Important suggestions for generating reliable HPLC results

Each instrument component contributes to high reliability of results
Each instrument component contributes to high reliability of results
Each instrument component contributes to high reliability of results

HPLC has a marked presence in analytical research and quality control laboratories. Important decisions concerning both manufacturing processes as well as publication of research findings are based on decisions which can be arrived at only if high degree of reliability can be placed on the data generated by the HPLC system.

HPLC is a multi component system so the accuracy and reliability of results is dependent on the performance level of each component part. The present article offers some suggestions which will contribute to greater authenticity of results. For simplicity the contribution of each component part is discussed under a separate heading.

Mobile phase

The principal role of mobile phase is to carry the injected sample to the column and after separation in the column to transfer the individual components to the detector for their identification and quantification. Ideally a mobile phase should be prepared fresh before start of analysis and HPLC grade solvents should be used. The solvents should be free of any suspended particles. Initially filter the solvents under vacuum using 0.45 μ filter. Further use of online filters is recommended to remove any residual suspended particles. The mobile phase solvents should be fully miscible with one another and the injected sample should be miscible as well. When using buffers as mobile phase components never allow the buffer to dry out inside the system as salts formed on drying will deposit and cause hindrance to free flow of mobile phase in addition to damage to pump components.


Like mobile phase solvents it is a good practice to filter the sample before injection. Large volume injections will cause peak shape distortions so columns should not be overloaded by making large volume injections. Whenever a new method is to be developed it is advised to check the compatibility of the sample with the mobile phase. In case the solubility is low try a solvent or combination of solvents matching the polarity of the mobile phase mixture.


 A column is a key component of HPLC system. Its proper care and use will go a long way in providing high reliability of results.

Operating flow rates and pressures should be changed gradually so that such changes do not disturb the stationary phase packing. Use of guard column will also prevent impurities from gaining entry to the main column. As the flow of mobile phase is dependent on temperature use of a column oven is strongly recommended so as to maintain a constant column temperature  for consistency of flow rate.


The pump is required to provide consistency of flow of mobile phase and maintain the solvent composition throughout the analysis run. In case of gradient analysis a pump is required to maintain the composition of mobile phase as per the software  program. Such conditions are achieved by preventing damage to pump components and timely replacement of such components on noticing any visible damage. Gradually flush the pump by allowing rinsing intermediate polarity solvents or pure water as the case may be.


Consistency of injection volumes is a prime need for reproducibility of results. Manual injectors comprise of fixed volume sample loops and in case of automated modes the injection syringe maintains consistency of volume without operator intervention.

Special care needs to be taken to prevent contamination from previous analysis. In case of manual injectors always flush the injection loop with sufficient sample and in case of automated systems make sure that intermediate wash cycles are part of the analysis program. A validation of the cleanliness of sample and standard reusable vials is vital before commencement of analysis.

Light sources

The majority of HPLC analysis can be carried out using UV light source. Such light sources have a useful life span. Lamp replacement as per recommendations of supplier will contribute to a high degree of reproducibility of absorbance values.


A detector is the ultimate part of the HPLC system which confirms the presence of different sample components as well helps in their quantification.

It is recommended that the detector be cleaned periodically from time to time off-line and flushed with water and mobile phase before being re- used. The operating pressure range should never be exceeded to prevent possible damage to detector cell walls.

The suggestions offered in the article will certainly improve the quality of results but it goes without saying that there is no substitute for periodic calibration of the system against reference compounds.

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