Understanding the C18 Columns: Activation, Maintenance, and Usage Tips

High-Performance Liquid Chromatography (HPLC) is integral to various fields, including pharmaceutical diagnostics, food safety, chemical engineering, environmental monitoring, biomedicine, forensic analysis, and energy research.

At the heart of chromatographic techniques lies the HPLC column, where the selection of stationary phase significantly impacts analytical outcomes.

To accommodate diverse sample analyses, engineers have developed an array of bonded phases, such as C18, C8, phenyl, amino, chiral stationary phases, size exclusion, and ion exchange phases.

Among these, the C18 column stands out as the most widely used. Proper usage and maintenance of C18 columns are thus critical for optimal performance.

Pre-use activation is necessitated when columns are found dried out. The reason for column dry-out varies from the loosening of end caps to the loss of storage solvent. Activation involves wetting the column with the appropriate storage solvent at a low flow rate.

For new columns, the purity of storage solvents can vary significantly across brands, leading to increased noise and baseline drift during analyses. To minimize these effects, it’s crucial to replace any storage solvent before first use. Various reasons like the loosening of end caps may also cause solvent evaporation and column drying during shipping and storage.

Activation of new columns typically last 1 to 4 hours according to their respective manufacturer’s instructions. If peak shapes remain abnormal after a short activation, extend the activation to overnight at a flow rate of 0.2-0.3 mL/min using the specified solvent from the instruction.

For used columns, activation is also required if not used for over a month, especially if left connected to the system for more than a week.

To ensure the longevity and efficiency of C18 columns, follow these guidelines:

• Use HPLC-grade organic solvents and high-purity water. All additives should also be of HPLC or analytical grade.
• Filter mobile phases using a 0.45 µm or smaller membrane before use. Choose filters and membranes that match solvents. For instance, use organic membranes for organic solvents and avoid aqueous filters that can dissolve in organic phases.
• Ideally, prepare mobile phases for use within 24 hours to prevent microbial contamination.
• Before using mobile phases that contain buffer salt (e.g phosphate), transition the high organic storage phase using 10% methanol (or a similar transition phase) first. Similarly, replace the buffer salt phase using transition phases after use and then flush out with high organic storage phase.
• Before using mobile phases that are insoluble in water (e.g dichloromethane), transition using a solvent that dissolves in both phases (isopropanol in the above example) first.
• Prevent sudden temperature fluctuations. Connect the column and allow the mobile phase to circulate before gradually increasing the temperature, and vice versa.

During routine analyses, particularly with complex herbal samples, contaminants may adsorb onto the column, leading to increased backpressure and reduced separation efficiency. Besides using guard columns, regeneration can restore column performance.

For columns contaminated by complex samples, reverse the column flow (not recommended if the mobile phase pH exceeds 7) and flush with:

1. 10% organic solvent to remove buffering salts
2. Pure acetonitrile at 1 mL/min for 30 minutes
3. Isopropanol at 0.2 mL/min for at least 4 hours (overnight is ideal)
4. Finally, flush with pure acetonitrile at 1 mL/min for another 30 minutes.

For columns contaminated by biological samples or affected by microbial growth, flush overnight with a low-speed acetonitrile-water-TFA (50-50-0.1) mixture.