From: Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  To  : Gresham Weatherly <gresham@unc.edu>
  Date: Mon, 6 Dec 1999 19:31:04 -0600 (CST)

Re: XLA monochromator

> 
> I am trying to collect data at two different wavelengths during an
> equilibrium sedimentation experiment. The monochromator does not always
> collect the data at the correct wavelength, sometimes the header file shows
> that it has collected + 1 nm from the desired wavelength.  This is okay
> because I can discard these files that have been collected at the wrong
> wavelength.
> However, sometimes subsequent scans are offset even though the wavelength
> in the header file is the same.  The attached file shows three scans that
> have supposedly been collected at one wavelength, but appear to be
> collected at different wavelengths. The data collected at the second
> wavelength overlay, so I believe the system is at equilibrium.  
> My question is when the monochromator collects five scans and averages them
> does it collect all five scans at the same wavelength, and is there
> anything I can do to make the XLA collect data at the right wavelengths?
> 
> Thank you for any help you can give me,
> 
> Gresham Weatherly

Gresham,

This is an old problem with all XL-A's I believe. There is no guarantee
that the monochromator will reset to exactly the same wavelength when
you switch the wavelength from scan to scan (and often it will not
do so). This is particularly problematic if you scan on an absorbance peak
shoulder, as would be the case with 230 nm, measuring proteins. There, a
difference of just a single nanometer can have a large effect in change
of absorbance, thus making this problem more pronounced.

The cure, as Olin already mentioned, is to do all scans at the first 
wavelength, and then perform all subsequent scans at the second wavelength.
This way you only switch the wavelength once, and not back and forth.
You can do that with equilibrium data, since the data doesn't change once
you are at equilibrium, so you have lots of time to do all the scanning
you want. However, for velocity runs it is advisable to run a complete
run at one wavelength and then shake up the cells and rerun the same
sample at a different wavelength, with a different file name for the data
collection.

Hope that helps, -Borries