[RASMB] fringe deviation

[Tom Laue]

Dear Barbara,
I concur with the replies of others, but wish to add a couple of quick 
comments. As John Philo mentions, you working at concentrations where 
both thermodynamic and hydrodynamic nonideality are significant. The two 
forms of nonideality are different and have different (often opposite) 
effects on the raw data, making them difficult to separate. If you stick 
with equilibrium analysis, you will eliminate hydrodynamic nonideality, 
and can get a better fix on the effects of thermodynamic nonideality.
Regarding the optical systems, it is important to note that it is the 
*gradient* in the concentration that results in Weiner skewing, not the 
concentration per se. If you can limit the steepness of your gradients 
(or trim away the data from steep gradients), you will limit these effects.
Be aware that the absorbance optics are subject to the effects of 
refractive gradients, too. If you have a steep concentration 
(refractive) gradient, light can be deviated sufficiently that it will 
not be detected by the absorbance detector. No adjustment of the 
wavelength will overcome this problem. In fact, the tell-tale test for 
this problem is to scan your sample at a wavelength where no absorbance 
occurs- you will see a positive going peak in the data (the derivative 
of the concentration profile, it turns out) where the gradient is steep. 
You can help minimize the problems by using shorter pathlength cells and 
reducing concentration gradients.
Best wishes,
Tom

lelj wrote:

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>Hello!
>
>I am trying to determine the association constant for a monomer-dimer equilibrium. I know the association occurs because I've seen it by velocity experiments.
>
>I run equilibrium at 3 concentrations and 3 speeds, my conc. are high and therefore I used interference optics. I have a fringe displacement at equilibrium of up to 50 fringes. I fit my data with WinNonlin and at the best I can get a SQUARE ROOT OF VARIANCE=9.3317E-02.  Since I have such a big fringe displacement when I plot deviation vs. indipendent variable, in the worst case I have a deviation of -0.25 to 0.375 fringes, is it reasonable?
>
>thanks 
>barbara
>
>---
>Barbara Lelj Garolla Di Bard
>Dr. Mauk's Lab
>Dept. of Biochemistry and Molecular Biology
>University of British Columbia
>2146 Health Sciences Mall
>Vancouver, B.C. V6T 1Z3
>Phone: (604) 822-2526
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Department of Biochemistry and Molecular Biology
University of New Hampshire
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Phone: 603-862-2459
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E-mail: Tom.Laue at unh.edu
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