<div dir="ltr"><div>Hello Andrew,<br><br><br></div><div>Thanks for the tip, it make sense. I didn't try that yet, but will do it tomorrow.<br><br></div><div>Best regards<br></div><div>Igor<br></div></div><div class="gmail_extra"><br><div class="gmail_quote">On Sun, Feb 22, 2015 at 8:20 PM, Andrew Leech <span dir="ltr"><<a href="mailto:andrew.leech@york.ac.uk" target="_blank">andrew.leech@york.ac.uk</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">Hi Igor,<br>
<br>
Have you tried loading the cell upside down (or backwards), to put the<br>
sample in the reference side and vice versa? Alternatively load the<br>
two sectors with grossly different volumes (only spin at 3000 rpm to<br>
scan). I think you are seeing the reference sector twice somehow.<br>
<br>
All the best,<br>
<br>
Andrew<span class=""><br>
<br>
On 21/02/2015 22:09, John Philo wrote:<br>
</span><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><span class="">
Igor, I agree with Tom that possibly the wavelength is very different<br>
from what is reported. For that reason it would perhaps be better to<br>
test with something having very broad absorbance rather than sharp<br>
peaks. On the other hand the wavelength intensity scans you sent earlier<br>
seem to indicate the wavelength is getting selected (i.e. the grating<br>
has not fallen out) and not grossly out of calibration.<br>
<br>
Is there any possibility the instrument is somehow reporting data for<br>
the wrong cell? You have told the GUI that you are using a 4-hole rotor,<br>
not an 8-hole, correct? What was in the other cell positions when you<br>
recorded these benzene data?<br>
<br>
John<br>
<br></span>
*From:*RASMB [mailto:<a href="mailto:rasmb-bounces@list.rasmb.org" target="_blank">rasmb-bounces@list.<u></u>rasmb.org</a>] *On Behalf Of *Igor<br>
Perevyazko<br>
*Sent:* Saturday, February 21, 2015 11:24 AM<br>
*To:* Laue, Thomas<br>
*Cc:* RASMB List<br>
*Subject:* Re: [RASMB] XLI absorbance problem<span class=""><br>
<br>
Dear Thomas,<br>
<br>
Thank you very much for your reply.<br>
<br>
Well, the same intensities points out the problem we are dealing with<br>
(nonetheless, at much higher concentration (~ 30 mg/ml for proteins, for<br>
example) we do observe some level of optical densities ~0.1-0.2 OD). As<br>
I mentioned previously we have checked our monochromator on another XLI<br>
and it was working absolutely fine (please see the first email in the<br>
conversion list, there is also information about the lamp intensity and<br>
its comparison with the intensities of dye solution). The mismatched<br>
position of menisci, you have mentioned, I believe is simply related to<br>
a slightly different filling volumes.<br>
<br>
The long -pass filter was in 0 zero position.<br>
<br>
Best regards<br>
<br>
Igor<br>
<br>
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><br></span><span class="">
<mailto:<a href="mailto:Tom.Laue@unh.edu" target="_blank">Tom.Laue@unh.edu</a>>> wrote:<br>
<br>
Hi-<br>
The sample and reference intensities are nearly identical. There is<br>
no way they are 1.2 and 0.6 OD.<br>
Is there any possibility that your monochromator is malfunctioning?<br>
If the actual wavelength were significantly different from what is<br>
being reported, you would get the sort of scans you are seeing. The<br>
menisci are mismatched, demonstrating that there is not a leak<br>
across the septum, so a wrong wavelength makes some sense.<br>
One last possibility- is the 400 nm long-pass filter in (lever in<br>
the horizontal position)? That would block the signal at the shorter<br>
wavelengths. The instrument would respond by setting the gain as<br>
high as it could, leading to a noisier signal (all dark current).<br>
Tom<br>
<br></span>
------------------------------<u></u>------------------------------<u></u>------------<br>
<br>
*From:*RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org" target="_blank">rasmb-bounces@list.rasmb.org</a><br>
