What Causes Pollution of HPLC Column?
Usually, the sample contains something that is not of interest to the analyst. Salt, lipids, lipid-containing substances, humic acid, hydrophobic proteins, and other biological substances are some substances that may interact with the HPLC Column ( such as CN HPLC Column, C18 Low pH HPLC Column ) in use.
These substances have less or greater retention values than the analyst’s target. Substances with smaller retention values, such as salts, are generally washed into excellent spectral columns as empty volumes. The interference of these non-target products can be detected by detectors and can form chromatographic peaks, bubbles, baseline upward shifts, or negative peaks.
If the sample composition is strongly retained in the HPLC column and the mobile phase solution composition is not enough to elute these substances, after repeated sampling, the substances adsorbed on the surface of the column will usually accumulate on the column header. These behaviors are usually detected only through parallel experiments. Samples with moderate retention values can be washed out slowly and exhibit broad peaks, baseline disturbances, or baseline drift.
Sometimes these adsorbed sample components accumulate to a certain extent to enable them to start forming new stationary phases. The analyzer can interact with these impurities to form a certain separation mechanism. Retention time will fluctuate and tailing will occur. If enough pollution is produced, the back pressure of the HPLC column can exceed the maximum pressure that the pump can withstand, making the column unable to work and generating an empty volume in the blocked place.
Pollution of an HPLC column can significantly affect the accuracy and reproducibility of your results. There are several factors can contribute to column pollution:
Sample Contaminants: The nature of the sample being analyzed can lead to contamination of the column. Particulate matter, dissolved impurities, and compounds that are difficult to remove from the column can accumulate over time.
Matrix Effects: Complex samples often contain components that can interact with the column packing material. These interactions can lead to retention time shifts, peak broadening, and overall deterioration of column performance.
Sample Solvents: Incompatible solvents or solvents that are not properly filtered can introduce impurities or particulates into the column. This can result in clogging and reduced separation efficiency.
Mobile Phase: The composition and quality of the mobile phase can have a significant impact on column performance. Using mobile phases that are not properly filtered, degassed, or prepared can introduce contamination.
Injection Port Contamination: If the injection port is not properly cleaned between injections, sample residues can accumulate and lead to carryover effects. This can affect the reproducibility of results.
Column Maintenance: Inadequate maintenance procedures, such as not properly flushing the column at the end of a run or not storing the column under suitable conditions, can contribute to contamination.
Column Incompatibility: Using a column for a sample type that it was not designed for can lead to contamination and poor separation. Ensure that the column’s specifications match the intended use.
Inadequate Cleaning: After completing an analysis, thorough cleaning of the column is essential to remove any residual compounds and contaminants. Failing to clean the column properly can lead to carryover and decreased separation efficiency.
Column Age: Over time, columns naturally degrade due to use and exposure to various samples and mobile phases. An old or heavily used column might exhibit poorer separation and increased contamination.
Column Quality: Poor-quality or defective columns can be more prone to contamination. It’s important to use reputable column manufacturers and to properly store and handle columns.
To minimize column pollution, it’s crucial to follow good laboratory practices, including proper sample preparation, filtration of solvents, appropriate mobile phase preparation, and regular maintenance of the HPLC system and column. Regularly flushing the column with suitable solvents and cleaning it according to the manufacturer’s recommendations can also help extend its lifespan and maintain optimal performance.