From: Borries Demeler/Biophysics <demeler@selway.umt.edu>
  To  : rasmb@bbri.eri.harvard.edu
  Date: Thu, 5 May 1994 12:06:38 -0600 (MDT)

Re: XL-A absorption optics

Hi everybody:

One of the most important, easily overlooked problems in measuring 
absorbances with the XL-A is the fact that the 
light is NOT monochromatic across the cell (only a laser gives you 
monochromatic light). What that means is that the wavelength actually
changes along the radial dimension in the cell. If you measure your
sample at a wavelength corresponding to the shoulder absorbance of your
sample and not the peak absorbance, you will actually get a different
absorbance at the top of the cell vs. the bottom of the cell. This
effect is rather striking if the shoulder is very steep. A difference
of only 0.5 nm can result in a noticeable change of absorbance  across
the length of the cell.
 
 This problem is most pronounced at low wavelength, since the pi to pi*
 absorption band of the peptide bond can be quite sharp and have a steep
 shoulder.

My recommendation is to make a wavelength scan of the sample and find the exact
peak of the absorption. If the sample is too concentrated for that 
peak wavelength, dilute it.

You can find out the effect for yourself by finding the point of the steepest
slope in the absorption band and measure a scan across the entire cell 
by running it at a slow speed so it doesn't sediment. The larger the initial
concentration of your sample, the more pronounced the effect.
For velocity runs it is usually not as important to watch out for that as
for equilibrium scans.

Good luck, and I hope this info can help you solve your problem.

Regards, -Borries