high performance liquid chromatography, broad peaks

HPLC buffer selection guide

The high-performance liquid chromatography separates the molecules in which the stationary phase and mobile phase are significant factors, in which the buffers with the organic phase are also used in the analysis. Here are mentioned some buffer selection tips for HPLC.

  • The selection of suitable buffers for analysis depends on characteristics of the buffer for example, pH range, pKa, and UV cutoff.
  • As a common rule pH buffer must be used within +/- 1 compounds pKa value. Buffers within this range resist the change in pH.
  • If the phosphate buffer mobile phase pH more than 7 it will accelerate the dissolution of silica, resulting in damage to the column or shorten the column lifespan. Typically the pH range of RP-HPLC on silica-based packing is 2.00 to 8.00.
  • Preferably the lowest concentration should be chosen that produces reproducible results. Normally, a 10 to 50 mM buffer concentration is enough for the separation of components.
  • If the amount of organic modifiers in the buffers is low or not, then the microbial growth can occur quickly, this growth will accrue in the inlets of the HPLC column and may cause damage as well as decrease the performance of the column. Therefore, freshly prepared buffer solutions are used.
  • The buffer solution should be filtered through a 0.5-micron nylon filter.
  • The mobile phase of HPLC should be degassed.
  • The solubility of phosphate is in water, methanol, acetonitrile, and tetrahydrofuran (THF).
  • Some salt buffers are hygroscopic, which could changes chromatography results.
  • The solubility of ammonia salts is usually in water or organic mobile phases.
  • Trifluoroacetic acid (TFA) can degrade over time, absorbs at lower wavelengths UV detector.
  • Ammonium bicarbonate buffers are generally prone to changes in pH and are usually stable for up to 48 hours.
  • Commonly used HPLC buffers are Ammonium acetate, Ammonium format, potassium dihydrogen phosphate or Phosphoric acid, Ammonium hydroxide, Trifluoroacetic acid, Formic acid, etc.
  • Today, more common issues associated with compatibility with mass spectroscopy detection, format, and acetate buffers are ideal options.


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