From: H. Olin Spivey <ospivey@bmb-fs1.biochem.okstate.edu>
  To  : RASMB@BBRI.ORG
  Date: Thu, 18 May 2000 16:15:29 -0500

Summary and Resolution of Problem

<fontfamily><param>Times</param><bigger><bigger>Dear RASMBers,


   Summary of suggestions and resolution of problem with variable
intensities over the radial range.  Problem reported on April 4, 2000.


   Thank you for the suggestions concerning our problem reported on
April 4, 2000.  Our service rep., Hugh Nichols, came and achieved a
quick fix this time.  A new photomultiplier (PM) completely removed the
large dip in intensity near the middle of the radial range.  Hugh was
very surprised to find this fix so quickly since he was sure he had
tried a new PM earlier along with many other items.  Hugh thought that
he could see evidence (deposits on the glass surface) that the
photocathode had an abnormally high emission near its outer
circumference.  Thus our theory is that the light area illuminating the
photocathode moves significantly during a radial scan.  If the
photocathode has considerably variable sensitivity over its surface,
this will generate the variable intensities we were observing.  We
would be happy to know if you have a better theory.  This idea is
consistent with John Gunther's experience with a similar problem.  They
 "de-tuned" (de-focused?) the lamp to recover a reasonably flat
baseline at the expense of lower intensities.


   Thus our intensities before the repair were abnormally high at the
two ends of the radial scan.  The new PM gives a lower intensity, but
adequate.  John Philo is correct, we didn't need the extra energy.  We
gathered data from 9 different concentrations of BSA over the
absorbance range of 0 - 2.0 at 3,000 rpm.  We pooled the standard
deviations, stdev. (16 repetitions at each radius), and plotted these
stdev.  vs. the A280.  The best-fit line shows only a slightly larger
noise/signal ratio after the repair - perhaps within the data error. 
(Actually, our stdev vs. A280 are nonlinear beyond A280 approx. 1.75,
having a larger slope beyond this point).  However, we expect the
frequency of problems with non-overlapping reference and sample
intensities to improve saving us considerable time.  We acknowledge
Allen Minton for his published suggestion to fit to stdev. vs.
Absorbances to obtain a better (global) estimate of these standard
deviations and their dependence on Absorbance.  This gives more
reliable weighting factors for data analyses than using the reported
standard deviations at each radii (based on much fewer points).  This
procedure also gives a nice report of the signal/noise ratio for each
experiment, which provides a useful monitor of the instrument over time
and from one lab to another.


   John Philo mentioned that cleaning the optics doesn't take them
long.  They use an eraser to scrub the lamp.  We were taught to use a
toothbrush and toothpaste for this cleaning, but our rep. agreed with
the eraser method as well.  We will try the erasure next time.  I'm
sure John is more skilled than we at cleaning, but we found it took
several attempts and often hours to get our instrument cleaned (lamp
and slits) to where the sample and reference intensities overlapped
again.


   Thank you for your suggestions,

   Olin

</bigger></bigger></fontfamily>
H. Olin Spivey                       Phone: (405) 744-6192

Dept. Biochem. & Molec. Biology      Fax:   (405) 744-7799

246 NRC                              Email:
OSpivey@Biochem.Okstate.Edu

Oklahoma State University

Stillwater, OK 74078-3035