[RASMB] XLI absorbance problem
Igor Perevyazko
i.perevyazko at gmail.com
Sun Feb 22 09:12:13 PST 2015
Hello Andrew,
Thanks for the tip, it make sense. I didn't try that yet, but will do it
tomorrow.
Best regards
Igor
On Sun, Feb 22, 2015 at 8:20 PM, Andrew Leech <andrew.leech at york.ac.uk>
wrote:
> Hi Igor,
>
> Have you tried loading the cell upside down (or backwards), to put the
> sample in the reference side and vice versa? Alternatively load the
> two sectors with grossly different volumes (only spin at 3000 rpm to
> scan). I think you are seeing the reference sector twice somehow.
>
> All the best,
>
> Andrew
>
> On 21/02/2015 22:09, John Philo wrote:
>
>> 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
>>
>>
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> --
> Dr Andrew Leech * Laboratory Head
> Technology Facility * Molecular Interactions Laboratory
> Department of Biology (Area 15) * Tel : +44 (0)1904 328723
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