In daily HPLC analysis, obtaining high-quality data requires the HPLC system to deliver reliable and reproducible experimental results. Below are some considerations and recommendations for routine liquid chromatography applications:
01 The quality of reagents
In gradient elution, non-polar impurities from solvents A and B can accumulate at the head of the column and gradually elute during the subsequent gradient process. These impurities may appear during the testing of blank and actual samples. For this reason, it is necessary to use HPLC-grade reagents when performing gradient experiments. Low-quality reagents may be suitable for isocratic elution, but even the slightest impurity can cause abnormal issues during gradient elution. To obtain optimal experimental results, it is essential to ensure the use of only HPLC-grade reagents in all HPLC experiments.
Water-based reagents and buffer solutions should be regularly replaced to avoid contamination caused by microbial growth. Impurities in the water, especially, can lead to various issues.
02 System Cleaning
As important as the quality of reagents, maintaining the cleanliness of the instrument helps reduce ghost peaks. It is recommended to flush the instrument regularly. If any other issues arise, such as liquid overflow, leaks, or other potential sources of contamination, they should be promptly cleaned up.
03 Solvent Degassing
Although some HPLC systems can operate without degassing the mobile phase, using degassed solvents makes the operation of all systems more reliable. Degassing the solvent helps to significantly reduce the presence of residual bubbles and gas overflow phenomena, ensuring a more stable performance across all operations.
Additionally, there might be residual gases in the HPLC system. It is a good practice to flush the pump and solvent delivery lines daily. This can be achieved by opening the purge valve and running the pump at a higher flow rate (e.g., 5 mL/min) for a few minutes to remove any bubbles present in the pump and lines.
04 Specialized Chromatography Columns
Each analytical method should have a dedicated chromatography column. It is not advisable to use the same column across different methods because irrelevant peaks from one method may become interfering peaks in another method. Sample components can alter the selectivity of the chromatography column, or one method may cause column degradation, affecting subsequent analyses.
Before each analysis, the chromatography column should be equilibrated to the same extent to prepare it for subsequent separations. Complete equilibration is crucial, especially for gradient elution experiments, to ensure consistent and reliable results.
06 Initial Injection
Performing several “initial” injections before the first sample injection can lead to improved results in some methods. These injections with standard solutions or mock samples help to saturate the active sites on the chromatography column, resulting in better reproducibility of experimental outcomes. Initial injections are particularly beneficial for the separation of complex biological samples. Occasionally, system suitability injections can also serve as initial injections.
07 Ignore the First Injection
As some methods require the use of an initial injection procedure, and the equilibration state of the first injection may differ from subsequent injections, it is advisable to avoid using the first injection for quantitative analysis. The second and subsequent injections are more reliable than the first injection. System suitability tests conducted before formal sample injections can also serve this purpose.
08 System Suitability
Many methods regulated by authorities such as FDA, EPA, OECD, USP, etc., require conducting system suitability tests before analyzing samples. System suitability is crucial for verifying that the equipment and analytical methods can produce reliable experimental results.
The requirements for system suitability testing vary depending on the specific regulations and standards. Therefore, relevant personnel should seek guidance from applicable regulatory sources to help select appropriate testing methods. Many operators utilize one or a combination of parameters such as retention time and peak area reproducibility, chromatographic peak response (detection sensitivity), peak width, tailing factor of chromatographic peaks, resolution, and column pressure as part of the system suitability testing. These tests are conducted to ensure the stability and performance of the analysis system and to guarantee the reliability and accuracy of the experimental results.
System suitability samples can be diluted standard solutions, mock samples extracted from the matrix, or selected samples used to demonstrate system performance. One important principle is that the chosen system suitability samples must be capable of testing whether the intended objectives of the method can be achieved. Whether or not system suitability testing is required, performing such tests before routine analysis is a wise choice, even if it is just injecting a standard sample to verify if the retention time and peak size match the expected values.
09 Standard Samples and Calibration Standards
For quantitative analysis, it involves comparing the response of an unknown sample with the response of known concentrations. The concentration range of standard solutions, the number of replicate tests, and the injection sequence depend on the specific experimental objectives. Both external standards and internal standards can be used. In any case, at least one standard sample should be injected before analyzing unknown samples to ensure that the analytical method is functioning correctly prior to sample analysis.