Liquid chromatographs are mainly divided into two types based on the stationary phase: liquid‑liquid chromatography (LLC) and liquid‑solid chromatography (LSC). Commonly used liquid chromatography systems today generally include key components such as a high‑pressure pump, an injection device, a temperature control system, a column, a detector, and a signal recording system. Compared with traditional liquid column chromatography equipment, modern systems offer clear advantages in analysis speed, separation efficiency, and detection sensitivity, helping laboratory personnel complete analytical work more efficiently.
In liquid chromatography analysis, the daily cleaning and proper storage of the column are particularly critical, as they directly affect the stable performance of the column. Therefore, these two aspects must be handled carefully.
I. Cleaning of Liquid Chromatography Columns
After each sample analysis, the column must be cleaned promptly; otherwise, sample residues or impurities will accumulate in the column, affecting its performance over time. The specific cleaning method depends on the chromatographic mode used and the composition of the mobile phase – there is no one‑size‑fits‑all approach.
Cleaning methods for different chromatographic modes
Reversed‑phase columns: In reversed‑phase chromatography, the more polar components elute first, leaving less polar substances behind in the column. In this case, the column should be flushed with a strong elution solvent, such as 100% methanol, isopropanol, or tetrahydrofuran (THF). Flushing with about 20 column volumes of such a solvent is usually sufficient to remove residual substances.
Cleaning after using buffer‑containing mobile phases: If a buffer salt was used in the mobile phase, the column must be thoroughly flushed with distilled water before switching to an organic solvent – this step cannot be skipped. Skipping it may cause salt crystals to precipitate and clog the column or the system flow path, making later cleaning very troublesome. After flushing with distilled water, continue flushing with an appropriate solvent.
Gel columns: Gel filtration columns mostly use buffer solutions as the mobile phase. After each use, flush the column with distilled water as soon as possible. If the column is to be used continuously and you plan to leave it overnight, you may store the column with buffer solution, but keep the flow rate low to prevent salt precipitation. Special note: if the mobile phase contains corrosive components such as halides, even if stored only overnight, the column must be thoroughly flushed with distilled water to avoid corrosion of the column and the system.
Common cleaning problems and solutions
Problem 1: Column backpressure remains high after cleaning
This is often caused by residual difficult‑to‑elute impurities or small particles blocking the bed. Try increasing the elution solvent strength (e.g., for reversed‑phase columns, replace methanol with isopropanol), extending the flushing time, or flushing at a low flow rate (0.2‑0.3 mL/min) for 30‑60 minutes to allow the solvent to better contact the impurities. If the blockage is caused by particles, disconnect the detector and back‑flush the column with filtered solvent (check the column manual to confirm whether back‑flushing is allowed) to help dislodge the particles.
Problem 2: Peak broadening or decreased resolution after cleaning
This may be due to a sudden change in solvent composition causing the stationary phase to shrink or become damaged. For example, switching directly from a high‑aqueous phase to pure organic solvent can cause the stationary phase in a reversed‑phase column to dehydrate and shrink. The solution is to increase the proportion of organic solvent gradually (10%‑20% increments) during the next cleaning to allow the stationary phase to adapt. If the peak shape change is significant, try flushing at a low flow rate with a low‑strength mobile phase (e.g., 5% methanol‑water) to help the stationary phase slowly recover.
Problem 3: White crystals appearing inside the column due to buffer salt residues
This is a typical problem caused by switching to organic solvent without thoroughly flushing out the salt. Once crystals appear, disconnect the detector and flush the column slowly with distilled water at a low flow rate (0.1‑0.2 mL/min) to dissolve the crystals gradually (do not use a high flow rate, as it may scratch the bed). The flushing time depends on the amount of crystals, usually 1‑2 hours, until the backpressure returns to normal. Then clean the column according to the standard procedure.
II. Storage of Liquid Chromatography Columns
When the column is not in use for a while, the storage conditions directly affect its service life and performance stability, so storage must also be handled carefully.
Storage methods for different durations
Short‑term storage (a few days)
After routine cleaning, tighten the column ends with the appropriate sealed plugs. Be sure to use matching plugs; otherwise, poor sealing will allow air to enter and solvent to evaporate. Then store the column at room temperature in a clean environment, avoiding heat sources or direct sunlight.
Long‑term storage (more than a few weeks)
If the storage period exceeds a few weeks, simply sealing the ends is not enough to maintain column efficiency. In this case, fill the column with a suitable storage solvent recommended in the column manual.
Reversed‑phase columns: Methanol is generally recommended as the storage solvent; it keeps the stationary phase wetted and inhibits microbial growth.
Normal‑phase columns: Organic solvents such as hexane or heptane can be used to prevent the stationary phase from absorbing moisture.
Gel columns: Do not store in pure water, as this can promote microbial growth and affect column performance. It is recommended to flush the column with 0.05% sodium azide (NaN₃) solution before sealing and storing. Sodium azide effectively inhibits microbial growth, but protective measures should be taken during handling to avoid skin contact.
Regardless of the column type, before long‑term storage, ensure the column is filled with the appropriate storage solvent and that both ends are tightly sealed. If the solvent evaporates, the stationary phase may dry out and shrink, leading to a significant loss of column efficiency or even permanent damage – a costly consequence.
Common storage problems and solutions
Problem 1: Bubbles appear inside the column after short‑term storage
This is usually caused by loose sealing plugs allowing air to enter. Connect the column to the system, flush with the storage solvent (e.g., methanol for reversed‑phase columns) at a low flow rate (0.2‑0.3 mL/min) to expel the bubbles. After flushing for 10‑15 minutes, disconnect the column and retighten the plugs. Before the next storage, check that the plugs are intact and tighten them with moderate force to avoid damaging the column ends.
Problem 2: Flocculent material (microbial growth) appears inside a gel column after long‑term storage
This occurs because the storage solvent lacked an antimicrobial agent or the concentration was insufficient. First, discard the old solvent, flush the column with distilled water for 1 hour, then flush with 0.1% sodium azide solution at a low flow rate for 30 minutes to kill the microbes. Then fill the column with fresh 0.05% sodium azide solution and reseal it. If a large amount of flocculent material is present, the column may already be damaged; test its performance with a standard. If the efficiency has dropped by more than 30%, the column is unlikely to function properly.
Problem 3: Significant loss of resolution in a normal‑phase column after long‑term storage
This is likely due to the stationary phase absorbing moisture from a high‑humidity storage environment. Connect the column to the system and flush with a hexane‑isopropanol mixture (95:5) at a low flow rate for 30 minutes to remove moisture. Then replace with pure hexane, seal the column, and store it in a dry environment (adding a small amount of desiccant is advisable). For future storage, it is best to keep normal‑phase columns in a sealed desiccator to prevent moisture absorption.
In summary, cleaning and storing columns according to standard procedures not only extends their life but also ensures the repeatability and accuracy of analytical results – a crucial aspect of quality management in liquid chromatography experiments.
