From: Thomas M Laue <tml@christa.unh.edu>
  To  : RASMB group <rasmb@bbri.eri.harvard.edu>
  Date: Fri, 29 Apr 1994 13:57:54 -0400 (EDT)

Gradients and "nonideality" with absorbance

Hi-
I just received the replies to Joachim's question about the linearity of 
the XLA optics. My experience matches that of John Philo's exactly- the 
most common cause is too little light. There are a couple of other tricks 
that I have found useful-
	1) if the gradient is steep, the refractive bending of the light 
distorts the cell image. This is particularly severe at shorter 
wavelengths where the refractive index is climbing sharply. The 
consequence (if the light source is properly aligned, as John pointed 
out)is what appears as nonideality. This can be tested by determining the 
molecular weight at different wavelengths and different rotor speeds, but 
with the same concentration of material. If the molecular weight is 
dependent on the concentration gradient, but not on the rotor speed, then 
this is one possible cause. There was an example of this with a peptide- 
the only recourse is to work at lower gradients.
	2- Another quick and dirty trick is to measure the ratio of the 
absorbances at two different wavelengths and over a range of absorbances. 
For a pure substance, this will be constant and equal to the ratio of the 
extinction coefficients (if you know them). If there is some problem, 
then there will be a systematic shift in this ratio as the concentration 
increases. Some common problems are: a) Stray light- we've found that the 
absorbance system on one XLA loses linearity above 1.3 OD at 230 nm (but 
is quite linear to 2.1 OD at 280), b)cruddy sample (not as pure as 
advertised, including sedimentable buffer components), c) low light 
resulting in poor linearity (use the Xenon peaks to get the best signal 
to noise) and d) poor light source alignment, causing problems similar to 
those described above.

Finally, I have a new e-mail address:

tml@christa.unh.edu

Best to all-
Tom Laue