[RASMB] Absorbance Scan Question

Peter Edward Prevelige Jr prevelig at uab.edu
Tue Jan 31 10:40:41 PST 2012


Thanks everyone -

I've learned quite a bit and have some leads to check out. We do have a cell with a holium oxide filter so I can check the monochrometer alignment as per Tom and Walter's suggestion. That would seem to be pretty important to be certain of even if I ultimately stick to using just one wavelength for the analysis.

Peter  

Peter E. Prevelige Jr.
Professor
Dept. of Microbiology, BBRB 416/6
Univ. of Alabama @ Birmingham
845 19th St. South
Birmingham AL. 35294-2170
Phone 205 975-5327
FAX 205 975-5479
prevelig at uab.edu
http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm


-----Original Message-----
From: Tom Laue [mailto:Tom.Laue at unh.edu] 
Sent: Tuesday, January 31, 2012 12:00 PM
To: Cole, James
Cc: Peter Edward Prevelige Jr; RASMB
Subject: Re: [RASMB] Absorbance Scan Question

Hi-
A couple of thoughts- at 236 nm, you are nearer the sharp 230 nm spike 
in the Xe-lamp intensity than at 240 nm. The optical bandwith of the 
monochromator is ~5 nm. This should mean that you would have a lower 
apparent absorbance at 236 nm than at 240 nm if stray light were the 
problem.
You can test whether Walter's suggestion is correct (I believe it may 
be) using a holmium oxide filter mounted in a cell housing sector, and 
scanning the cell at 3000 rpm at a wavelength where the filter spikes in 
absorbance. If the absorbance changes along the length of the cell, the 
monochromator is out of mechanical alignment. Beckman used to sell an 
appropriate cell for this, or your field service agent may have one.
Best wishes,
Tom

On 1/31/2012 12:13 PM, Cole, James wrote:
> Hi Peter
> It could be heterogeneity but I suspect that you are seeing nonlinearity  as you  go to high absorbance. If the 20% TFA has significant OD at short wavelengths then the total absorbance at the bottom of the cell will be out of the linear range, even if the sample-buffer absorbance is below 1 OD.  I would not expect that the ratios you obtain from a spectrum recorded on the XLA would match those in the plot because the wavelength  reproducibility of the monochromator  is lousy and you are looking at a steeply rising portion of the spectrum.
> Jim
>
>
> On Jan 31, 2012, at 11:32 AM, Peter Edward Prevelige Jr wrote:
>
> Hi All -
>
> I'm doing an equilibrium run on a  ~3 KDa synthetic peptide lacking aromatics in 20% TFE. I  thought I might use multiple wavelengths to extend the concentration range that I could follow.
>
> I thought I'd be clever and obtain the ratio of extinction coefficients by dividing the absorbance at one wavelength (236 nm) by the other  (240 nm) across the cell (rather than using the spectrum).
>
> I calculated the ratio using only the points that were at the same radial distance and plotted them vs radial distance. It is pretty apparent that the ratio is changing across the cell.
>
> My question is whether this is likely to be an optical artifact or whether it most likely represent heterogeneity in the sample. It is probably worth noting that the ratio from the spectrum is 1.43, intermediate between the extremes seen across the cell which to me suggests heterogeneity.
>
> Thanks
>
> Peter
>
> Peter E. Prevelige Jr.
> Professor
> Dept. of Microbiology, BBRB 416/6
> Univ. of Alabama @ Birmingham
> 845 19th St. South
> Birmingham AL. 35294-2170
> Phone 205 975-5327
> FAX 205 975-5479
> prevelig at uab.edu<mailto:prevelig at uab.edu>
> http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm
>
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
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