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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>Igor, I agree with Tom that possibly the wavelength is very different from what is reported. For that reason it would perhaps be better to test with something having very broad absorbance rather than sharp peaks. On the other hand the wavelength intensity scans you sent earlier seem to indicate the wavelength is getting selected (i.e. the grating has not fallen out) and not grossly out of calibration.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>Is there any possibility the instrument is somehow reporting data for the wrong cell? You have told the GUI that you are using a 4-hole rotor, not an 8-hole, correct? What was in the other cell positions when you recorded these benzene data?<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'>John<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial",sans-serif;color:blue'><o:p> </o:p></span></p><p class=MsoNormal><b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'>From:</span></b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>Igor Perevyazko<br><b>Sent:</b> Saturday, February 21, 2015 11:24 AM<br><b>To:</b> Laue, Thomas<br><b>Cc:</b> RASMB List<br><b>Subject:</b> Re: [RASMB] XLI absorbance problem<o:p></o:p></span></p><p class=MsoNormal><o:p> </o:p></p><div><div><div><div><div><p class=MsoNormal style='margin-bottom:12.0pt'>Dear Thomas,<o:p></o:p></p></div><div><p class=MsoNormal>Thank you very much for your reply.<o:p></o:p></p></div><p class=MsoNormal>Well, the same intensities points out the problem we are dealing with (nonetheless, at much higher concentration (~ 30 mg/ml for proteins, for example) we do observe some level of optical densities ~0.1-0.2 OD). As I mentioned previously we have checked our monochromator on another XLI and it was working absolutely fine (please see the first email in the conversion list, there is also information about the lamp intensity and its comparison with the intensities of dye solution). The mismatched position of menisci, you have mentioned, I believe is simply related to a slightly different filling volumes. <o:p></o:p></p></div><p class=MsoNormal>The long -pass filter was in 0 zero position. <o:p></o:p></p></div><div><p class=MsoNormal><o:p> </o:p></p></div><p class=MsoNormal>Best regards<o:p></o:p></p></div><p class=MsoNormal>Igor<o:p></o:p></p></div><div><p class=MsoNormal><o:p> </o:p></p><div><p class=MsoNormal>On Sat, Feb 21, 2015 at 9:18 PM, Laue, Thomas <<a href="mailto:Tom.Laue@unh.edu" target="_blank">Tom.Laue@unh.edu</a>> wrote:<o:p></o:p></p><blockquote style='border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-right:0in'><div><div><p class=MsoNormal><span style='color:black'>Hi-<br>The sample and reference intensities are nearly identical. There is no way they are 1.2 and 0.6 OD.<br>Is there any possibility that your monochromator is malfunctioning? If the actual wavelength were significantly different from what is being reported, you would get the sort of scans you are seeing. The menisci are mismatched, demonstrating that there is not a leak across the septum, so a wrong wavelength makes some sense.<br>One last possibility- is the 400 nm long-pass filter in (lever in the horizontal position)? That would block the signal at the shorter wavelengths. The instrument would respond by setting the gain as high as it could, leading to a noisier signal (all dark current). <br>Tom<o:p></o:p></span></p><div><div class=MsoNormal align=center style='text-align:center'><span style='color:black'><hr size=3 width="100%" align=center></span></div><div><p class=MsoNormal style='margin-bottom:12.0pt'><b><span style='font-family:"Tahoma",sans-serif;color:black'>From:</span></b><span style='font-family:"Tahoma",sans-serif;color:black'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org" target="_blank">rasmb-bounces@list.rasmb.org</a>] on behalf of Igor Perevyazko [<a href="mailto:i.perevyazko@gmail.com" target="_blank">i.perevyazko@gmail.com</a>]<br><b>Sent:</b> Saturday, February 21, 2015 11:49 AM<br><b>To:</b> <a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@uni-duesseldorf.de</a>; <a href="mailto:jphilo@mailway.com" target="_blank">jphilo@mailway.com</a><br><b>Cc:</b> RASMB List<br><b>Subject:</b> Re: [RASMB] XLI absorbance problem</span><span style='color:black'><o:p></o:p></span></p></div><div><div><div><div><div><div><div><div><p class=MsoNormal style='margin-bottom:12.0pt'><span style='color:black'>Dear John, Luitgard<o:p></o:p></span></p></div><p class=MsoNormal><span style='color:black'>Attached you can find radial intensity scans with an optical density of ~0.6 OD (KNO3, с = 0.07 dl/g, l = 302 nm) and ~1.2 OD (pyrene dye, 2.5micrM, l = 338 nm). I am using 4-hole rotor. <o:p></o:p></span></p></div><p class=MsoNormal><span style='color:black'>Regarding Luitgards points: Concerning benzene, this is correct, but for our system, previous experiments with such solvent /polymer combination resulted in a satisfactory optical densities. The large concentration of BSA in this case was chosen, as a model/test system to represent the problem - that even at such a high BSA content the optical density is at zero level.