[RASMB] XLI absorbance problem

[Igor Perevyazko]

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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