Normal and Reverse-Phase High Pressure Liquid Chromatography (NP-HPLC and RP-HPLC) and LC-UV

High pressure liquid chromatography (HPLC) is a liquid chromatographic technique in which the sample is forced through the column under high pressure. In normal phase (NP-HPLC), the mobile phase is non-polar and the stationary phase is polar. In reverse-phase (RP-HPLC), the polarity of the stationary and mobile phases is reversed, allowing only hydrophobic interactions with the analytes. Polar analytes elute first followed by non-polar. The benefit of RP-HPLC, which is the more commonly used technique, is that small changes in mobile phase composition (e.g. salts, pH, organic solvents), or in temperature, can profoundly affect the separation characteristics, making it a sensitive and flexible technique.

RP-HPLC coupled with UV spectrophotometry detection, LC-UV, is used in many applications:

  • Amino Acid Analysis (AAA), Extinction Coefficient estimation or determination

  • Liquid chromatographic pattern 'fingerprinting', as recommended in the ICH Q6B guidelines

  • N-terminal sequencing , following Edman chemistry

  • Proteomics research

  • Analysis of aldehydes in airborne samples; samples are collected on-site in a sampling tube and returned to SGS M-Scan for analysis. Formaldehyde is commonly analysed using this method.

  • Preparative LC-UV is used to collect pure fractions of components which can be used for offline molecular weight or structural elucidation studies e.g. 1HNMR analysis

  • HPLC coupled with mass spectrometry is a very powerful technique, used in many applications. See LC-MS, LC-MS/MS, nano LC-MS/MS, (LC) APCI-MS.