<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:black'>Looking forward to any of your suggestions.<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p></div><p class=MsoNormal><span style='color:black'>Best regards<o:p></o:p></span></p></div><p class=MsoNormal><span style='color:black'>Igor<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><div><p class=MsoNormal><span style='color:black'>On Fri, Feb 20, 2015 at 9:12 PM, Luitgard Nagel-Steger <<a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@uni-duesseldorf.de</a>> wrote:<o:p></o:p></span></p><blockquote style='border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-right:0in'><p class=MsoNormal><span style='color:black'>Dear Igor,<br><br>I am wondering whether benzene is a good choice of a solvent if your pyrene dye is expected to absorb in the range between 250 and 350 nm (shown in the attached graph 1), since it absorbs itself rather strongly in the UV range.<br><br>Also 4 mg/ml of BSA are a pretty large amount of protein for the optics in the AUC. Assuming you are using standard cells with 1.2 cm optical path length this should give an absorbance at 280 nm of 4 OD. The upper limit is at about 1 OD.<br><br>Best regards,<br><br>Luitgard<br><br>Am 20.02.2015 um 14:20 schrieb Igor Perevyazko:<o:p></o:p></span></p><blockquote style='border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-right:0in'><div><div><p class=MsoNormal style='margin-bottom:12.0pt'><span style='color:black'>Dear RASMB members,<br><br>We are having a problem with absorbance optics on XLI centrifuge(XLI_1<br>on the images attached). Hopefully you can suggest something that will<br>help. The problem is that optical density of any samples (which<br>definitely absorb, including for example BSA (c = 4mg/mL) or pyrene dye<br>(c = 10µM )) recorded by the AUC is within the zero level.<br>Nevertheless, at much higher concentrations (approximately 10 -20 times<br>higher) the optical density reaches some level of absorbance (~0.1-0.2<br>OD). The same solutions checked by normal UV-Vis spectrophotometer and<br>on another XLI centrifuge (XLI_2), have adequate values of the<br>absorbance level. We have as well checked our monochromator on XLI_2<br>and it was working ok. Recorded lamp intensities (air to air) at 230 nm<br>and 530 nm are at the normal level. If we perform the absorbance scan of<br>a sample (for example Pyrene dye at c = 10µM) in the intensity mode, we<br>received the same level of intensities for both reference and sample<br>chamber! The corresponding comparison of the intensities is shown on the<br>image attached. Any of your suggestions about the possible problem<br>and/or the solution will be highly appreciated.<br><br><br>Best regards<br><br>Igor Perevyazko<br><br><br>PhD Igor Perevyazko<br>Physics Department, Polymer Physics<br>St.Petersburg State University<br>Ul. Ulyanovskaya 1, Petrodvorets<br>St.Petersburg, Russia, 198504<br>Tel.: (812)4284382<br>Fax.: (812)4287240<br><br><br><o:p></o:p></span></p></div></div><p class=MsoNormal style='margin-bottom:12.0pt'><span style='color:black'>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@list.rasmb.org" target="_blank">RASMB@list.rasmb.org</a><br><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org" target="_blank">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><o:p></o:p></span></p></blockquote><div><div><p class=MsoNormal><span style='color:black'>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@list.rasmb.org" target="_blank">RASMB@list.rasmb.org</a><br><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org" target="_blank">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><o:p></o:p></span></p></div></div></blockquote></div><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p></div></div></div></div></div></div></div></blockquote></div><p class=MsoNormal><o:p> </o:p></p></div></div></body></html>