From: Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  To  : jphilo@mailway.com
  Date: Sun, 21 Nov 1999 17:48:00 -0600 (CST)

Re: Help... what's going on?

> What you are seeing is a consequence of the fact that the monochromator is
> not coming back to the same wavelength (there is an uncertainty of +/- 1 nm
> or so every time it moves). The different extinction coefficients cause the
> amplitudes of the scans to jump up and down, particularly when you are on a
> steep part of the absorbance spectrum. It only *looks* like the scans are
> being taken at the wrong times.
> 
> Basically, for this reason you should never do velocity runs at multiple
> wavelengths. If you need multiple wavelengths you simply have to do separate
> runs. In principle, if all scans for any run are at the same wavelength then
> the monochromator doesn't move and this problem goes away (but see my recent
> message to RASMB and the responses to that).

Also, it seems to me that the noise level is rather substantial - maybe
move to 230 nm, where the lamp has maximum emission - that should improve
the  noise level somewhat.

> Because of this problem you really won't be able to analyze your data by any
> method that uses global analysis of multiple scans, since really no two
> scans are at the same wavelength. You can, however, analyze a *single* scan

Actually, one global method that can deal with shifting plateaus just
fine is van Holde - Weischet, at least as implemented in UltraScan. The
deltaC/deltaT implementation in UltraScan is also immune to jumping
plateaus resulting from shifting wavelengths. This of course is only
true if this shifting is really due to improper monochromator resetting.
Nonetheless, if you were running at a single wavelength, there is probably
something else going on and you should try to get this problem fixed by
Beckman service.

-Borries