High-Performance Liquid Chromatography (HPLC) is a technique used for the separation, identification, and quantification of components in a mixture. It provides rapid separation and high-accuracy results. Despite the advantages, interferences might occur and can affect the samples.
References:
High Performance Liquid Chromatography. (2020, August 16.). Retrieved from https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumentation_and_Analysis/Chromatography/High_Performance_Liquid_Chromatography
Co-elution of an unrelated compound with your compound of interest can, for example, affect detection/quantitation, as the signal you're looking at is representative of multiple species.
Among the various refining techniques, the most widely used chromatographic methods call for a column that can charge the resin with great performance and reproducibility. The current HPLC preparation column does, however, have a hydraulic for controlling the moving stopper and compressed chemical ingredients. It always results in the column performing erratically.
Possible problems stemming from HPLC Columns: Broad peaks due to issues with the mobile phase, leaks, contamination in the column or guard column, temperature issues, low buffer concentration, issues with detector settings, high detector time constant, or a void at the column inlet. Loss of resolution due to contamination of the mobile phase, obstruction of the analytical or guard column, or a column that needs to be replaced. Peaks that are too small or too large due to issues with detector attenuation, injection size, or an improper recorder connection. If these issues are not resolved, it could produce inaccurate results of the samples.
권오영, 박형석, 오석근, & 홍대희. (2019). Automation Control System for Servo Prep Column of High-Performance Liquid Chromatography Based on Fuzzy Inference Control. Journal of the Korean Society for Precision Engineering, 36(12), 1135-1140.
Interference from sample matrix is common. If any matrix component give response at same retention time as that of the compound of interest, we can't get actual result. Interference may also come from structurally related components of the material of interest.
Sometimes, HPLC column can itself be the reason of some interference. Proper column selection is required.
Different brands of reagents and solvents that are used for mobile phase preparation, can be reason of interference.
For this method, interferences can either result from pressure, leakage, or chromatography problems.
In relation to the pressure problem, there are four common errors that could arise. First is the abnormal pressure, which is caused when there is a leak or air trapped inside the pump head resulting in no flow. Next is the high backpressure which is caused by a flow rate that is set too high. Another one would be low back pressure. With this, the flow rate is set too low, and can also be caused by having a high temperature, system leak, and controller malfunction. Lastly, the pressure cycling is caused by a faulty valve, system leak, seal failure in the pump, use of gradient elution, and insufficient degassing.
The problem with leakage can occur in different settings such as leakage in fittings, pumps, injectors, HPLC columns, and in detectors. While for chromatography problems, these could arise due to different peaks, drifts, retention time changes, and resolution loss.
Science Unfiltered, 2022. Common HPLC Problems & How to Deal With Them. Retrieved from https://phenomenex.blog/2022/01/04/hplc_problems/#:~:text=Using%20an%20improper%20HPLC%20column,the%20use%20of%20gradient%20elution.
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