[RASMB] Absorbance Scan Question

[Cole, James]

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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