(342g) 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_swas 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.