HPLC stands for high performance-liquid chromatography. HPLC analysis is one of the most vital bioanalytical techniques in drug development and clinical sciences. The primary objective of the HPLC test is to separate individual compounds from a given study mixture. HPLC labs employ this technique to assess drug compounds, their metabolites, and other analytes in complex study matrices.
HPLC analysis includes passing a crystallized liquid solvent containing the sample mixture through a stationary column comprising a solid adsorbent surface. Each component has a unique flow rate depending on the interaction with the solid surface. The differences in individual flow rates cause components to elute and flow out of the HPLC column at different times. However, HPLC analysis is a sophisticated technique requiring good lab practices. Hence the current article shares tips and tricks for accurate and reliable HPLC analysis for a new drug.
Tips and Tricks for HPLC Tests
Identifying the right HPLC column is the primary step during HPLC method development. There are several HPLC columns, but determining an ideal one needs thorough consideration. The toolkit approach is a robust and effective technique for identifying the application to investigate a wide range of sample components. However, this selection of HPLC columns is based on the required selectivity.
HPLC selectivity largely depends on additives, modifiers, gradient profiles, and the characteristic of the stationary phase. One must consider features and supporting elements of a stationary phase as they are crucial factors driving assay selectivity.
Visual evaluation of HPLC column performance is critical. Researchers should have an in-depth understanding of critical relationships when designing an HPLC experiment. Considering peak capacity is essential to avoid explosion.
UV detectors are frequently employed in HPLC analysis as they produce reliable data and are easy to use. However, UV detectors should be set up in a specific way so that they can perform unusual tasks to achieve better sensitivity and reproducibility.
Assay sensitivity and peak efficiency are directly related to the volume and type of flow cell. Keeping the slit at a narrow width improves the resolution of bandwidth, spectral, and references. However, it is critical to note that by using the slit with a higher range, the width bandwidth has a noise reduction.
Crude samples can impact HPLC retention times and peak shapes. The overload effect can be controlled by injecting the sample volume lower than the peak value by 15%.
Buffer selection in HPLC analysis depends on several factors, such as mobile phase pH and buffer volatility. A wrongly chosen buffer will require an addition of higher concentrations for the HPLC analysis to perform well. But addition will, in turn, cause issues with HPLC robustness.
Peak tailing is a type of distortion in HPLC analysis. An asymmetry higher than 1.2 is referred to as a peak. The primary reason for peak tailing is the presence of multiple reactions. Researchers can avoid peak tailing by ensuring the HPLC system has a lower pH to reduce secondary interactions.
Finally, wrongly prepared HPLC mobile phases can alter the retention times. Researchers can achieve reliable retention time by using correct pH calibrators, the right assay buffer and avoiding vacuum separation while premixing mobile phases.