Expert Tips To Accurately Perform Your HPLC Method Development & Sample Analysis

A wide variety of operational parameters involving columns, equipment, and eluents generates an adequate level of complexity around high-performance liquid chromatography (HPLC) method development and sample analysis. The nature of an analyte largely influences the HPLC testing protocol. HPLC method consists of five steps, which are; HPLC method and system selection, selection of primary conditions, optimization of selectivity, optimization of the system, and method validation.

Depending on the analytes and samples, some steps involved in HPLC testing may not be even necessary. For instance, if a satisfactory separation is achieved in step 2, the next two steps may not be required. The extent of HPLC method development and validation is performed depending on the intended purpose.

Expert tips on HPLC testing

Before initiating HPLC testing, it is mandatory to check whether the chromatograph is in perfect working condition. Regular maintenance and OQ tests are needed, but not enough for HPLC analysis. Staff should be well-trained and experienced enough to locate and sort out signs of trouble in HPLC testing. Some signs of trouble or basic knowledge include; any buffer or salt crystal on the outside of the instrument, or knowledge of normal backpressure when the system is running. It could be beneficial to have all troubleshooting solutions as a part of the standard operating procedure.

During preparing the mobile phase, it is crucial to prepare enough of the fresh mobile phase to last the whole day. If kept for a day or two, the mobile phase may have significant microbial growth. Therefore, the mobile phase should always be filtered and freshly prepared for the analysis. Before introducing the mobile phase solution in the pumps, they should be continuously degassed for better performance. If not degassed continuously, issues such as drift and baseline instability may cause poor quality integration. Ideally, at the start of the day, each HPLC channel should be flushed with a fresh degassed mobile phase.

The next thing to do would be to learn more about the sample and determine the liquids in which the compound may be soluble. The stationary phase is the most significant influencer of HPLC selectivity. Therefore, selecting the right mode of chromatography and column is the most crucial phase of HPLC method development. Once selected, test and establish data to demonstrate that it meets both manufacturer’s specification and method requirements. First, avoid using additives as they may cause problems later. If needed, add them later. Employ a gradient method to detect all the compounds and elution conditions. If compounds are visible under a UV/VIS detector, use a Diode Array Detector. Shun the use of single or multi-wavelength detectors, as they invite errors and raise analysis costs.


HPLC method development and validation are two different processes, but at times are performed simultaneously for faster method validation. If HPLC is used in quality control, it is critical to ensure an acceptable level of confidence that results are viable. Similar to LC-MS analysis, HPLC or lcms testing should be performed within good laboratory practice and good manufacturing practice environments. As both the FDA and USP refer to ICH guidelines, it is crucial to develop HPLC analysis using ICH recommendations.

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