[RASMB] XL/I Absorbance Problem (follow-up)

[Borries Demeler]

Dear Colleagues, 

> we've used the intensity data acquisition for a long time now after we 
> discovered that the noise structure of log(raw intensity) data is 
> similar to that of interference optical data and can be treated with the 
> same algebraic noise elimination technique.  However, this also 
> introduces the same disadvantages that we have in interference optical 
> detection from the lack of an optical reference baseline.  Therefore, we 
> only use it for doubling the rotor capacity if necessary.  One should 

As I indicated in my previous mail, it is sufficient if at least
one of the channels in one of the cells is used as a reference, this
provides a reference baseline for all cells simultaneously, as long as
no non-sedimenting material is present, but in that case you probably
want to know about it anyway. Any remaining baseline offsets are simply
calculated during the 2-dimensional spectrum analysis with TI noise removal.

> mention one particular downside of the use of different samples in the 
> two sectors, which is that one can't have a high absorbance in the 
> reference sector at 6.5 cm, because that's a reference point for the 
> photomultiplier voltage adjustment.

We have not seen any problems with this as long as we keep the absorbance
values below 0.6-0.7 OD. Above this absorbance, you can get different gain
settings in the early scans, so keep the OD somewhat lower and you will be OK.

> So far I did not notice a marked improvement of signal/noise ratio when 
> using pseudo-absorbance instead of absorbance data.  Marc Lewis has 

Roughly a factor of square-root of 2 is all you can hope for, but that
is better than nothing at all.

> If you use TI noise subtraction from the regular absorbance data, you 
> can get excellent signal/noise ratio, as well.  I'm puzzled by the 0.3 
> OD example, which is actually well in the range of OD values that can be 

This observation is independent of absolute OD. I merely showed 0.3 as
an example for the quality of data that can be observed. The instrument
I tried normally produces an RMSD of 0.005 - 0.004 in absorbance mode,
while typically dropping to 0.0025-0.003 when using pseudo absorbance.
You can find more information about the effect of noise on the data
analysis in the latest online issue of Colloids and Polymer Science:
http://www.springerlink.com/content/43x3208363v45032/?p=0e109a93d39948f2ba2351ad504c6e8e&pi=2
(Demeler B. and E. Brookes, Monte Carlo analysis of sedimentation
experiments).

Regards, -Borries