[RASMB] interference optics

John Philo jphilo at mailway.com
Sat Apr 5 11:25:30 PDT 2008


Fumio,

There is a limit on the maximum gradient that can be measured (fringes/mm)
due to Weiner (? spelling) skewing, and you are exceeding that limit.  As
Ariel said you should switch to 3 mm centerpieces, and then at some point to
get to higher concentrations you would have to drop the rotor speed too to
broaden the boundaries. If you look through the old RASMB postings you will
find a number of excellent posts from others about Weiner skewing and proper
optical alignment to minimize distortion of strong gradients.

But probably the more important question is what are you going to be able to
do with such data even if you collect it? Remember, in velocity there is
hydrodynamic non-ideality as well as thermodynamic non-ideality, so the
non-ideality effects will kill you at much lower concentrations than in
equilibrium. To my knowledge the only model available to analyze a velocity
experiment at 10 mg/mL is a single non-ideal species. At high concentrations
you cannot even correctly measure the fractions of different components
(e.g. aggregates) in a multi-component mixture due to the Johnston-Ogston
effect.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [ <mailto:rasmb-bounces at rasmb.bbri.org>
mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Fumio Arisaka
Sent: Saturday, April 05, 2008 4:20 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] [Fwd: interference optics]

Dear RASMBers,

This concerns measurement of high concentrations of IgG with interference
optics. I have not much experience with IF, but have started to use it for
the necessity to measure high concentration samples. Concentrations less
than 5 mg/mL had no problem.
Attached jpg file is to show the problem at 10mg/mL. Somehow, the boudaries
tend to go horizontally before reaching the plateau.

I thought this was due to the misalignment of optics or something, because
the fringe pattern is not so good as I expect, but the BeckmanCoulter person
who came to fix it could not make it better.

I would like to know what is the common highest concentration that you could
measure by IF. As I understand one could measure SE at higher concentrations
than 100 mg/mL, but when the boundary gets too steep in SV measurement, the
fringes get too much squeezed to count precisely.

Any comments and advice are highly appreciated.

Best wishes, Fumio






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