[RASMB] XLI absorbance problem

[Luitgard Nagel-Steger]

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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