High-performance liquid chromatography (HPLC) is a robust technique with multiple applications. HPLC method includes a column of adsorbent material through which the study sample passes under pressure. Each component will interact uniquely with the column and have a different flow rate. Therefore, individual components of the sample mixture will elute from the column at distinct times and be collected individually. Quantitative and qualitative HPLC analysis can help researchers identify individual parts and the overall composition of the study sample.
HPLC technique has several pharmaceutical applications, such as purity checks, formulation evaluations, and monitoring alterations during scale-up or process adjustments. During HPLC method development, it is vital to consider different factors based on intended applications. The current article focuses on HPLC labs. It discusses the critical equipment used in HPLC testing. However, robust assay qualification and validation remain vital for delivering reproducible and accurate results.
Although HPLC is versatile, one must identify the correct method and equipment for specific applications. Several factors need to be considered while using the HPLC method. In pharmaceutical testing, regulatory functions are considered while identifying the method, specification, and equipment. Multiple factors affect the quality of HPLC separation. These variables, such as column temperature, injection parameters, column and mobile phase properties, and instrumental design, need to be thoroughly understood before beginning with HPLC method development.
HPLC is one of the most widely used analytical tools in pharmaceutical testing. It can characterize potential drug candidates and ensure it is manufactured consistently and safely. Besides, it is an ideal tool for elucidating the chemical properties of small drug compounds by assessing the hydrophobicity of the molecule to sugar structures affecting immune responses.
HPLC is a unique technique with several options to choose from. Normal and reverse phases are the two most common approaches in HPLC testing. Both these approaches employ separation based on its polarity. In a normal phase setting, the solvent is nonpolar, while the adsorbent material is polar. Hence, lesser polar components are eluted first in the normal phase HPLC method. On the other hand, the reverse phase employs an opposite setup, resulting in more polar compounds eluting quickly.
Besides, there are other HPLC techniques, such as chiral for separating enantiomers and ion exchange-based techniques for ionic charges. Additionally, ultra-high-performance liquid chromatography employs smaller particles and columns than traditional HPLC, making the process more efficient.
Moreover, HPLC can be combined with different detectors, such as fluorescence, mass spectrometry, and UV-visible spectroscopy. These alterations make HPLC testing even more versatile. For example, mass spectrometers can help identify a molecule’s mass-to-charge ratio and molecular weight. UV-visible spectroscopy is ideal for determining the concentrations of molecules once they elute from the column. Moreover, fluorescence detectors are sensitive and can identify analytes in parts per billion detection range.
The HPLC method is critical in pharmaceutical analysis. Its utility and accuracy make it an ideal tool for different stages of drug discovery and development studies. Although multiple factors should be considered for an HPLC analysis, its flexibility and compatibility make it a perfect choice for different applications.