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<TITLE>Re: [RASMB] fringe deviation</TITLE>
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<BLOCKQUOTE>Hi Tom -<BR>
<BR>
Obviously we shared the same thought about <FONT COLOR="#800080">'schlieren effects</FONT>' within a few seconds of each other!<BR>
<BR>
Your points are well made. Just <FONT COLOR="#FF0000">one little thing</FONT>, though, which prompts an extra comment from me. I cannot see that you get a 'better fix' on non-ideality in SE than you do in SV. In both cases there are ball-park values you can use for globular systems (e.g. ks ~ 5-6; 2BM ~ 8-10). But for SV you can <I>compute</I> ks from molecular parameters (frictional ratio etc), which you certainly cannot do for 2BM. The latter is also more prone to charge effects. <BR>
<BR>
I have used SV to probe or get Kd values and published results over the years. But right now, with software on my desktop which happily gives me estimates for both BM and Ka from SE, I think it is a complex issue as to which approach is best. <BR>
<BR>
Regards<BR>
<BR>
Arthur<BR>
<BR>
<TT><BR>
Dear Barbara,<BR>
I concur with the replies of others, but wish to add a couple of quick <BR>
comments. As John Philo mentions, you working at concentrations where <BR>
both thermodynamic and hydrodynamic nonideality are significant. The two <BR>
forms of nonideality are different and have different (often opposite) <BR>
effects on the raw data, making them difficult to separate. <FONT COLOR="#FF0000">If you stick <BR>
with equilibrium analysis, you will eliminate hydrodynamic nonideality, <BR>
and can get a better fix on the effects of thermodynamic nonideality.<BR>
</FONT>Regarding the optical systems, it is important to note that it is the <BR>
*gradient* in the concentration that results in Weiner skewing, not the <BR>
concentration per se. If you can limit the steepness of your gradients <BR>
(or trim away the data from steep gradients), you will limit these effects.<BR>
Be aware that the absorbance optics are subject to the effects of <BR>
refractive gradients, too. <FONT COLOR="#800080">If you have a steep concentration <BR>
(refractive) gradient, light can be deviated sufficiently that it will <BR>
not be detected by the absorbance detector.</FONT> No adjustment of the <BR>
wavelength will overcome this problem. In fact, the tell-tale test for <BR>
this problem is to scan your sample at a wavelength where no absorbance <BR>
occurs- you will see a positive going peak in the data (the derivative <BR>
of the concentration profile, it turns out) where the gradient is steep. <BR>
You can help minimize the problems by using shorter pathlength cells and <BR>
reducing concentration gradients.<BR>
Best wishes,<BR>
Tom<BR>
<BR>
lelj wrote:<BR>
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>Hello!<BR>
><BR>
>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.<BR>
><BR>
>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?<BR>
><BR>
>thanks <BR>
>barbara<BR>
><BR>
>---<BR>
>Barbara Lelj Garolla Di Bard<BR>
>Dr. Mauk's Lab<BR>
>Dept. of Biochemistry and Molecular Biology<BR>
>University of British Columbia<BR>
>2146 Health Sciences Mall<BR>
>Vancouver, B.C. V6T 1Z3<BR>
>Phone: (604) 822-2526<BR>
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<BR>
-- <BR>
Department of Biochemistry and Molecular Biology<BR>
University of New Hampshire<BR>
Durham, NH 03824-3544<BR>
Phone: 603-862-2459<BR>
FAX: 603-862-0031<BR>
E-mail: Tom.Laue@unh.edu<BR>
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