The full name of C18 chromatography column is octadecylsilane bonded silica gel chromatography column, which is a reversed-phase chromatography column. "C18" refers to the chemical structure of the stationary phase, that is, the chromatographic column packing is an octadecylsilane bonded phase. The stationary phase of this chromatographic column is adsorbed on the silica matrix through chemically bonded octadecyl functional groups. . The principle of C18 chromatographic column is based on the difference in hydrophilicity and hydrophobicity of compounds on the surface of C18 packing material, and the separation of compounds is achieved through solvent gradient elution. Specifically, the non-polar carrier surface has hydrophobic characteristics and can adsorb non-polar compounds, while the polar mobile phase usually contains organic solvents and buffers, which are highly polar and play an important role in separation.
1. New column activation 1. Flushing: Before use, the new column needs to be flushed with pure organic reagents (such as methanol, acetonitrile) at a low flow rate. The flushing volume is preferably 20 times the column volume, so that the stationary phase can be fully wetted and the carbon chain can be fully wetted. Stretch to maximize column performance. 2. Anti-shelling treatment: Immerse the new column in pure methanol or acetonitrile solution, rotate or otherwise make the surface fully contact for at least 30 minutes. After washing away most of the residual organic matter, rinse with pure water or 75% methanol or acetonitrile 2 to 3 times until the output liquid is colorless and odorless. 3. Acid-alkali washing treatment: After the anti-shelling treatment is completed, use 0.11% acetic acid solution to drop on the surface of the chromatographic column and stay for about 30 minutes, and rinse thoroughly with DI water. Then use 0.11% ammonia solution to adsorb the amino-containing sample on the column and stay there for about 30 minutes, and then push it out with DI water again until the output is stable. 4. Gradient elution: Add a mixture of isopropyl alcohol and formic acid with a molar ratio of 1:1 into the column, and gradient elute at a flow rate of 0.5 mL/min (10% B, 3min; 40% B, 3min; 95% B, 3min, reset to 10% transition for three minutes) to allow faster recovery of open free colloids.
2. Daily use 1. Sample processing: Ensure that the sample is properly processed to remove impurities and interfering substances to improve the separation effect. 2. Mobile phase selection: Select the appropriate mobile phase composition and ratio according to experimental requirements. 3. Flow rate control: Control the appropriate flow rate to avoid excessively high or low flow rates affecting the separation effect. 4. Pressure monitoring: During use, pay attention to monitoring the pressure changes of the chromatographic column, and detect and deal with abnormal situations in time.
3. Maintenance 1. Flush the chromatographic column: After the daily experiment, the chromatographic column should be flushed, especially when the mobile phase contains acid or salt, the acid or salt in the chromatographic column needs to be flushed. It is usually recommended to use 10% methanol water. Flush 20 column volumes. 2. Avoid high water phase conditions: Since the stationary phase of the C18 column (except those with polar modification) is highly hydrophobic, try not to use high water phase conditions during use. If the water phase ratio is too high, it may easily cause the stationary phase to become unstable. Hydrophobic collapse leads to a rapid decline in column efficiency and even irreversible negative effects. It is recommended that the water phase ratio does not exceed 90% during use. 3. Regeneration treatment: When the chromatographic column pressure increases and column performance decreases, it is usually because the chromatographic column is contaminated. Regeneration of the chromatographic column can restore part of the column efficiency.
4. Long-term storage 1. Flushing: The chromatographic column should be flushed for more than 3 to 4 hours before long-term storage. 2. Storage solvent: Store in pure organic reagents or high-proportion organic reagent solutions (such as 90% methanol water). If it needs to be used again the next day, the daily storage solvent should be the same as this.
5. Precautions 1. Avoid violent vibration to avoid damaging the chromatographic column. 2. Prevent drying: When not in use for a long time, make sure the column is filled with appropriate solvent and seal the plugs at both ends to prevent drying and contamination. 3. Service life: The service life of the C18 chromatographic column is about 100 to 200 times. When the peak shape of the output result becomes blurred and the output stability decreases, please replace the column with a new one in time.
6. Common problems and solutions 1. Salting out problem: When using C18 chromatographic column, salting out phenomenon may occur. This is usually caused by salts in the mobile phase leaching out in the column. In order to solve this problem, measures such as optimizing the composition of the mobile phase, using a proportional valve that is resistant to high salt, or adding a column thermostat can be taken.
2. Chromatographic column clogging: Chromatographic column clogging is another common problem. This may be due to impurities in the mobile phase, insoluble materials in the sample, or buffer salts deposited in the column. To solve this problem, measures such as flushing with a high aqueous phase, using a guard column or a pre-column filter can be used to prevent clogging. If the chromatographic column is clogged, you can try to recover it by using methods such as backflushing or ultrasonic cleaning. However, please note that cartridge cores and some types
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