<html><head><base href="x-msg://12/"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">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><br><div>
<span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Arial; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Arial; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="font-family: Century; ">==============================================</span><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Century; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Century; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><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>617-658-7808<br>skype: w.stafford3<br>----------------------<br>"Be kind, for everyone you meet is fighting a hard battle." -Plato<br>------------------------------------</div><div><br></div></div><br></div><br><br></div></span></div></span></div></span></span>
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<br><div><div>On Jan 31, 2012, at 11:32, Peter Edward Prevelige Jr wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"><span class="Apple-style-span" style="border-collapse: separate; font-family: Arial; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><div lang="EN-US" link="blue" vlink="purple"><div class="WordSection1" style="page: WordSection1; "><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 –<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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.<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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).<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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.<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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. <o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></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<o:p></o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Peter E. Prevelige Jr.<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Professor<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Dept. of Microbiology, BBRB 416/6<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Univ. of Alabama @ Birmingham<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">845 19th St. South<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Birmingham AL. 35294-2170<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">Phone 205 975-5327<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; ">FAX 205 975-5479<o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; "><a href="mailto:prevelig@uab.edu" style="color: blue; text-decoration: underline; ">prevelig@uab.edu</a><o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><span style="font-size: 10.5pt; font-family: Consolas; "><a href="http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm" style="color: blue; text-decoration: underline; ">http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm</a><o:p></o:p></span></div><div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif; "><o:p> </o:p></div></div><span><Scan.pdf></span>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@rasmb.bbri.org" style="color: blue; text-decoration: underline; ">RASMB@rasmb.bbri.org</a><br><a href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb" style="color: blue; text-decoration: underline; ">http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</a><br></div></span></blockquote></div><br></div></body></html>