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Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) and LC-UV / DAD (Diode Array Detector)

High pressure liquid chromatography (HPLC) is a liquid chromatographic technique in which the sample is forced through the column under high pressure. In reversed-phase (RP-HPLC), separation of a peptide or protein mixture is achieved by only hydrophobic interactions between the analytes and the column material. By applying a gradient with an organic solvent like acetonitrile (most often used), polar analytes elute first followed by less polar compounds. The benefit of RP-HPLC 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 / DAD (diode array detector) and/or or fluorescence detection is used in many applications:

  • Amino acid analysis; extinction coefficient estimation or determination
  • LC pattern/profile; 'fingerprinting', as recommended in the ICH Q6B guidelines
  • N-terminal sequencing, following Edman chemistry
  • Preparative LC-UV / DAD is used to collect pure fractions of components which
    can be used for offline molecular weight or structural elucidation studies like
    mass determination, MSn or N-terminal sequencing.

HPLC coupled with DAD is very beneficial for locating for instance Trp-containing peptides in LC fingerprinting. HPLC coupled with mass spectrometry is a very powerful technique, used in many applications. 

SEC-HPLC (Size Exclusion Chromatography) is often used for the determination of possible aggregates present in biopharmaceuticals. The columns used work like a molecular sieve like in (the old) gel filtration technique, and analysis can be performed under physiological conditions. Aggregate analysis is important because of the risk of possible immunological reactions if a too high quantity of aggregates is present in human therapeutics.