277330 Operating Window Determination for Chromatography of Biomolecules On the Basis of the Iso-Resolution Curve
We have proposed a concept “iso-resolution curve” for linear (salt concentration) gradient elution of proteins in electrostatic interaction chromatography (ion-exchange chromatography, IEC). The iso-resolution curve was found to be useful for understanding the relationship between separation time and buffer (solvent) consumption .
We further extended the iso-resolution curve concept to stepwise elution chromatography, in which the mobile phase for elution is changed discontinuously . The following two model separation systems were chosen: polyphenol separations by polymer-based reversed phase chromatography (RPC) with ethanol-water mobile phase [2,3] and IEC of proteins.
The distribution coefficient values Kwere determined from linear gradient elution experiments by using our model. For both cases, the distribution coefficients depended strongly on the mobile phase modulator (ethanol for RPC and salt for IEC). HETP values as a function of mobile phase velocity were determined by linear gradient elution (LGE) experiments as well as isocratic elution experiments.
Based on the iso-resolution curve we can choose the mobile phase modifier concentration which can provide the desired separation time and buffer consumption. However, in the actual operations the acceptable operating windows must be considered as variations for the equipment such as pumps and mobile phase compositions are unavoidable. It was found that when the separation time is short and the buffer consumption is small, a small change in the mobile phase modifier results in significant variations. The operating window is small and the mobile phase modifier concentration must be carefully prepared.
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