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  From: JOHN PHILO <>
  To  : rasmb <>
  Date: 11 May 1994 08:20:33 U

sloping plateaus III

        Reply to:   sloping plateaus III
Geoff Howlett asked:

>How big are these sloping baselines and are they machine or sample 
>dependent?  i.e. is it a 10% slope from meniscus to base and is it only 
>apparent with a full cell? Is there a sample (say bromphenol blue) >everyone
has access to which would allow sloping baselines to be >checked on different

First, to get our terminology straight, what Jack Correia and I are seeing is
a sloping plateau, not a sloping baseline.  That is, the slope is only
apparent when the absorbance is non-zero.  The magnitude can be of the order
of a 10% difference in absorbance from meniscus to cell bottom when the cell
is full.  It is only obvious with a full velocity cell, because there is such
a long region where the absorbance should be constant, but there is no reason
to believe that the same effect does not exist for 6-channel cells or
partially-filled double sector cells.

This problem is (at least) machine, wavelength, time, and lamp dependent.  To
date, Jack and I have not heard from anyone else who obviously has this
problem, but it may be present to greater or lesser extents in all XL-As. 
The extent of slope seems to vary somewhat from run to run (but I am not
certain of this). The percentage slope seems always to be worse at
wavelengths where there is less absolute light intensity.  It therefore also
seems to grow worse as the lamps get dirty with use.

In principle, yes, bromphenol blue or any other small molecule absorber such
as the Beckman OD calibration solutions can be used to look for this problem.
 However, as Bo Demeler correctly pointed out, you must be careful to
distinguish this problem from the one due to wavelength shifts across the
cell, so you want to be sure you are working at an absorption peak or with
something with a very broad absorption spectrum.  The wavelength shift
problem can produce sloping plateaus, but the magnitude is probably only
1/10th that that Jack and I see.

Lastly, to update our experience, yesterday my service engineer put a new
lamp in my machine.  Preliminary testing seems to indicate that this has
reduced the problem, but I do not believe it is entirely gone.  However, the
new lamp also increased the light intensity, which always seems to decrease
the slope, so this does not necessarily imply that the lamp is the origin of
the problem.  Changing the lamp has clarified that the slope is not directly
related to the pattern of lamp intensity across the cell:  my old lamp always
gave a nearly linear drop in intensity from inner to outer by about 30%,
whereas the new one rises quite non-linearly by nearly a factor of 2 from
inner to outer.  This change in pattern has clearly NOT reversed the sign of
the slope.

I will update everyone when it becomes clearer to what extent this lamp
change has eliminated the problem.

Thanks to all who offered help and suggestions.

John Philo
Protein Chemistry,  Amgen

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