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 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 analyze 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 empty volume in the blocked place.
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