<mailto:<a href="mailto:rasmb-bounces@list.rasmb.org" target="_blank">rasmb-bounces@list.<u></u>rasmb.org</a>>] on behalf of Igor Perevyazko<br>
[<a href="mailto:i.perevyazko@gmail.com" target="_blank">i.perevyazko@gmail.com</a> <mailto:<a href="mailto:i.perevyazko@gmail.com" target="_blank">i.perevyazko@gmail.com</a><u></u>>]<br>
*Sent:* Saturday, February 21, 2015 11:49 AM<br>
*To:* <a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@uni-<u></u>duesseldorf.de</a><br>
<mailto:<a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@<u></u>uni-duesseldorf.de</a>>;<br>
<a href="mailto:jphilo@mailway.com" target="_blank">jphilo@mailway.com</a> <mailto:<a href="mailto:jphilo@mailway.com" target="_blank">jphilo@mailway.com</a>><br>
*Cc:* RASMB List<br>
*Subject:* Re: [RASMB] XLI absorbance problem<span class=""><br>
<br>
Dear John, Luitgard<br>
<br>
Attached you can find radial intensity scans with an optical density<br>
of ~0.6 OD (KNO3, с = 0.07 dl/g, l = 302 nm) and ~1.2 OD (pyrene<br>
dye, 2.5micrM, l = 338 nm). I am using 4-hole rotor.<br>
<br>
Regarding Luitgards points: Concerning benzene, this is correct, but<br>
for our system, previous experiments with such solvent /polymer<br>
combination resulted in a satisfactory optical densities. The large<br>
concentration of BSA in this case was chosen, as a model/test system<br>
to represent the problem - that even at such a high BSA content the<br>
optical density is at zero level.<br>
<br>
Looking forward to any of your suggestions.<br>
<br>
Best regards<br>
<br>
Igor<br>
<br>
On Fri, Feb 20, 2015 at 9:12 PM, Luitgard Nagel-Steger<br>
<<a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@uni-<u></u>duesseldorf.de</a><br></span><div><div class="h5">
<mailto:<a href="mailto:Luitgard.Nagel-Steger@uni-duesseldorf.de" target="_blank">Luitgard.Nagel-Steger@<u></u>uni-duesseldorf.de</a>>> wrote:<br>
<br>
Dear Igor,<br>
<br>
I am wondering whether benzene is a good choice of a solvent if<br>
your pyrene dye is expected to absorb in the range between 250<br>
and 350 nm (shown in the attached graph 1), since it absorbs<br>
itself rather strongly in the UV range.<br>
<br>
Also 4 mg/ml of BSA are a pretty large amount of protein for the<br>
optics in the AUC. Assuming you are using standard cells with<br>
1.2 cm optical path length this should give an absorbance at 280<br>
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:<br>
<br>
Dear RASMB members,<br>
<br>
We are having a problem with absorbance optics on XLI<br>
centrifuge(XLI_1<br>
on the images attached). Hopefully you can suggest something<br>
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<br>
pyrene dye<br>
(c = 10µM )) recorded by the AUC is within the zero level.<br>
Nevertheless, at much higher concentrations (approximately<br>
10 -20 times<br>
higher) the optical density reaches some level of absorbance<br>
(~0.1-0.2<br>
OD). The same solutions checked by normal UV-Vis<br>
spectrophotometer and<br>
on another XLI centrifuge (XLI_2), have adequate values of the<br>
absorbance level. We have as well checked our monochromator<br>
on XLI_2<br>
and it was working ok. Recorded lamp intensities (air to<br>
air) at 230 nm<br>
and 530 nm are at the normal level. If we perform the<br>
absorbance scan of<br>
a sample (for example Pyrene dye at c = 10µM) in the<br>
intensity mode, we<br>
received the same level of intensities for both reference<br>
and sample<br>
chamber! The corresponding comparison of the intensities is<br>
shown on the<br>
image attached. Any of your suggestions about the possible<br>
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>
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</span></blockquote><div class="HOEnZb"><div class="h5">
<br>
-- <br>
Dr Andrew Leech * Laboratory Head<br>
Technology Facility * Molecular Interactions Laboratory<br>
Department of Biology (Area 15) * Tel : +44 (0)1904 328723<br>
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