Why Reversed Phase Chromatographic Columns Are Polluted?
Reversed phase chromatography is the most widely used technology in HPLC, which is mainly because it is suitable for the analysis of a large number of non-polar substances and many ionizable compounds.
The reversed-phase HPLC column (RP-HPLC column), represented by the C18-HPLC column, is often based on silica gel with a relatively weakly bonded phase bonded to the surface. The mobile phase used in the RP-HPLC column is more polar, usually water, a mixture of buffer and methanol, and nitrile. The order in which the sample flows out of the column is that the most polar combination is first and the less polar component is next. The commonly used reversed-phase fillers are C18 (ODS), C8 (MOS), C4 (B), C6H5 (Phenyl), etc.
And what causes the pollution of reversed phase chromatographic columns?
Usually, the sample contains something Unnecessary. Substances like salts, lipids, lipid-containing substances, humic acids, hydrophobic proteins, and other biological substances may interact with HPLC columns in use. These substances may have less or greater retention value than the analyst’s target. Those with lower retention values, such as salts, are generally washed out of the column when the volume is empty. And the interference of these non-target products can be detected by the detector and the experimenters will obtain the shift-up of chromatographic peak, bubble, and baseline or the negative peak.
If the composition of the sample is strongly retained in the column and not eluted thoroughly by the mobile phase solution, the substances adsorbed on the surface of the column will usually accumulate in the column head after several times of sampling.
This can be found only through parallel experiments. Samples with moderate retention values can be slowly washed out and exhibit broad peaks, baseline perturbations, or baseline drift.
Cleaning of HPLC Reversed Phase Column
When the HPLC Reversed-Phase column is contaminated, its chromatographic behavior will be different from the uncontaminated ones. There would be back pressure problems happened. Therefore, the contaminated reversed-phase columns must be cleaned.
Common Cleaning Method
Generally, the contamination is caused by the accumulation of strongly retained materials during the repeated sampling process. Basically, their chromatographic behavior could be restored with simple procedures to remove the contaminants. After multiple operations, the column could be rinsed with 90-100% solvent B (the stronger solvent in the dual solvent reversed-phase system) to remove contaminants. If a buffer system is employed, do not directly switch to a strong solvent.
Suddenly switching to a strong organic solvent may precipitate the buffer in the HPLC flow system, which can cause larger problems, such as blockage of the column, pump Leakage, or injection valve shaft failure. In general, the solvent used is increased based on the strength of the solvent, and often the last solvent is very hydrophobic (such as ethyl acetate), which can be used to dissolve the non-polar substances, such as lipids and oils.
Protein Residue Cleaning Method
If a biological material on a reversed-phase column, such as plasma or serum accumulates, the operator must use a different cleaning procedure. In most cases, pure organic solvents such as acetonitrile or methanol could not dissolve the polypeptide and protein, which means it could not effectively clean the column. However, a mixture of an organic solvent with a buffer, an acid, an ion-pairing agent or the like can be used to effectively dissolve the proteins.