[RASMB] AUC interference concentration limits

[Laue, Thomas]

Hi all-

Re Weiner skewing: It is my observation that the Beckman service personnel do not have the equipment needed to focus properly at the 2/3 plane. What is used is a ruled disc positioned at the 2/3 plane. However, there is no liquid in the cell, so the disc position is off slightly. It is difficult to know when you have the proper focus... the fringes always look just fine.

Also, you need to focus at the 2/3 plane of any centerpiece type. If you are focused above this plane, you will observe that the fringes are too steep, below it, they will be less steep than what is correct.

Lee Gropper outlined a nice way to focus the interference system (albeit at the mid-plane rather than the 2/3 plane... the only difference is where the ruled disc is positioned in the cell). Note, however, the procedure requires the schlieren optical system, which is not available on the XLI.

Tom


From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Walter Stafford
Sent: Tuesday, October 20, 2015 6:32 PM
To: John Sumida
Cc: rasmb at list.rasmb.org
Subject: Re: [RASMB] AUC interference concentration limits

As said by others. the limiting thing here is not the concentration, but the concentration gradient. There really is no practical limit to the max concentration. The limit is the ability to resolve the fringes in the radial direction. The steepness for the fringes and the radial resolution of the camera determines that. The vertical distance between fringes is constant and is something like 22 pixels/per fringe, and in the radial dimension the distance between fringes is about 0.007 cm per pixel. So 1 fringe displacement over one pixel in the radial direction correspond to an upper limit of 142 fringes per cm. The way to avoid the problem you are seeing, if it's feasible, is to run at a slower speed and wait for the boundary to spread enough so that the fringes can be resolved. The actual practical limit is probably about half that, or about 70 fringes per cm, or a gradient of about 21 mg/(mL-cm).

Walter Stafford
wstafford3 at walterstafford.com<mailto:wstafford3 at walterstafford.com>

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On Oct 20, 2015, at 15:32, John Sumida <jpsumida at uw.edu<mailto:jpsumida at uw.edu>> 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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