[RASMB] XL/I absorbance problem

Fri Jun 15 10:59:01 PDT 2007

Hi Andrew,
I have some points of your issue with the absorbance scans.
(1) Run scans on an empty hole at different wavelenths, 220 nm, 250 nm
and 280 nm
(2) Check the position of the monochromator filter lever, for work
below 400 nm, it should parallel to the stem. Interestingly this
position could affect the RMSD a lot.
(3) I totally agree with other remarks, you'd better try a new slit.
Even some tiny dirt could block it  which you cannot see by eyes.

Good luck!
Joy
> >
> > -----Original Message-----
> > From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
> > Behalf Of Leech, AP
> > Sent: Wednesday, June 13, 2007 7:03 AM
> > To: rasmb at server1.bbri.org
> > Subject: [RASMB] XL/I absorbance problem
> >
> >
> > Hello all,
> >
> > I'm not rally happy with the noise I'm seeing on my XL/I's absorbance scans,
> > and I have a few queries. I'd be grateful for any comments.
> >
> > 1. Noise seems excessive even on reasonable samples. Scans on an empty hole
> > at 280 nm give an overall flat profile (in A280) but peak noise is +/-0.05.
> > More peculiar is that the noise seems to be spikes, randomly distributed
> > along radius and not in the same place on successive scans. (For comparison
> > our old XL/A has peak to peak noise +/- 0.015 under the same conditions)
> >
> > 2. Using the request data intensity option, the overall profile has a dip at
> > r=6.3, but also numerous spikes, mostly upwards of about 600 counts on a
> > background of 6000. Cleaning if anything has made this worse. (Cf about 3000
> > for the XL/A which does need
> > cleaning.)
> >
> > 3. A wavelength intensity profile looks normal though the maxima are not
> > great (16000 at 230 nm after cleaning, 11000 before).
> >
> > 4. What does the "request data incident" option in the GUI do? For the XL/A
> > this produces very flat lines with some spikes upward or downward, of
> > constant size; for the XL/A reasonably flat lines with random noise
> > superimposed (4000 +/-100). As I don't know what this is measuring, I'm not
> > sure what to expect.
> >
> > I'll try and post some graphs later.
> >
> > Many thanks,
> >
> > Andrew
> >
> >
>
> --
> Department of Biochemistry and Molecular Biology
> University of New Hampshire
> Durham, NH 03824-3544
> Phone: 603-862-2459
> FAX:   603-862-0031
> E-mail: Tom.Laue at unh.edu
> www.bitc.unh.edu
> www.camis.unh.edu
>
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 13 Jun 2007 18:45:38 +0100
> From: "Leech, AP" <apl3 at york.ac.uk>
> Subject: Re: [RASMB] XL/I absorbance problem
> To: rasmb at server1.bbri.org
> Message-ID: <46702D42.7090604 at york.ac.uk>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello again,
>
> Many thanks to Virgil, John, Borries and Tom for such timely and
> informative replies.
>
> I have now attached two plots showing actual data: the first
> (IncidentIntensityComp.gif) shows the data from 00001.RN2 scan
> 280 nm velocity scan on an empty hole. The top trace is the
> incident and the bottom the intensity as seen through the empty
> hole. The second graph shows two incident (.RNx) plots. These
> were both at 280 nm; the top one from an old scan long before
> this problem arose and the bottom one from a recent scan. Note
> the different scales.
>
> I agree with Virgil that we may have a slit problem, but I think
> this is causing the broad dip in the intensity profile. The marked
> difference between the old and new incident light profiles makes
> me think the problem is with the lamp and/or reference detector.
>
> I've tried re-aligning the lamp (no difference) but I hadn't
> thought of swapping between machines. I'm not sure we can do this
> as the lamp housing is rather different on the old XL/A and I don't
> think it would fit.
>
> What I did wonder is whether it is permissible/advisable to try
> the XL/A monochromator in the XL/I? It would appear to fit, though
> it has less pins. But I don't know if this would do something
> nasty e.g. short out the laser power supply at the socket!
>
> Any further comments are appreciated - I will let you know the
> resolution (if any) of the problem.
>
> Best to all,
>
> Andrew
>
> --
> Dr Andrew Leech                   *  Laboratory Manager
> Technology Facility               *  Molecular Interactions Laboratory
> Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
> University of York                *  Fax   : +44 (0)1904 328804
> PO Box 373,  York  YO10 5YW       *  Email : apl3 at york.ac.uk
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> ------------------------------
>
> Message: 3
> Date: Wed, 13 Jun 2007 13:55:51 -0500
> From: Virgil Schirf <schirfv at biochem.uthscsa.edu>
> Subject: Re: [RASMB] XL/I absorbance problem
> To: Virgil Schirf <schirfv at biochem.uthscsa.edu>
> Cc: rasmb at server1.bbri.org
> Message-ID: <46703DB7.8000604 at biochem.uthscsa.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Andrew,
>
> There shouldn't be a problem switching out the monochromator to
> troubleshoot. I've done it before without incident. You will have to do
> a wavelength calibration if you do switch out the monochromators and
> that sometimes can take several scans to get the 230/567 nm peaks in the
> correct positions.
>
> Attached is a picture of, what we termed, the dreaded comb similar to
> what you are seeing. This particular problem was caused by bad circuitry
> atop the heat sink after the instrument overheated causing major damage;
> however, other factors mentioned earlier can cause the same effect -
> most notably a bad or dirty slit drive.
>
> Good luck,
>
> Virgil
>
>
>
> --
>
> Virgil Schirf
> Manager, CAUMA <http://cauma.uthscsa.edu> & CLS <http://cls.uthscsa.edu>
> Department of Biochemistry, Rm 4.523U
> The University of Texas Health Science Center
> 7703 Floyd Curl Dr.
> San Antonio TX 78229-3900
> Voice mail: 210 567-6590
> Fax: 210 567-6595
> Email: schirfv at biochem.uthscsa.edu <mailto:schirfv at biochem.uthscsa.edu>
>
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> End of RASMB Digest, Vol 21, Issue 4
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