[RASMB] interference & meniscii

[Holger Strauss]

Dear all,

recently, I started to work more with the interference optics of the Xl-I,
and there are some points not clear to me yet:

a) how do you "precisely" determine the mensiscus and bottom position of
the cell? I started with the recommended procedure by Beckmann, but now I
include a larger region in the scanning, but still, things are not clear.
Kristian Schilling suggested in this forum that you can only get the
precise meniscii positions from the intensity data, and not from the
interference alone? (I'd rather measure that parameter than have it
estimated.)

b) There seems to have been a problem with the timing of the laser pulses
at speeds > 50 K a couple of years ago. How's about that (our machine is
from '99)?

c) When using Lamm-equation modelling to evaluate the data, you need an
estimate of c0; what is the best way to get (again, measure) this
parameter? At low loading concentrations and 3-mm CPs I guess you run
into trouble when simultaneaously estimating for both meniscii and c0?
There is a paper by Babul-J et al. (1969), Anal Biochem 28: 216, but the
procedure descibed in there seems to be unsuitable for low (<0.15 mg/mL of
protein/peptide) concentrations. (And again, I'd rather fix that
parameter, since it should not be so much of a problem to include that as
prior knowlegde into the fitting?) Calculating the TI- and RI-noise and
substracting it from a scan at 3 K?
The other way of asking the question is of course how do get the baseline
offset, when you let the SedVel continue until SedEq is established; but
if I remember there is no way of measuring that parameter unless you can't
deplete the meniscus (which I can't, in this special case).

d) Is there a suggested procedure to test wether the change in
concentration measured is really only due to transport of solute and not
of anything else (or otherwise, how do you establish that chemical
equilibrium of the low-molecular weight components really has been
reached in the dialyzate, when you can't deplete the meniscus of your
solute?)? I can't use absorption optics simultaneaously with the
interference (small peptide, no absorption >240 nm).

e) Is there a numerical value of fringe shifts, where the signal is not
linear any more with the change in concentration? And of fringe
shifts/radial position?

f) Finally, is there somewhere a good standard protocol out how to plan,
set up and evaluate experiments with interference optics?

Na. Marvelling at the commplexity of things, all the best. Holger

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Holger Strauss 

Forschungsinstitut fuer Molekulare Pharmakologie (FMP)
Robert-Roessle Strasse 10

13125 Berlin/Germany

Tel: +49 (0)30 94793 - 223 (office)
                     - 316 (lab)

Fax: +49 (0)30 94793 - 169 


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Science is spectrum analysis; art is photosynthesis.

                                                    Karl Kraus