23
(5) Continue stepping up the pressure in small increments of 250 psi while
continuing to observe the mixing process as noted by the cloudy nature of the
sample.
(6) Around 3,500 – 4,000 psi the sample should become noticeably more clear. At
this point the experience of the user becomes important. Most samples will not
completely solubilize so some flocculence may be apparent in even a good sample.
Stop the stirring process and allow the insoluble particles to settle. If the previous
steps were followed, and a mixing transition was observed a transparent sample
usually indicates it is close or at the encapsulation point. The alternative is to
increase pressure some more, but doing will increase the total viscosity of the
sample and defeat the purpose of encapsulating proteins in ethane. However, it
may be necessary to do so to achieve stable samples so a judgment must be made.
A sample that is properly encapsulated will not become clearer with the addition of
more pressure.
Increasing the pressure in the chamber directly using ethane is not always the best
option since this would have negative consequences on the sample viscosity as well
as the pressure could not be subsequently reduced as this would draw sample out of
the chamber. However, the pressure on the piston can be used to increase the
chamber pressure without diluting the sample or making irreversible additions. This
procedure is
optional
, and is presented only as another method available for finding
the proper encapsulation pressure. To perform this piston pressurization routine
refer to Figure 13 and perform the following:
i)
Close
the
HP ALKANE
valve to isolate the mixing chamber.
ii)
Open
the
HP ALKANE TO BOOSTER
valve.
iii)
Open
the
PISTON BOOSTER
valve to expose the piston to the pressure
source.
iv)
Using the syringe pump increase the pressure on the piston to a target
pressure above the internal chamber pressure.
v)
Watch for additional clearing of the sample.
vi)
After testing is complete, reduce the syringe pump pressure to below the
internal chamber pressure (if possible – see (ix))
vii)
Close
the
HP ALKANE TO BOOSTER
and
PISTON BOOSTER
valve.
viii)
Resume preparing the sample with the newly obtained data.
ix)
Optional: It may be necessary to release the piston pressure to reduce
the chamber pressure back to what it was originally. If necessary, be sure