[RASMB] AUC interference concentration limits

[John Philo]

John, I think you are almost certainly exceeding the gradient that can be
tracked (at least on your instrument). That screenshot may not accurately
reflect the true camera image but one can't really see any fringes in the
middle of the boundary. If you want to go that high in concentration IMHO
you need to use 3 mm centerpieces (which of course don't put the sample at
the 2/3 plane, but they will make a huge difference nonetheless).

The Yphantis paper is of course talking about work done in a Model E, not an
XL-I, and you can be sure that instrument was aligned as best as could be
done. I believe there are other old threads that discuss aligning the XL-I
at the 2/3 plane. Jeff Lary at UConn used to talk about alignment at the
Advanced Workshops when they were held there so you may want to contact him.

John

 

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: Tuesday, October 20, 2015 12:33 PM
To: RASMB <rasmb at list.rasmb.org>
Subject: [RASMB] AUC interference concentration limits

 

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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