[RASMB] XLI absorbance problem
Andrew Leech
andrew.leech at york.ac.uk
Thu Feb 26 09:19:25 PST 2015
Hello all,
I should learn to do the experiment before making pronouncements!
The counterbalance radial intensity scan on my XL/I gives about
150 counts where the light is coming through at the edges, so not
too different from Igor's system.
Andrew
On 24/02/2015 10:29, Andrew Leech wrote:
> 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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>>
>> http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org____
>>
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>> __ __
>>
>> __ __
>>
>>
>>
>>
>>
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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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