[RASMB] XLI absorbance problem
Andrew Leech
andrew.leech at york.ac.uk
Tue Feb 24 02:29:44 PST 2015
Hi Igor,
Those numbers (80 counts) look really low. I only recall seeing
such small numbers when we had a photomultiplier problem, but your
other scans rule that out. I think the light is not going through
the counterbalance holes and you are seeing stray light.
All the best,
Andrew
On 24/02/2015 08:01, Igor Perevyazko wrote:
> Dear Titus,
>
> Please find attached the radial intensity scan of the counter balance.
>
> Best regards
> Igor
>
> On Sun, Feb 22, 2015 at 9:09 PM, Titus M. Franzmann
> <titus.franzmann at outlook.com <mailto:titus.franzmann at outlook.com>> wrote:
>
> Can you send a radial scan of the counter balance.
>
> Sent from my Windows Phone
> ------------------------------------------------------------------------
> Von: Igor Perevyazko <mailto:i.perevyazko at gmail.com>
> Gesendet: 22.02.2015 18:06
> An: jphilo at mailway.com <mailto:jphilo at mailway.com>; rasmb at rasmb.org
> <mailto: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 <mailto:0.6OD at 302> nm ** Radial intensity scan
> of Pyrene (2.5 microM), with ~1.2OD at 3 <mailto: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
> <mailto: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
> <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
University of York * Fax : +44 (0)1904 328804
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