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<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2>Arthur's suggestion of running the same sample in multiple labs may
be a good one, but I'm not so certain the results will clearly indicate
differences in temperature calibration, at least not unless it is all
thought through carefully in advance.</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff size=2>The
problem is to distinguish real differences in temperature from other effects
that can change the values of the sedimentation coefficient:</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff size=2>(1)
systematic errors due to differences in the software used to evaluate the
data;</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff size=2>(2)
systematic errors due to how the meniscus position is defined (this is a bigger
problem than most people realize);</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff size=2>(3)
potential systematic differences between absorbance and interference data (has
anyone really looked at this carefully?);</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff size=2>The
obvious way to deal with the first two would be to have one person do all the
data analysis, but that could be a burden (no, I'm NOT
volunteering!).</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2>Lastly, there is simply the problem of precision of the values from any
one lab. How many labs have really evaluated their reproducibility,
for example for running the same sample in triplicate in the same run, and from
run-to-run? If the run-to-run precision is say 1%, then that translates into a
0.4 degree temperature uncertainty. </FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2>Overall then you really need VERY high quality data to evaluate
the temperature calibration issue. A casual effort is potentially a waste of
everyone's time.</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2>Lastly, I might suggest that this may be something that should be handled
under the auspices of the Molecular Interactions Research Group of the
Association of Biotechnology Resource Facilities (ABRF), who have already run
(and published) tests involving both BIAcore and AUC studies of the same protein
in multiple labs. Preston Hensley and Ed Eisenstein are, I believe,
currently part of that group so perhaps one of them could comment on
this.</FONT></SPAN></DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=931551815-16122004><FONT face=Arial color=#0000ff
size=2>John</FONT></SPAN></DIV>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV></DIV>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left><FONT
face=Tahoma size=2>-----Original Message-----<BR><B>From:</B>
rasmb-admin@server1.bbri.org [mailto:rasmb-admin@server1.bbri.org] <B>On
Behalf Of </B>Arthur Rowe<BR><B>Sent:</B> Monday, December 13, 2004 7:47
AM<BR><B>To:</B> John Correia; mchien@beckman.com;
rasmb@server1.bbri.org<BR><B>Subject:</B> Re: [RASMB] Re: XL-A/I temp
control<BR><BR></FONT></DIV>Hi Everyone<BR><BR>I entirely agree that the
temperature on machines needs to be calibrated. Equally, I agree with Jack's
statement that "most users . . . never calibrate the temperature". As to
whether it is actually easy to do same using the Stafford & Liu method,
opinions might differ. We ourselves find that the integration required to get
the area under the curve is easy enough to estimate with data from the spec,
but less easy (baseline definition, noisier values) with data from the XL-A.
<BR><BR>The suggestion that a quick set of standard runs be undertaken on
multiple machines would serve to clarify as to whether it all really matters,
at the level of doing careful calibration - if one is talking at the
<I>precision </I>(not accuracy) level of the instrument. After all, with a
(presumably standardised) production line, one might have supposed that any
errors at a given temperature might have been pretty much the same from one
instrument to another. The sketchy data which we have to hand suggests that
that is not the case.<BR><BR>Measuring an s value of a given solute via an
agreed procedure is about as objective a procedure as one can define.
Variations are easily transformed into temperature uncertainties. If that is
what we are looking for, then this is the easiest way to find it. The numbers
of instruments volunteered to date (10) looks enough to give a pretty good
idea of what the variation level actually is - but any more would bolster the
stats nicely . . . .<BR><BR>Regards to all<BR><BR>Arthur<BR><BR>--
<BR>*************************<BR>Arthur Rowe<BR>Lab at Sutton
Bonington<BR>tel: +44 115 951 6156<BR>fax: +44 115 951
6157<BR>*************************<BR>
<BLOCKQUOTE><BR><B>From: </B>"John Correia"
<jcorreia@biochem.umsmed.edu><BR><B>Date: </B>Fri, 10 Dec 2004
11:16:43 -0600<BR><B>To: </B><mchien@beckman.com>,
<arthur.rowe@nottingham.ac.uk>,
<rasmb@server1.bbri.org><BR><B>Subject: </B>Re: [RASMB] Re: XL-A/I
temp control<BR><BR></BLOCKQUOTE><BR>
<BLOCKQUOTE><FONT size=2>I suspect other will give technical responses about
the ability of these temperature controllers to function at the +/- 0.1 C
level. My take is slightly different. <BR></FONT><BR><FONT
size=2>What matters is not the set point but knowing the actual
temperature.<BR></FONT><BR><FONT size=2>To my knowledge most users never use
the Stafford & LUS method to calibrate the temperature on their machine.
