Chromatograph Maintenance - Troubleshooting the Seventh Chromatographic Retention Time Drift
Chapter 7 Troubleshooting Chromatographic Retention Time Drift The retention time is not heavy in two different situations: retention time drift and retention time fluctuations. The former means that the retention time changes only in one direction, while the latter refers to the fluctuation of the retention time without a fixed regularity. It is often helpful to distinguish between the two situations to find the cause of the problem. For example, the drift of retention time is often caused by column aging; while column aging is unlikely to cause irregular fluctuations in retention time. In fact, most of the retention time drift is due to column aging of different mechanisms, such as loss of stationary phase (eg by hydrolysis), column contamination (caused by sample or mobile phase). The most common reasons for retention time drift are as follows:
One Column Equilibrium If we observe retention time drift, we should first consider whether the column is fully equilibrated with the mobile phase. Usually a balance of 10-20 column volumes of mobile phase is required, but if a small amount of additive (such as an ion pair reagent) is added to the mobile phase, it takes a considerable amount of time to equilibrate the column.
Mobile phase contamination may also be one of the reasons. A small amount of contaminants dissolved in the mobile phase may slowly enrich on the column, causing drift in retention time. It should be noted that water is a mobile phase component that is easily contaminated.
Stability of the two stationary phases The stability of the stationary phase is limited, and the stationary phase will slowly hydrolyze even when used within the recommended pH range. For example, the silica gel matrix has the best hydrolytic stability at pH 4. The rate of hydrolysis is related to the type of mobile phase and the ligand. The bifunctional ligand and the trifunctional ligand are more stable than the bonded phase of the monofunctional ligand; the long chain linkage is more stable than the short chain bonding phase; the alkyl linkage is much more stable than the cyano bonded phase.
Frequent cleaning of the column also accelerates the hydrolysis of the column stationary phase. Other silica matrix bonded phases may also undergo hydrolysis in aqueous solutions, such as amino bonding.
Three-column contamination Another common cause of retention time drift is column contamination. The HPLC column is a very effective adsorptive filter that filters and adsorbs any material carried by the mobile phase. Sources of contamination can be: mobile phase itself, mobile phase vessels, connecting tubes, pumps, injectors and instrument gaskets, and samples. The source of the contamination can usually be determined experimentally.
If there is a strong component remaining on the column in the sample, it may be a potential source of drift in retention time. These roots are usually the sample matrix. Such as: excipients in the drug, protein and lipid compounds in biochemical samples (such as serum), starch in food samples, humic acid in environmental water samples, etc. Usually, the strongly retained component in the sample has a higher molecular weight, in which case the retention time drifts or there is an increase in back pressure. The effect of the sample matrix can be removed by using a sample preparation method such as solid phase extraction (SPE).
The easiest way to avoid column contamination is to prevent it from happening. In contrast, finding the problem and designing an effective cleaning step to remove contaminants is much more difficult. Strong solvents are usually used under the given chromatographic conditions, but not all contaminants can be dissolved in the mobile phase. For example, THF removes many of the contaminants in the reversed-phase column, but the protein does not dissolve in THF. DMSO is often used to remove proteins from reversed phase columns.
Using a guard column is a very effective method. Recoil columns are only a last resort.
Four mobile phase composition The slow change in mobile phase composition is also a common cause of retention time drift. For example, the volatile components in the mobile phase are volatilized and the flow in circulation is equal.
Five-hydrophobic collapse When a reverse-phase packing column with a small pore size and good end-end sealing uses nearly 100% water as the mobile phase, sudden loss of separation and significant or no retention of analyte retention sometimes occur. Hydrophobic collapse. This phenomenon is caused by the fact that the mobile phase does not wet the surface of the stationary phase. The salvage method achieves infiltration of the stationary phase with a mobile phase containing a large amount of organic components, and then equilibration with a mobile phase having a high water content. This phenomenon can also occur due to long-term storage of the column. Collapse can also be avoided by using reversed-phase columns with embedded polar groups (such as Waters SymmetryShield RP columns) or non-end-sealed columns (such as Waters Resolve columns)
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