| In conventional stopped-flow mixers, relatively large
volumes are used. As a consequence, the mixing time typically lies in the millisecond time
range. Regenfuss and co-workers demonstrated that the mixing dead time can be dramatically
reduced by passing two solutions into a small aperture of ~10 micrometers where they are
forced to meet; this generates turbulence causing the solutions to mix rapidly by
diffusion within small eddies. The mixed solution coming from the aperture forms a
continuous free jet where the time evolution of the sample can be followed by probing at
different positions down stream from the mixer. This type of mixer has three limitations:
first, the free jet is not always perfectly stable making spectroscopic measurements
difficult; second, because the solutions are forced to pass through a very small aperture,
a shear force is introduced in the mixed solutions that can affect some biological
samples; and third, the samples can not be maintained in an oxygen-free atmosphere which
may be crucial for some experiments. To overcome
these limitations, a new family of submillisecond mixers was designed as illustrated below
(Ref: "Folding of Cytochrome c Initiated by Submillisecond Mixing" S. Takahashi,
S.-R. Yeh, T. K. Das, C.-K. Chan, D. S. Gottfried & D. L. Rousseau, Nature Struct, Biol. 4, 44-50, 1997). |
| The two solutions to be mixed enter the "T"
shaped nozzle with high velocity from the opposite ends of a 100 micrometers wide and 25
micrometers deep channel and meet at a point directly below the entrance of an observation
cell with a 250 x 250 micrometers cross section. The nozzle and the cell are separated by a ~50
micrometers thick teflon sheet with a 250 micrometers wide hole in the center. As the
solutions pass from the narrow flow channels into the 250 micrometers wide mixing chamber
at the center of the "T", turbulence is generated which results in mixing. The
mixed solution continuously flows through the observation cell where the progression of
the reaction can be probed. The time evolution of the reaction can be determined from the
flow rate and the distance between the mixing point and the point being probed. The mixing efficiency and the mixing deadtime of the "T"
mixer were evaluated by resonance Raman scattering and
fluorescence quenching experiments. It was found that the solutions were >95% mixed at
the first observable point. It should be noted, however, that the mixing efficiency
depends on the flow rate. Only at a flow rate of >2 mm/ms (0.125 ml/s) were we able to
achieve complete mixing; at a flow of 0.5 mm/ms, the mixing efficiency dropped to 80% at
the first observable point in the cell. The mixing deadtime was measured to be 100 microseconds,
consistent with the estimated value from the calculated dead volume. |
