[RASMB] XLI absorbance problem

Sat Feb 21 14:09:23 PST 2015

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.

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?

John

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Igor Perevyazko
Sent: Saturday, February 21, 2015 11:24 AM
To: Laue, Thomas
Cc: RASMB List
Subject: Re: [RASMB] XLI absorbance problem

Dear Thomas,

Thank you very much for your reply.

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. 

The long -pass filter was in 0 zero position. 

Best regards

Igor

On Sat, Feb 21, 2015 at 9:18 PM, Laue, Thomas <Tom.Laue at unh.edu <mailto:Tom.Laue at unh.edu> > wrote:

Hi-
The sample and reference intensities are nearly identical. There is no way they are 1.2 and 0.6 OD.
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.
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). 
Tom

  _____  

From: RASMB [rasmb-bounces at list.rasmb.org <mailto:rasmb-bounces at list.rasmb.org> ] on behalf of Igor Perevyazko [i.perevyazko at gmail.com <mailto:i.perevyazko at gmail.com> ]
Sent: Saturday, February 21, 2015 11:49 AM
To: Luitgard.Nagel-Steger at uni-duesseldorf.de <mailto:Luitgard.Nagel-Steger at uni-duesseldorf.de> ; jphilo at mailway.com <mailto:jphilo at mailway.com> 
Cc: RASMB List
Subject: Re: [RASMB] XLI absorbance problem

Dear John, Luitgard

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. 

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.

Looking forward to any of your suggestions.

Best regards

Igor

On Fri, Feb 20, 2015 at 9:12 PM, Luitgard Nagel-Steger <Luitgard.Nagel-Steger at uni-duesseldorf.de <mailto:Luitgard.Nagel-Steger at uni-duesseldorf.de> > wrote:

Dear Igor,

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.

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.

Best regards,

Luitgard

Am 20.02.2015 um 14:20 schrieb Igor Perevyazko:

Dear RASMB members,

We are having a problem with absorbance optics on XLI centrifuge(XLI_1
on the images attached). Hopefully you can suggest something that will
help. The problem is that optical density of any samples (which
definitely absorb, including for example BSA (c = 4mg/mL) or pyrene dye
(c = 10µM )) recorded by the AUC is within the zero  level.
Nevertheless, at much higher concentrations (approximately 10 -20 times
higher) the optical density reaches some level of absorbance (~0.1-0.2
OD). The same solutions checked by normal UV-Vis spectrophotometer and
on another XLI centrifuge (XLI_2), have adequate values of the
absorbance level. We have as well checked our monochromator   on  XLI_2
and it was working ok. Recorded lamp intensities (air to air) at 230 nm
and 530 nm are at the normal level. If we perform the absorbance scan of
a sample (for example Pyrene dye at c = 10µM)  in the intensity mode, we
received the same level of intensities for both reference and sample
chamber! The corresponding comparison of the intensities is shown on the
image attached. Any of your suggestions about the possible problem
and/or the solution will be highly appreciated.

Best regards

Igor Perevyazko

PhD Igor Perevyazko
Physics Department, Polymer Physics
St.Petersburg State University
Ul. Ulyanovskaya 1, Petrodvorets
St.Petersburg, Russia, 198504
Tel.: (812)4284382
Fax.: (812)4287240

_______________________________________________
RASMB mailing list
RASMB at list.rasmb.org <mailto:RASMB at list.rasmb.org> 
http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

_______________________________________________
RASMB mailing list
RASMB at list.rasmb.org <mailto:RASMB at list.rasmb.org> 
http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20150221/9e64676c/attachment.htm>