[RASMB] XLI absorbance problem
Andrew Leech
andrew.leech at york.ac.uk
Sun Feb 22 08:20:58 PST 2015
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
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Department of Biology (Area 15) * Tel : +44 (0)1904 328723
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