[RASMB] XLI absorbance problem

Sat Feb 21 11:23:33 PST 2015

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> 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] on behalf of Igor Perevyazko
> [i.perevyazko at gmail.com]
> *Sent:* Saturday, February 21, 2015 11:49 AM
> *To:* Luitgard.Nagel-Steger at uni-duesseldorf.de; 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> 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
>>>
>>>
>>>
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>>> RASMB at list.rasmb.org
>>> http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org
>>>
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>>
>
>
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