Molecular and Pore Diffusion Coefficients of DNAs and Modified Proteins for Process Chromatography

As the performance of chromatography is governed by pore diffusion, it is important to know the pore diffusion coefficient especially for separation of large bioproducts such as proteins and DNAs.

However, compared with the diffusion coefficient of globular proteins (molecular weight 10,000- 100,000), the diffusion coefficients of DNAs and modified proteins have not been measured extensively. Consequently, it is difficult to predict the diffusion coefficient in the solution (molecular diffusion coefficient, D_m), which is needed for understanding the pore diffusion coefficient. D_s.

In this study, D_m was determined by the Taylor method, which uses a pulse response curve from a narrow tube at laminar conditions. Single strand and Double stranded DNAs were used as the sample. D_s was determined also by using a pulse response experiment with agarose-based ion-exchange chromatography particles, Sepharose HP under non-binding conditions.

D_m of DNA was correlated with the molecular weight M_W as follows.

Single strand DNA    : D_m =1.94x10^-8M_w^-0.57

Double strand DNA  : D_m =1.33x10^-8M_w^-0.49

Compared with the established correlation for proteins (D_m is proportional to M_w^-0.33), D_m changes much more strongly with M_W. This is because of the shape of DNA molecules.

D_s of single stranded DNA was proportional to M_w^-0.99, which indicates that diffusion of single strand is highly hindered in the pore. This also suggests that separation of DNA molecules with conventional porous particles is difficult and the use of convection-aided media such as monoliths is preferred.