[RASMB] Lamp Diagnostics

[Tom Laue]

Hi-
There is a spec on the minimum intensity and on the maximum allowed 
variation in intensity along the radial axis. My recollection (which 
very well may be wrong) is that at 230 nm with a blank rotor hole, the 
minimum intensity is ~10,000, and the variation is < 20%. Again, I may 
have the numbers wrong, but there is a spec. Contact Beckman to get the 
correct values.
John is right about the signal magnitude being relatively unrelated to 
the signal to noise until the signal gets quite small (<1000 counts). 
You do want the lamp positioned to minimize the variation in intensity 
(usually sloped) along a cell, so after you have tweaked the lamp 
position to get a strong signal, you need to do an intensity scan to 
make sure the signal does not vary too much.
Best wishes,
Tom
John Philo 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: mitrana at mail.utexas.edu [mailto:mitrana at mail.utexas.edu] 
> Sent: Thursday, April 03, 2008 6:00 PM
> To: jphilo at mailway.com
> 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 <jphilo at mailway.com>:
>
>   
>> 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: rasmb-bounces at rasmb.bbri.org 
>> [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of 
>> mitrana at mail.utexas.edu
>> 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
>>     
> spot'
>   
>> 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 _______________________________________________
>> RASMB mailing list
>> RASMB at rasmb.bbri.org
>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>>
>>     
>
>
> --
> Mitra S. Rana
> Graduate Student
> Institute for Cellular and Molecular Biology 2500 Speedway, UT-Austin
> Austin, TX-78712
>
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb
>
>   

-- 
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