[RASMB] AUC interference concentration limits

[Borries Demeler]

John,

If the fringes get too steep you will get refractive artifacts and
lens effects such as light bending, up to the point where the light is
actually bent away from the camera, or it least from the radial position
where you expect to see it.
The best way to deal with it is to switch to 3 mm centerpieces.
Tom once mentioned to me that he ran a velocity experiment by using a 
seal for the aluminum centerpieces as a centerpiece. Not sure how 
well that works, but that would give you about a 1 mm "centerpiece",
but may not follow sector shape exactly. But reducing the pathlength 
is the best way to eliminate such affects. Other things you may try 
is to switch to heavy water, which has a different scattering factor
that may reduce refractive index artifacts. Not sure how well this 
works with proteins, but may be worth a try.

-b.




On Tue, Oct 20, 2015 at 12:32:51PM -0700, John Sumida wrote:
> Dear RASMB,
> 
>  
> 
> I have been reviewing the thread posted on RASMB between September 1 2008
> and September 15 2008 under the subject heading "upper concentration limit
> AUC".   
> 
>  
> 
> We attempted to perform an SV measurement of a sample using interference
> detection at a sample concentration of 17 mgs/ml.  
> 
>  
> 
> At this concentration however I observe a gap in the boundary data that
> resulted from the spin (see bmp image attached); this behavior was not
> observed at lower concentrations up to 2-5 mgs/ml.  
> 
>  
> 
> The gap in the data appears to be correlated with the absence of a fringe
> pattern in the ccd image.  Adjustment of the duration setting does not
> resolve the issue with the data, but it does improves the ccd image so that
> I can make out the fringes in the region where I'm observing the gap in the
> data.  Counting the fringes/mm in this region, I estimate the fringe
> gradient to be approximately 50 fringes/mm. This is less than the 76
> fringes/mm noted in the RASMB thread but my count may be off.
> 
>  
> 
> On a separate note, since I suspect that the artifact I'm seeing is
> associated with the observed steep fringe gradient I had wondered if our
> instrument was focused at the 2/3rds plane or not.  I have been informed, by
> the vendor, that the process of focusing the interference optics at the
> 2/3rds optical plane is not something that is possible in the "real world"
> (their words, not mine), and that this is that this is only something that
> is only "theoretically" possible.  This appears to be in direct
> contradiction to the descriptions provided on pages 311 and 312 of Yphantis
> et. al. Biochemistry 1964 vol.3  (see attached) where concentrations of 12.5
> and higher were measured (if my reading is correct) where measurements were
> performed by using the 2/3rds plane focusing.
> 
>  
> 
> Questions.
> 
> Is the displacement in the fringe data along the radial axis consistent with
> a concentration gradient that is too steep?  
> 
> What is the best way to measure the fringe/mm value?
> 
> Are concentrations up to 30mgs/ml possible in a 1.2 cm cell using
> interference optics, and if so how should the optics be focused?
> 
>  
> 
> Thank you in advance for your time, comments and your advice.
> 
>  
> 
> Best regards,
> 
> John Sumida 
> 
> University of Washington
> 
> Molecular Engineering & Sciences
> 
>  
> 



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