[RASMB] What is wrong with my XLA?

[Borries Demeler]

> 
> Steve,
> 
> I am with John on the meniscus issue - can you plot the first, middle
> and last scan in three different colors, it would be nice to see if
> there is a pattern to the meniscus drift.
> 
> Secondly, the scans in the velocity scan seem to jump around and
> apparently seem to have different scalings. Assuming you collected scans
> from both reference and sample channel, are you sure that the absorbance
> of the sample plus baseline absorbance in the reference cell did not
> exceed 0.5 OD? It almost looks like it rescaled on you. If not that,
> perhaps it was acquired at different wavelengths. The scans you show do
> not show a regular velocity pattern. Something is screwed up either with
> your expt.  design or your instrument, there is not enough information to
> determine which.  You should run some diagnostics on your machine and call
> Beckman service for assistance - it is not clear what is going on here,
> but too high a concentration in the reference channel will cause a reset
> of the gain settings for the PM tube, and you want to avoid this. Even if
> your sample OD is 0.3 or less, if you have baseline absorbance from buffer
> or reductants, this can seriously mess you up. I just assume you are not
> running DTT in your sample, correct?
> 
> -b.

OK, a followup discussion with Steve revealed that indeed PMT resetting
during the run was the problem. If you make intensity measurements and
load sample into the reference channels you must make sure the OD does
not exceed 0.3-0.4 in the reference channel, otherwise the PMT WILL rescale
the data, and not just in the reference channel. If you can't measure 
the OD of your sample before running in the reference in intensity mode,
and confirm it to be below 0.4 OD, just load water in the reference
to avoid the rescaling issue Steve is encountering. OD levels in the sample
channel should stay within the dynamic range of the detector, but can be
higher. I recommend a highest OD of 0.9 - 1 for the sample channels, or
a bit higher for measurements at high intensity wavelengths, say 230 nm.

-b.