attention should be paid to the connection method and to prevent contaminants from contaminating the detection cell. 2. Reverse connection: Reverse the chromatographic column and flush it with acetonitrile and other solvents at a low flow rate for a period of time (such as 6 hours). Reverse connection is the last struggle before the column is scrapped. After reverse connection, it cannot be used in normal connection.
4. Precautions 1. Solvent selection: Make sure the solvent used is chromatography grade, and remove bubbles by ultrasonic before use. 2. Flow rate control: During the flushing process, the appropriate flow rate should be selected based on the pressure resistance of the chromatographic column and the viscosity of the solvent. 3. Column temperature control: During the cleaning process, the column temperature can be appropriately increased to enhance the cleaning effect. 4. Avoid salt contamination: Avoid using mobile phases containing salt during long-term storage, because salt may cause damage to the chromatographic column during the storage process. 5. Regular maintenance: Clean and maintain the chromatographic column regularly to extend its service life and maintain the separation effect.
Cleaning of C18 packing is a key step to ensure its long-term stability and efficient separation performance. By selecting appropriate cleaning agents, optimizing cleaning conditions, and using appropriate cleaning methods, we can effectively remove contaminants and residues on the surface of the packing and restore its original separation capacity and service life.
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