(can we vote on the RASMB in some way?) It is no more tedious than
waiting three hours before starting a run. I have checked the
calibration on my machine twice, once in '93 when I got it, and again in
1999 when a result caused me to question the temperature accuracy. A
series of measurements up to 40 C and back down to 4 C (with parallel
measurements in a spec) takes about two days. It does not take three
hours to go from 20 to 25 C, as verified by the stability of the area under
the CoCl2/ethanol curve. At a setting of 4 C I get 3.6, at 20 I get
19.7, at 40 I get 39.8. The values drifted by 0.1 C in 7 years.
So if we mean by accuracy +/- 0.5 C of setting, my machine is within
spec. When I fit data I use the actual temperature, not the set point.
& I trust the setting on the screen at low vacuum and hit start when it
reports the set temperature.<BR></FONT><BR><FONT size=2>Comparisons between
uncalibrated machines honestly make no sense to me. Calibrate & be
done with it. (Arthur, if you do that in Nottingham & still get
different values of S between machines I would be concerned, & amazed.)
<BR></FONT><BR><FONT size=2>I also use the calibrated temperature
values when I measure density in an Anton Paar DMA 5000. It has a
peltier cell good between 0 - 80 C, so I dial in 19.7 instead of 20 C and
measure away. I suspect the error from a calculated density (viscosity
& Vbar) is larger than (albeit coupled to) an assumed temperature.
<BR></FONT><BR><FONT size=2>At this point the inquiring "student"
should assume errors in various parameters and propagate them into S or MW
by the appropriate equations - my favorite book for teaching this is
Bevington, "Data Reduction and Error Analysis for the Physical
Sciences".<BR></FONT><BR><FONT size=2>PS - why Beckman has never joined up
with Anton Paar to bundle Density Meters into XLA/XLI quotes amazes
me.<BR></FONT><BR><FONT size=2>PPSS - Walter claims to keep the original
solution around, in a cell, from the CoCl2 calibration work, and years later
it still gives the same results. Quick checking may not be as
difficult as one might
think.<BR></FONT><BR><BR><BR>-------------------------------------------------------------------<BR>Dr.
John J. "Jack" Correia<BR>Department of Biochemistry<BR>University of
Mississippi Medical Center<BR>2500 North State Street<BR>Jackson, MS
39216<BR>(601) 984-1522
<BR>fax
(601) 984-1501
<BR>email
address: jcorreia@biochem.umsmed.edu <BR>homepage
location: http://biochemistry.umc.edu/correia.html<BR>dept homepage
location:
http://biochemistry.umc.edu/<BR>-------------------------------------------------------------------<BR><BR><BR><BR><BR>>>>
Arthur Rowe <arthur.rowe@nottingham.ac.uk> 12/10/04 08:17AM
>>><BR>Hi Everyone <FONT color=#000080>{this is a second
(</FONT><FONT color=#ff00ff>now 3rd!</FONT><FONT color=#000080>) try at
getting this mail out - first attempt got lost in cyber-space, it
seems}<BR></FONT><BR>Mei-Ling Chien gives us a very useful review of the
nature of the temperature measurement and control system in the XL-I/A
instrument. However, I do not think that this fully addresses the problems
which one has in determining what the absolute temperature of one's sample
actually <I>is</I> when it is going round in the rotor at speed.<BR><BR>It
is, of course, only a worry to those (very limited) number of people for
whom an absolute s value is of importance, normally for hydrodynamic
modelling purposes (although formulation issues should not be forgotten).
When I raised this issue on RASMB a week or so back, my concern was not
"<FONT color=#ff0000><TT>to ensure their operation within the
published specification".</TT></FONT> . I am trying to get
the <U>accuracy</U> of the temperature read-out to be close to the
<U>precision</U> of which the system is capable. I have no evidence at all
to suggest that the accuracy is outside the quoted spec of 0.5º. It is just
that I - in my greedy way - want 0.1º.<BR><BR>Even the method mentioned
(equilibrate for 3 hours - under vacuum - and then check "<FONT
color=#ff0000><TT>with a calibrated external temperature sensing device to
verify accuracy"</TT></FONT> is not unambiguous in what it will
yield. Quite apart from matters such as adiabatic effects when one releases
the vacuum to use an "external temperature sensing device", can one be sure
that the thermal emissivity of a spinning rotor surface, averaged over
everything that is passing by, is equal to that of a piece of the rotor
surface 'seen' in a stationary rotor? <BR><BR>None of these are new
concerns, and I certainly lay no claim to the IPRs! I imagine, from what
Mei-Ling Chien has communicated, that we at least know clearly that the
±0.5º refers to the accuracy of the temperature <U>as measured by the
defined procedure</U>. Walter Stafford's colorimetric method (Stafford &
Liu) did not suggest the presence of errors outside the stated accuracy
limit, and is surely a valid way to approach the absolute temperature issue.
