high performance liquid chromatography, broad peaks

Method development by HPLC

Method development is the procedure of confirming that an analytical method is suitable for applying in the research laboratory to separate, identify and quantification of samples. Analytical methods must be applied within the GLP and GMP surroundings and have to be developed with the acceptance criteria and protocols as per the guidelines of ICH.When developing the analytical method you have to keep some things in mind, like develop an analytical method simple as possible do not make it complicated, try to use of most common HPLC grade solvents and stationary phase, develop a cost and time-saving method.

Before starting the method development on HPLC Instrument.

  • Always use HPLC Grade solvents.
  • Filter the mobile phase through a 0.45 μ nylon membrane filter.
  • Filter the sample through a 0.2 μ nylon membrane filter.
  • Sonicate the mobile phase and samples to remove dissolved gases.
  • Purge the all solvent reservoir and flush autosampler for removing air bubbles and replace the solvents.
  • Set the desired parameter in the system.
  • Condition the column with a mobile phase for sufficient time.
  • Check column pressure, it should be minimum.
  • Start the analysis.
  • Integrate and Keep the data

How to start method development in HPLC

  1. Nature of sample (chemical and physical properties of the sample)
  2. Number of compounds present
  3. Structure of analyte
  4. pKa value
  5. solubility of analyte
  6. Molecular weight
  7. UV spectrum (λ max)
  8. Selection of the HPLC method, initial conditions, and optimization.
  • Reverse phase HPLC - use of water, the organic solvent as mobile phase, Column C18, C8, phenyl, TMS and Cyano.

Primary option for the majority of samples particularly neutral and nonionized compounds that soluble in water and organic mixture.

  • Ion Pair HPLC – use of water, the organic solvent as mobile phase, Buffer for control the pH and as the ion-pair reagent. Column C18, C8, and Cyano.

A suitable option for the ionic/ ionizable compounds particularly bases /cations.

  • Normal Phase HPLC – mobile phase are as mixture of organic solvents, Columns Cyano, diol, amino, silica.

Next option while reverse phase and ion-pair HPLC do not work, primary option for samples of lipophilic that not soluble well into water and organic mixtures, primary option for combinations of isomers.Other than the theoretical part you should refer the literature to observe if there are already methods are exists for the same sample or a similar one. You must check journals with reference books for HPLC methods.

Whereas developing HPLC method follow the ICH guidelines like tailing factor, Asymmetry factor, Resolution, Capacity factor, theoretical plate’s number. You should try with methanol and water in while doing method development or according to your sample's solubility it should start with 50 % and according to the peak retain you can change the percentage of mobile phase until you get the Sharpe peak, If peak is not retained on mobile phase of Methanol / Acetonitrile /water after that you can use buffer.

It should be phosphate or acetate. Phosphate buffer has commonly used a buffer for its easy availability and PKa value. Phosphate buffer is stable and provides the additional Hydrophilic effect.Buffers are used when you analyze ionizable analyte with reverse phase chromatography and to maintain the pH stable of the mobile phase. Once you obtain better peak shape after that go for peak sharpness.

Keep in mind the method should be economy and time-saving.Peak shape should be within the range of as per the ICH guidelines for tailing factor, Asymmetry factor, Resolution, Capacity factor, theoretical plate number, Peak response.It is a challenging task to be logical before changes any parameter after method development method validation should be done as per the guidelines of ICH. 


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