[RASMB] XLI absorbance problem

Sun Feb 22 09:22:34 PST 2015

Dear Titus,

Sure, I don't have at hand in the moment and will send it in the morning.

All the best
Igor

On Sun, Feb 22, 2015 at 9:09 PM, Titus M. Franzmann <
titus.franzmann at outlook.com> wrote:

>  Can you send a radial scan of the counter balance.
>
> Sent from my Windows Phone
>  ------------------------------
> Von: Igor Perevyazko <i.perevyazko at gmail.com>
> Gesendet: ‎22.‎02.‎2015 18:06
> An: jphilo at mailway.com; rasmb at rasmb.org
> Betreff: Re: [RASMB] XLI absorbance problem
>
>   Dear John,
>
> Thank you very much for your reply.
>
> You wrote:
> > 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?
>
> Yes,I was using 4-hole rotor and GUI was checked for it as well.  The last
> two experiments (* Radial intensity scan of KNO3 (0.7 g/dl), with
> 0.6OD at 302 nm ** Radial intensity scan of Pyrene (2.5 microM), with ~1.2
> OD at 3 <0.6OD at 302>38 nm) were conducted separately, with only one cell
> loaded. I don't think that the instrument reporting data for the wrong
> cell. For some reason the photomultiplier does not see the difference in
> intensities/absorbance for samples which definitely absorb, unless a real
> big concentration(that is how we can also proof  that the instrument
> doesn't mismatched the position of a scanning cell) is used.
> As I mention previously, the absorbance of samples was checked on regular
> Spectrofotometer and on working XLI_2. We have as well checked the
> monochromator of our XLI_1 on the working XLI_2 (please, see the initial
> message). This makes us think, that monochromator is ok.
>
> Best regards
>  Igor
>
>
>
> On Sun, Feb 22, 2015 at 2:09 AM, John Philo <jphilo at mailway.com> 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> 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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