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The encapsulation pressure is dependent on the dielectric constant of the
medium. The elevated pressure is required to raise the dielectric of ethane to
match the dielectric of the surfactant.
The encapsulation pressure can be lowered through the use of additives.
This serves to increase the dielectric constant of the bulk solvent. Examples
of additives are pentane, dichloromethane, and carbon disulfide.
Encapsulation conditions found in pentane in general are translatable to
ethane with minor changes. For example CTAB sample may require 1.5%
more hexanol in ethane than pentane.
Water loading is the ratio of molar water to molar surfactant in the reverse
micelle sample. It has a large impact on the size of the particle.
Past studies using reverse micelles have worked to keep the water loading
low; typically in the range of 10-15 to keep the particle as small as possible.
However, larger proteins likely require more water to maintain the proper
hydration shell. Thus water loadings of 20-25 should be considered for
proteins larger than 40 kDa. At this size the protein is dominating the size of
the particle so the excess water has less impact.
Ethane can support much higher water loadings than are common in pentane.
This will require slightly higher encapsulation pressures.
CALIBRATING THE SYSTEM
The mixing chamber and components are designed to provide an internal volume
near 1.65 ml. Of this volume only 1.1 ml can be displaced by the piston. The actual
volume of the mixing chamber and the amount displaced are important numbers.
The former being important for the initial reagent calculations, and the latter for
determining the over pressure requirements for the transfer step to the NMR cell.
These numbers can be determined by a simple protocol. Use protonated alkanes
for these measurements as it will be vented from the mixing chamber. The gas
booster reservoir should be charged with CO
2
to the normal operating pressure.
(1) Setup the mixing chamber as would be done for a typical preparation process.
Include the reagent plate and stir bar.
(2)
Open
the
HP ALKANE
valve just enough to let in a little alkane gas then
close
the valve. The syringe pump
OUTLET
valve should be
open.