Molecule HPLC Band Broadening

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

Polymer-based adsorbents are not widely used in the HPLC analysis of small molecules, mainly because of the presence of micropores in the structure of polymer resins [29]. These micropores may trap small molecules, and their relatively slow diffusion leads to signihcant band broadening and overall loss of separation efficiency. 

The porosity of particles suitable for packing HPLC columns depends on the size of molecules to be separated. Totally porous particles with a pore size of 7-12 nm and specific surface area of 150-400 m"/g are suitable for the separation of small molecules, but wide-pore particles with a pore size of 15-100 nm and relatively low specific surface area (10-150 nr/g) are required for the separation of macromolecules to allow easy access to the interactive surface within the pores. Packings with perfusion particles contain very broad pores (400-800 nm) throughout the whole particle interconnected by smaller pores. The mobile phase flows through the pores in the particle, which minimises both band broadening and column backpressure (111. Perfusion materials have been designed especially for the separation and isolation of biopolymers. 

The development of a theory of retention and band broadening for macro-molecular HPLC is intended primarily for improving such separations. We need to relate various separation goals to (1) experimental conditions, (2) the choice of column type and HPLC method (RPLC, SEC, etc.), and (3) the nature of the sample. The present model allows os to do this 1 prediction, rathor than by experiment Our main requirement is usuaOy to achieve adequate separation, or some minimum resolution R, betw adjacent bands of interest. In this section we will show that the separation of macro-molecular samples by either isocratic or gradient elution is understandable and controllable, using the same concepts that we use for optimizing the isocratic separation of small molecules. 

The only factor contributing to band broadening is, unlike HPLC, longitudinal diffusion. The spread in the the axial direction down the center of the capillary for ions and molecules introduced as a tight plug at the inlet to the column can be described by the Einstein equation ... 

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