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Hi-<br>
The variation in lamp intensity with wavelength is less at 400 nm than
at 230 nm. <br>
If you are interested in the uniformity of the light intensity across
the cell due to the alignment of the lamp, spatial variation in PMT
sensitivity, cleanliness of the various optical components, it would be
better to use 400 nm. If you want to include variation due to the
coincidence of the radial axis with the wavelength dispersion of the
light coming out monochromator (i.e. the light along one side of the
rectangle of light is a slightly different color than the light on the
opposite edge), then use the sharper peak at 230 nm. Using the holmium
oxide filter test is an even better way to get at the latter effects,
but my experience is that the wavelength dispersion effects are small
compared to the other effects.<br>
Tom<br>
<br>
<a class="moz-txt-link-abbreviated" href="mailto:mitrana@mail.utexas.edu">mitrana@mail.utexas.edu</a> wrote:
<blockquote
cite="mid:1207425875.47f7db53bf051@webmailapp4.cc.utexas.edu"
type="cite">
<pre wrap="">According to the manual the wavelength to set for the intensity variation is 400
nm. Is there any particular reason to do this at 400 nm instead of 230 nm.
-Mitra
Quoting John Gunther <a class="moz-txt-link-rfc2396E" href="mailto:jrg@mywdo.com"><jrg@mywdo.com></a>:
</pre>
<blockquote type="cite">
<pre wrap="">Here is the lamp intensity check procedure from a 10-2001 manual. I'd follow
this to step 8 then just look at it. The origin part is a waste of time in my
opinion.
John Gunhter
*********** REPLY SEPARATOR ***********
On 4/4/2008 at 10:57 AM John Correia wrote:
It is worth noting that Beckman has a service manual for the XLA XLI that
discusses all of these features and tests and specs. The spec for linearity
is 20% when measured at 280 nm on a windowless cell (or an empty hole) at
3000 rpm using OD values 1 cm apart. The old manual specified 6.0 and 7.0
cm. With the old lamp rotation of the lamp strongly influenced the intensity
across the cell; the newer lamp holder is now symmetric and do not vary as
much with rotation, in my experience. The old part # for this manual is
679045 but my copy is dated 1/92. The newest version I am aware of is called
Proteome Lab XLA & XLI Service Manual dated (10/3). Sorry I do not have a
part # - the relevant chapter is section 3. It describes wavelength
calibration and accuracy checks, Intensity checks, cleaning the optics,
radial position calibration, etc, etc. Obviously depends upon the software
you have as well. In my opinion it is well worth buying or getting a copy
for your service guy - in the long run it saves both of you time. Why this
isn't standard operation procedure with Beckman is a very good question.
-------------------------------------------------------------------
Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-1522
fax (601) 984-1501
email address: <a class="moz-txt-link-abbreviated" href="mailto:jcorreia@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</a>
homepage location: <a class="moz-txt-link-freetext" href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</a>
dept homepage location: <a class="moz-txt-link-freetext" href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</a>
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</pre>
<blockquote type="cite">
<blockquote type="cite">
<blockquote type="cite">
<pre wrap="">On 4/4/2008 at 10:18 AM, in message
</pre>
</blockquote>
</blockquote>
</blockquote>
<pre wrap=""><C7B1B01A69FC4164B0213B9168C3CBAA@79B7XD1>, "John Philo" <a class="moz-txt-link-rfc2396E" href="mailto:jphilo@mailway.com"><jphilo@mailway.com></a>
wrote:
Mitra,
To my knowledge there is no specification for intensity variation across the
cell, but in my experience few instruments would vary as little as 10%.
Probably 20-30% is more typical. It is a common mistake to think that the
absolute intensities have a strong effect on the signal/noise. It is
doubtful that you would notice the effect of even a 50% drop in intensity
(assuming you are starting from a normal level). Further, positioning the
lamp to give the maximum possible intensity will not necessarily give the
best overall performance---quite often the maximum intensity position also
gives high stray light and compromises the linearity.
John
-----Original Message-----
From: <a class="moz-txt-link-abbreviated" href="mailto:mitrana@mail.utexas.edu">mitrana@mail.utexas.edu</a> [<a class="moz-txt-link-freetext" href="mailto:mitrana@mail.utexas.edu">mailto:mitrana@mail.utexas.edu</a>]
Sent: Thursday, April 03, 2008 6:00 PM
To: <a class="moz-txt-link-abbreviated" href="mailto:jphilo@mailway.com">jphilo@mailway.com</a>
Cc: 'RASMB'
Subject: RE: [RASMB] Lamp Diagnostics
John
I feel better already.
What I meant in 1. is that I take a wavelength scan at 5.9, 6.5, and 7.1 cm
and compare the 230 nm peaks. I guess a radial scan is definitely a better
way to check intensity variability across a cell. How much variability is
considered acceptable - somehow 10% seems stuck in my head.
Mitra
Quoting John Philo <a class="moz-txt-link-rfc2396E" href="mailto:jphilo@mailway.com"><jphilo@mailway.com></a>:
</pre>
<blockquote type="cite">
<pre wrap="">Mitra,
Points 2 and 3 are normal. Every time the monochromator moves there is
an uncertainty of +/- 1-2 nm (I believe the official specification is
actually
+/- 4 nm).
Also for wavelength scans the monochromator doesn't actually take data
at the exact wavelength interval you ask for. So sometimes it will
essentially step over the sharp peaks (if you double-click on the line
graph and ask it to mark the data points with symbols you can more
easily see there are no points where the peak should be).
I'm not sure I understand your point 1, but I think the intensity
variability may just be a consequence of the wavelength variability
discussed above. Normally one would try to adjust the lamp alignment
using radial scans, not wavelength scans, and typically the wavelength
intensity scans to check the strength of the 230 nm peak or wavelength
calibration are run at 6.5 cm.
John
-----Original Message-----
From: <a class="moz-txt-link-abbreviated" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a>
[<a class="moz-txt-link-freetext" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] On Behalf Of
<a class="moz-txt-link-abbreviated" href="mailto:mitrana@mail.utexas.edu">mitrana@mail.utexas.edu</a>
Sent: Thursday, April 03, 2008 2:58 PM
To: 'RASMB'
Subject: [RASMB] Lamp Diagnostics
Hello Spinners
Our AUC started giving some problems recently. Did a wavelength scan
and here's a summary of the results -
1. Lamp intensity varies at the 5.9 cm position although at the 'sweet
</pre>
</blockquote>
<pre wrap="">spot'
</pre>
<blockquote type="cite">
<pre wrap="">obtained from the lamp alignment this variation is about 9-10%.
2. Sometimes it seems the peaks are cut-off and I get a plateau
instead of a peak with of course a lower intensity count.
3. The peak oscillates 1-2 nm (227-230 nm and 526-528 nm) on
successive scans at a single radial position.
It seems like a monochromator problem to me but I'd like to hear some
comments from experts here. I am particularly worried about the peaks
getting cut-off.
Our facility did not renew the service contract this year (I know,
extremely bad idea but too many size exclusion column huggers around)
and I'd rather know the problem instead of being told by the Beckman
Service person that it's within specs while charging $290.00 per hour.
Mitra
--
Mitra S. Rana
Graduate Student
Institute for Cellular and Molecular Biology 2500 Speedway, UT-Austin
Austin, TX-78712 _______________________________________________
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</pre>
</blockquote>
<pre wrap="">
--
Mitra S. Rana
Graduate Student
Institute for Cellular and Molecular Biology 2500 Speedway, UT-Austin
Austin, TX-78712
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