But is is pretty tedious to use as a procedure, and certainly as a routine
QA method is not feasible. <BR><BR><FONT color=#008000><I>As an approach to
the size of the problem, would there be support for Borries Demeler's
suggestion (a single sample to be circulated and multiple users on multiple
machines to report an s value under defined conditions)? After all, the NCMH
+ Borries's Lab gives us 6 machines for starters.<BR></I></FONT><BR>Any way,
we here keep trying here to locate the holy grail - a simple, cheap,
effective method for determining the in-cell temperature to ±0.1º
<BR><BR>Regards to all (and many thanks to Mei-Ling
Chien)<BR><BR>Arthur<BR><BR>-- <BR>*************************<BR>Arthur
Rowe<BR>Lab at Sutton Bonington<BR>tel: +44 115 951 6156<BR>fax: +44 115 951
6157<BR>*************************<BR>
<BLOCKQUOTE><BR><B>From: </B>mchien@beckman.com<BR><B>Date: </B>Fri, 3 Dec
2004 10:11:46 -0800<BR><B>To: </B>"'rasmb@rasmb-email.bbri.org'"
<rasmb@server1.bbri.org><BR><B>Subject: </B>[RASMB] Re: XL-A/I temp
control<BR><BR></BLOCKQUOTE><BR>
<BLOCKQUOTE><TT>----------------------------------------------------------------------------------<BR>The
older archived RASMB emails can be found
at:<BR>http://rasmb-email.bbri.org/rasmb_archives<BR>and current archives
at<BR>http://rasmb-email.bbri.org/pipermail/rasmb/<BR>Search All the
Archives
at:<BR>http://rasmb-email.bbri.org/rasmb_search.html<BR>----------------------------------------------------------------------------------<BR><BR>Hi
All,<BR><BR>Below is response regarding XL-A/I temperature control from
our Technical<BR>Support
Department.<BR><BR>******************************************************<BR>Mei-Ling
Chien PhD<BR>Staff Development Scientist, Centrifugation<BR>Platform &
Automation Business Center<BR>Beckman Coulter
Inc.<BR><BR>mchien@beckman.com<BR>(650)
859-1948<BR>******************************************************<BR><BR><BR>The
basis for temperature control specifications were instrument
design<BR>specifications for temperature control and dynamic system
testing during the<BR>prototype phase of the product.<BR><BR>If there is a
discrepancy in temperature control and measurement between<BR>instruments
of the same design then a dynamic calibration check should be<BR>performed
on both instruments <FONT color=#ff0000>to ensure their operation within
the published<BR>specification.<BR></FONT><BR>First the physical condition
of components within the temperature control and<BR>vacuum system should
be verified through inspection. Then an electronic<BR>calibration
for temperature control and vacuum can be performed. Lastly
a<BR>dynamic test or rotor dunk test is performed (rotor should be
precooled or<BR>preheated to avoid testing delay). The rotor and its
contents must be allowed<BR>to equilibrate for up to 3 hours or more.
When set temperature equals indicated<BR>temperature at the
instrument interface, the rotor temperature is then checked<BR><FONT
color=#ff0000>with a calibrated external temperature sensing device to
verify accuracy</FONT>.<BR><BR>If the checks fall out of specification
then appropriate troubleshooting is<BR>required to isolate the electronic
or mechanical fault in the temperature<BR>control or vacuum system.
Once the fault is corrected the temperature control<BR>checks are
performed again.<BR><BR>Quote from Bob Giebeler, Analytical
Ultracentrifugation in Biochemistry<BR> and Polymer Science,
1992,16-25 for the Optima XLA/I.<BR>"Temperature control is
considerably more stable, provides more rapid cool-down<BR>and
heat-up rates, is thermally more uniform, and has
equivalent accuracy as<BR>compared to previous models including the
Model E. This control system uses an<BR>isothermal radiometer
temperature-sensing system to sense the temperature of the<BR>rotor
that is emissivity-independent ad view
factor-corrected in software.<BR>Heating and cooling of the
rotor are accomplished by the refrigeration can that<BR>surrounds
the rotor, which is in turn
heated and cooled by thermoelectric<BR>modules. This
environment is very isothermal, and at equilibrium, irrespective<BR>of
speed or temperature, rotor temperature is within
about one degree of the<BR>refrigeration can temperature.<BR><BR><BR>The
control system that regulates rotor
temperature, as monitored by the<BR>radiometer, is
highly software-intensive. This software
encompasses triple<BR>proportional-integral-differential control
algorithms and proportional-integral<BR>smoothing algorithms.
In addition, radiometer view factors are measured during<BR>rotor
cool-down to allow more rapid
rotor cool-down and more
accurate<BR>temperature monitoring during
cool-down. While at equilibrium, refrigeration<BR>can
temperature fluctuation does not
typically exceed +0.5C, and
the<BR>corresponding rotor temperature fluctuation is less than
+0.2C.<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>_______________________________________________<BR>RASMB
mailing
list<BR>RASMB@rasmb-email.bbri.org<BR>http://rasmb-email.bbri.org/mailman/listinfo/rasmb<BR></TT></BLOCKQUOTE><TT><BR></TT><BR><BR>This
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