Three most common problems regard to solid phase extraction (SPE)
With regard to solid phase extraction (SPE), there are three most common problems: low recovery, poor reproducibility, and insufficient sample extract.
To solve the problem of recovery and reproducibility, we first need to verify that the analysis system is operating properly.
Reproducibility problems can be caused by residual samples, detector problems, or defective autosamplers.
It is possible to verify whether the response factor of the analytical instrument has changed by injecting an extraction solvent containing a known standard sample into the instrument. Repeated injections of pure standards can verify the reproducibility of the injection, while repeated injections of different concentrations of standards (concentrations from high to low, even to very low levels) can verify residual problems.
Each step of the SPE operation then needs to be evaluated, as a loss of analyte anywhere can result in lower recovery.
The evaluation begins with a standard process for the extraction process, then collects the fractions from each of the proposed steps and finally analyzes to determine if the analyte is being lost. If it is found in the loading process or the cleaning step, check that the solvents used, the conditions used, or the pre-equilibration are correct, as the effects of these factors are retained.
Try changing the sample solvent or washing step to increase the retention mechanism of the adsorbent.
If the analyte is retained on the column but not well eluted, then it is necessary to indicate that the solvent is correct and then the elution strength of the eluent needs to be increased. In addition, there may be secondary interactions between the analyte and the adsorbent, so a suitable elution solvent should be chosen to solve this problem. Finally, it may be necessary to replace an adsorbent that has weak retention of the target component.
If the SPE operation is all correct, it is likely that the analyte is not adsorbed on the column or directly destroyed after entering the column, so no analyte is detected in the fraction.
The former problem may be caused by the instability of the analyte in the sample; and if the sample pretreatment process involves protein precipitation and the target analyte will bind to the protein, then the analyte will precipitate with the protein and cause the above problem.
In addition, the resulting protein-bound analyte may pass directly through the SPE column without being adsorbed and is therefore not likely to be detected in subsequent analysis steps.
When using liquid chromatography-mass spectrometry (LC-MS), the matrix will inhibit or enhance the signal of the analyte of interest. Therefore, if the interfering substances cannot be well eliminated by SPE technology, then other pretreatment steps are particularly necessary. For example, liquid-liquid extraction techniques are commonly used to remove oil, fat or oil; ion exchange techniques are commonly used for desalination, or The inorganic salt is removed using a non-polar sorbent; proteins such as those that bind to the analyte are removed by adjusting pH, ultrafiltration or precipitation.
If there is a situation where the recovery rate is lower or more fluctuating than before, you should check the adsorbent used, which may be due to the different batches of adsorbent used.
When reproducibility is degraded, column failure (such as high back pressure or retention time changes), and quantitative inaccuracy, further purification of the sample is required.
The solution to this problem can be to change the original washing protocol, or to use other adsorbents with the same extraction mechanism, or to completely change the extraction method.
The wash solvent should have a mechanism to elute the maximum amount of interfering species while the analyte is not eluted.
The rinsing step can also be improved by using an analyte insoluble solvent – however, the surprising purification effect may be through the use of non-polar, water-immiscible solvents such as dichloromethane, ethyl acetate or hexane. to fulfill.
These solvents have strong non-polar mechanism elution characteristics (as they can elute many interferents), while the analyte will remain firmly on the adsorbent surface. The elution solvent can also be adjusted, and the weaker solvent may also result in a cleaner extract, but at the same time need to pay attention to whether the analyte recovery rate is affected.
If the desired pure analyte is not obtained even if the washing solvent is changed, an attempt can be made to change the adsorbent. For example, if C8 adsorbents, low-retention C4 or C2 adsorbents, etc. are used, their adsorption capacity to matrix components is relatively poor. However, you must also ensure that the sample is fully adsorbed on the stationary phase.
The final method to improve the purity of the sample is to completely change the extraction mechanism.
Suitable alternatives can be used to develop and determine methods with potentially available mechanisms. At present, a very effective technique is to use a hybrid mechanism extraction technology to replace the traditional single mechanism extraction technology. This method is most suitable for the extraction of analytes containing both polar and non-polar groups.