Steps for HPLC Method Development : Pharmaguideline

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Steps for HPLC Method Development

Know about the different steps of the HPLC analytical method development in pharmaceutical analysis.
Analytical method development is considered as a critical process in pharmaceuticals. Availability of the different types of columns, operating parameters, mobile phase composition, diluent and pH values make it critical to develop an analytical method. A good analytical method should be simple, used column, mobile phase and buffer should be common. It can be done easily step by step.

Following are the common HPLC method development steps.
1. Selection of HPLC Analytical Method
2. Selection of Chromatographic Conditions
3. Parameter Optimization

1. Selection of HPLC Analytical Method: First of all consult the literature that is available on the product. It will help you to understand the nature of the product that will help to select the different parameters.

HPLC SystemA. Sample Preparation: Select method to prepare the sample according to its solubility, filtration requirements, extraction requirements or other special requirements to make a clear solution of HPLC analysis.

B. Chromatography: Reverse phase chromatography is used for most of the samples but when acidic or basic molecules are present in the sample then reverse phase ion suppression (for weak acid or base) or reverse phase ion pairing (for strong acid or base) should be used. The stationary phase should be C18 bonded. Normal phase is used for low or medium polarity analyte especially when it is required to separate the product isomers. Choose cyano bonded phase for normal phase separations. Ion exchange chromatography is best to use for inorganic anion or cation analysis. If analyte has higher molecular weight than size exclusion chromatography is the best to use.

C Gradient/Isotonic HPLC: Gradient HPLC is helpful in the analysis of complex samples having a number of components. It will help to get higher resolution than isotonic HPLC having constant peak width while in isotonic HPLC peak width increases with the retention time. Gradient HPLC has great sensitivity, especially for the products having longer retention time.

D. Column Size: 100-150 mm columns are used for most of the samples. It reduces the method development and analysis time for the sample. Bigger columns are used for complex samples those take more time in separation. Initially, a flow rate should be kept between 1 and 1.5 ml/min and column particle size should be between 3 and 5 µm.

E. HPLC Detectors: If the analyte has chromophores that enable the compound to be detected by UV than it is better to use UV detector. It is always better to use a UV detector than others. Fluorescence and electrochemical detectors should be used for trace analysis. Samples having high concentration should be analyzed using refractive index detectors.

F. Wavelength: λmax of the sample has the greatest sensitivity to the UV light. It detects the sample components that have chromophores. A wavelength above 200 nm gives greater sensitivity than the lower wavelengths. Wavelengths lower than 200 nm gives more noise, therefore, it should be avoided.

2. Selection of Chromatographic Conditions: After selection of analytical method, different chromatographic conditions are selected.
The flow of the analytes through the column depends upon the concentration of the solvent in the mobile phase. The concentration of solvent is generally used to control the retention time. Mobile phase pH and ion pairing reagents also affect the retention time of the sample. Samples having a large number of components are analyzed using the gradient to avoid the large retention time while the samples containing one or two components are analyzed on an isotonic system.

3. Parameter Optimization: After taking the same sample runs some parameters including column dimensions, particle size, run time and flow rate are optimized. It is done to get the best resolution and minimum run time.

After proper optimization of the analysis method, it is validated to ensure the consistency of the analytical method. Analytical method validation is now done mandatory by all regulatory authorities.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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