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Analytical method development is considered an important process in pharmaceuticals. The availability of different types of columns, operating parameters, mobile phase compositions, diluents and pH values makes it important to develop an analytical method. A good analytical procedure should be simple, the column, mobile phase and buffer used should be common. It can be easily done 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 consult the literature available on the product. This will help you to understand the nature of the product which will help in selecting various parameters.
A. Sample preparation: Select the sample preparation method according to the sample’s solubility, filtration requirements, extraction requirements or other special requirements for clear solution of HPLC analysis.
B. Chromatography: Reverse phase chromatography is used for most samples but reverse phase ion suppression (for weak acids or bases) or reverse phase ion pairing (for strong acids or bases) should be used when acidic or basic molecules are present in the sample. To be used. The stationary phase should be C18 bonded. Normal phase is used for low or medium polarity analytes especially when product isomers need to be separated. Choose the cyano bonded phase for normal phase separation. Ion exchange chromatography is best used for analyzing inorganic anions or cations. Chromatography is best used when the analyte has a molecular weight greater than its size.
C Gradient/Isotonic HPLC: Gradient HPLC is useful in the analysis of complex samples containing several components. This will help to obtain higher resolution compared to the constant peak width of isotonic HPLC while isotonic HPLC peak width increases with retention time. Gradient HPLC has great sensitivity, especially for products with long retention times.
D. Column Size: 100-150 mm columns are used for most samples. This reduces method development and analysis time for samples. Larger columns are used for complex samples that take longer to separate. Initially, a flow rate should be kept between 1 and 1.5 ml/min and the particle size of the column should be between 3 and 5 µm.
E. HPLC Detector: If the analyte has chromophores that enable detection of the compound by UV, it is best to use a UV detector. It is always better to use UV detector than others. Fluorescence and electrochemical detectors should be used for trace analysis. High concentration samples should be analyzed using a refractive index detector.
F. Wavelength: λmax of the sample has maximum sensitivity to UV light. It detects sample elements that contain chromophores. A wavelength above 200 nm gives greater sensitivity than lower wavelengths. Wavelengths below 200 nm are more noisy, so should be avoided.
2. Selection of Chromatographic Conditions: After selecting the analytical method, various chromatographic conditions are selected.
The flow of analytes through the column depends on the concentration of the solvent in the mobile phase. Solvent concentration is usually used to control the retention time. Mobile phase pH and ion pairing reagents also affect sample retention time. Samples containing a large number of components are analyzed using gradients to avoid long retention times when samples containing one or two components are analyzed in an isotonic system.
3. Parameter Optimization: Some parameters including column dimensions, particle size, run time and flow rate are optimized after taking the same sample. This is done to get the best resolution and minimum run time.
After proper optimization of the analytical method, it is verified to ensure the consistency of the analytical method. Validation of analytical methods is now mandatory by all regulatory authorities.
