<html><head></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">I think your ratio is wrong for this to be effecting your data... so i make this comment because I am not absolutely sure about which way you did the ratio and this may possibly play into it- the Xenon spectrum has a pretty stark trough right at 240.. whereas 236 would be much more intense (as much as 2-3 fold). If my spectrum is correct. Its possible that with different enough input intensity you could be seeing non-linear behavior in one data set (remote...). It would be just my luck to do this exact experiment and run into something like this so I bring it up.<div>
<br></div><div>Matthew Hockin<br><div><br></div><div><br><div><div class="gmail_quote">On Tue, Jan 31, 2012 at 10:12 AM, Walter Stafford <span dir="ltr"><<a href="mailto:stafford@bbri.org">stafford@bbri.org</a>></span> wrote:<br>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word">This could be a monochromater alignment problem. If the m.c. is out of alignment as the scan proceeds the wavelength will vary from one end of the scan to the other ad that could account for the varying ratio.<div>
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<div><div><div>Walter F. Stafford, Ph.D.</div><div>Senior Scientist and</div><div>Director Analytical Ultracentrifugation Research Laboratory<br>Boston Biomedical Research Institute<br>64 Grove Street<br>Watertown, MA 02472<br>
<a href="tel:617-658-7808" value="+16176587808" target="_blank">617-658-7808</a><br>skype: w.stafford3<br>----------------------<br>"Be kind, for everyone you meet is fighting a hard battle." -Plato<br>------------------------------------</div>
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<br><div><div><div class="h5"><div>On Jan 31, 2012, at 11:32, Peter Edward Prevelige Jr wrote:</div><br></div></div><blockquote type="cite"><span style="border-collapse:separate;font-family:Arial;font-style:normal;font-variant:normal;font-weight:normal;letter-spacing:normal;line-height:normal;text-align:-webkit-auto;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;font-size:medium"><div lang="EN-US" link="blue" vlink="purple">
<div><div class="h5"><div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">Hi All –<u></u><u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">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.<u></u><u></u></div>
<div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
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).<u></u><u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">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.<u></u><u></u></div>
<div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
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. <u></u><u></u></div>
<div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
Thanks<u></u><u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
Peter<u></u><u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif"><u></u> <u></u></div><div style="margin-top:0in;margin-right:0in;margin-left:0in;margin-bottom:0.0001pt;font-size:11pt;font-family:Calibri,sans-serif">
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