[RASMB] XL/I Absorbance Problem (follow-up)

John Philo jphilo at mailway.com
Thu Aug 23 13:56:37 PDT 2007


Andrew,

Yes, when you are in continuous scan mode (normal velocity mode), at the
beginning of each scan the slit moves to 6.5 cm and I believe the intensity
is read for both reference and sample. The photomultiplier voltage is then
adjusted (and if necessary the programmable gain amplifier too) to try to
optimize accuracy/linearity assuming the entire sample has intensities
similar to those at 6.5 cm. I've never heard the details of the adjustment
algorithm but I assume it is derived from whatever Beckman uses for their DU
spectrophotometers that have photomultipliers. 

So yes this is exactly what creates some issues and artifacts when something
is sedimenting in the reference channel, and is one reason why if you load
the cell backwards you can't completely recover by simply multiplying the
data by -1.

When you are in step scan mode the photomultiplier and gain adjustments are
done at each new radius, which really slows things down.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Leech, AP
Sent: Thursday, August 23, 2007 9:53 AM
To: rasmb at server1.bbri.org
Subject: Re: [RASMB] XL/I Absorbance Problem (follow-up)


Hello John,

I'm not quite clear on this - does the XL before each scan move the slit to
6.5 cm, adjust the PMT voltage, then do the required radial scan? Presumably
this is only done on the reference sector?

I guess this could have a significant effect on attempts to do
"differential"  experiments, where there is something actually sedimenting
in the reference sector (not to mention cells which have sample and
reference in the wrong sectors!)

Kindest regards,

Andrew

John Philo wrote:
> I think a couple of points regarding the use of pseudo-absorbance were
> not sufficiently brought out by either Bo Demeler or Peter Schuck:
>  
> (1) With pseudo-absorbance there are vertical shifts of the scans from
> one scan to another, somewhat similar to the TIN or 'jitter' in 
> interference data. Treating this systematic noise as a true constant 
> vertical displacement certainly helps, but if you look closely at the 
> structure of these shifts they do not appear to be truly constant across 
> the cell. One source of this effect is clearly the adjustment of the 
> photomultiplier voltage based on the light levels at 6.5 cm, as Peter 
> mentioned, and thus the shifts tend to be largest as the boundary moves 
> past 6.5 cm.

[...]

> With respect to (1) it would be useful for Beckman to provide an
> option that suppresses the photomultiplier voltage adjustment after the 
> first scan of each cell, and that also drops the assumption that the 
> voltage should be initially optimized for measuring sample - reference.
>  
> Best regards,
>  
> John

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> RASMB at rasmb.bbri.org http://rasmb.bbri.org/mailman/listinfo/rasmb

-- 
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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