Over 3000 E-Learning topics / 5000 Articles & Applications
 
Developed
with the
Research Institute for Chromatography

Introduction and Benefits (Reversed Phase HPLC)

Reversed phase liquid chromatography (RPLC) finds a host of applications in protein biopharmaceutical analysis.  From analysis at the protein level of the intact species and major fragments – such as a monoclonal antibodies (mAbs) light chain/heavy chain (Lc/Hc) or fragment crystallizable/fragment antibody (Fc/Fab) - through to peptide mapping, and also at the amino acid level (Figure 1).

Typical Reversed Phase Chromatograms of a Protein Biopharmaceutical

Figure 1: Typical reversed phase chromatograms of a protein biopharmaceutical


Reversed phase HPLC offers numerous benefits over all other modes of chromatography.  It possess superior efficiency, such that large numbers of solutes can be analyzed in a reasonably short amount of time.  In addition, sensitivity increases alongside chromatographic efficiency, assuming no change in baseline noise, thus very low level determinations can be made.  As well as efficiency, a huge benefit of RPLC is the wide ranging selectivity which permits the retention and separation of a host of solute classes from hydrophobic (non-polar), through to hydrophilic (polar), and even charged species.  Efficiency and separation combine to generate resolution, the true arbiter of any chromatographic separation.  Separation, and to a lesser extent efficiency, are controlled and optimized by a plethora of parameters which the chromatographer has at their disposal, including mobile phase pH, polarity, and type of organic modifier, temperature, stationary phase chemistry, particle size, column dimensions, and volumetric flowrate.  This can make RPLC a little daunting and method development/optimization a little time consuming, however, no other mode of liquid chromatography allows the analysis of large numbers of varied analytes, while also being able to detect these species at very low concentrations.

Other key benefits of RPLC are the robustness this technique offers in terms of method and instrument precision and the predictable retention and elution of different solute classes.  RPLC is the mainstay of many analytical laboratories and is used almost universally in the discovery, development, and stability and release testing of conventional small molecule pharmaceuticals.  Vast amounts of research and development has been invested in understanding the separation process, optimizing the column (from the physical dimensions, through particle size/morphology, to the type of stationary phase support and how the stationary phase chemistry is bound to it), and developing instruments which have been carefully optimized in terms of their ability to operate at very high pressures, have reduced extra-column and dwell volumes, and detector scanning speeds which are able to detect very narrow, efficient peaks.

The predictable nature of the solute-stationary phase interaction mechanism(s) permits informed predictions to be made regarding retention order; this can be done by experienced method developers and has also led to the development of modelling software that facilitates in-silico method development.  This predictability allows 'generic' methods to be employed, which typically provide all the necessary operating parameters for separations; even complex analyses such as peptide or glycan mapping.  Generic methods are also column and instrument independent and standard conditions are generally acceptable, if not optimal.  Approaches to peptide and glycan mapping are discussed in detail in subsequent modules, with full descriptions not only of generic approaches but also the impact each parameter has on the separation, and how these parameters can be optimized.

Finally, the conditions employed in RPLC permit coupling directly to mass spectrometric (MS) detection.  RPLC and MS are by no means perfectly compatible, as the highly polar/aqueous mobile phases must be vaporized and pumped away such that only charged, gaseous solute ions enter the mass analyzing stages of the instrument without the high vacuum being compromised.  However, the mobile phases employed do actively promote and support the generation of charged species in solution – a pre-requisite for electrospray ionization (ESI).  ESI is the optimal mode of ionization for large macro/biological molecules as it permits multiply charging thus bringing huge species, well in excess of 100 kDa mass-to-charge ratio (m/z), into the working range of common mass analyzers.  MS detection can be useful in routine/release analysis in terms of offering an additional dimension of separation and enhancements in sensitivity, but the true advantage of LC-MS is in characterization, where 'unknown' solutes can be qualitatively identified/confirmed using a high accuracy mass analyzer such as time of flight (ToF) or Orbitrap.

loading data
loading data
loading data
loading data
loading data
loading data
loading data
 
 
 
 
 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data

 

loading data


loading data