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Greetings All:<br><br>
I've been watching and thinking how this discussion might apply to the
fluorescence detection system (FDS). The mobility of the rotor that
John refers to is due to the rubber mounts that support the drive
motor. We've all seen how the rotor wiggles around while taking it
in and out. The mounts stretch over time and occasionally need
replacing. The horizontal movements are manageable, and the
vertical movements probably don't affect the absorbance and interference
measurements too much as these beams are relatively well columnated and
insensitive to height. The FDS has the opposite optical design, a
fairly short focal path, and is sensitive to the sample height.
Therefore, for the FDS, I would add that the focus height should be
calibrated when rotor type is changed, as the weight difference could
cause a different position. <br><br>
Glen<br><br>
<br>
At 06:46 PM 1/7/2011, John Philo wrote:<br>
<blockquote type=cite class=cite cite="">Bo, that is indeed an
interesting idea for checking the counterbalances.<br><br>
I should perhaps clarify for everyone that while changing to a
different<br>
rotor or counterbalance can't directly alter the calibration of the
optical<br>
systems, simply taking any rotor in or out of the instrument can shift
the<br>
drive a little relative to the optical components. That is, both
calibration<br>
points on the counterbalance will shift in or out by the same amount as
the<br>
axis of rotation moves relative to each optical system (and the shift
seen<br>
by each optical system is not in general the same). <br><br>
Thus in principle one could argue that the radial calibration should
be<br>
re-done for each and every run. However in practice these shifts
don't<br>
really have a significant effects on the results. Sedimentation
coefficients<br>
(the thing we can measure most precisely) depend mostly on radial
position<br>
differences, and thus are independent of such shifts (to first order).
Even<br>
a huge shift of 1 mm would only change the apparent sedimentation<br>
coefficients by ~1.6%. Masses from equilibrium do depend more directly
on<br>
absolute radius values, but even this huge shift of 1 mm would only
change<br>
the apparent M by ~3%. As I recall these run-to-run shifts of the
drive<br>
relative to the optics are actually < 0.1 mm, so I doubt AUC users
will ever<br>
see significant errors arising from them. <br><br>
John<br><br>
-----Original Message-----<br>
From: Borries Demeler
[<a href="mailto:demeler@biochem.uthscsa.edu" eudora="autourl">
mailto:demeler@biochem.uthscsa.edu</a>] <br>
Sent: Friday, January 07, 2011 2:22 PM<br>
To: jphilo@mailway.com<br>
Cc: 'Xin-Ping Xu/FS/VCU'; rasmb@server1.bbri.org<br>
Subject: Re: [RASMB] counterbalance functions<br><br>
> <br>
> Xin,<br>
> <br>
> What Virgil said is mostly correct, but it is only part of the
story. <br>
> The counterbalance is also required for "delay"
calibration of the <br>
> absorbance optics (to set the timing of the lamp pulses). This <br>
> calibration happens every time you start a run or when the rotor
speed <br>
> is changed by more than 2000 rpm.<br>
> <br>
> Radial calibration needs only to be done intermittently (I recommend
<br>
> doing it only when the service tech has done something to the <br>
> instrument). Since the optical components are entirely independent
of <br>
> which rotor is used the radial calibration is independent of the
<br>
> rotor, so I have to disagree with what Virgil said about needing
to<br>
recalibrate for each rotor.<br>
> <br>
> If the counterbalances are machined accurately then they all should
<br>
> give the same radial calibration also. Again, remember the optical
<br>
> components do not move or change when you change counterbalances. If
<br>
> different counterbalances give different calibrations then it is
<br>
> impossible to say which one is more accurate. In that situation I
<br>
> would recommend adopting one of them as 'correct' and sticking to
its <br>
> calibration. Otherwise your results are potentially different from
one <br>
> experiment to another depending on which counterbalance you used
for<br>
radial calibration.<br>
> <br>
> If you are not using the absorbance optics then in fact the <br>
> counterbalance is not required and an additional interference or
<br>
> fluorescence sample can be run in that hole.<br>
> <br>
> John<br><br>
Yes, that is a chicken and egg sort of conundrum, if you cannot trust
your<br>
counterbalance you cannot trust your radial calibration. However, you
can<br>
get a little closer to the truth with some software I wrote.<br>
One way to check if the counterbalance is correctly calibrated is with
the<br>
UltraScan3 rotor calibration program. While the main purpose of the
program<br>
is to calculate the rotor's stretching function, you can also use it
to<br>
"measure" the counterbalance and cell housing and centerpiece
combinations. <br><br>
The way it works is that you would scan the counterbalance (or<br>
centerpiece) in intensity mode at multiple speeds, generating a
sharp<br>
boundary at each edge. The edge would move with each speed, as the
rotor<br>
stretches. If the edges are truly centered at 5.85 and 7.15 cm, then
the<br>
center should be at 6.5 cm, which is how the rotor has been
machined.<br>
The program will take the differences between the edges at successive
speeds<br>
and extrapolate these differences with a second order polynomial to
zero<br>
speed. The positions at rest should be 7.15 for the bottom then and 5.85
for<br>
the top, and the center (which is also calculated by the<br>
program) should then be at 6.5 cm. If the center is not at 6.5 cm, then
that<br>
is an indication that something is wrong with the
counterbalance.<br><br>
In addition to the counterbalance dimensions, you will get the rotor<br>
stretching function from this calculation. By scanning centerpieces
in<br>
different cell housings you will also get the true bottom of the cell
this<br>
way for each cell housing/centerpiece combo. This is helpful if you
want to<br>
do mass conservation calculations in equilibrium experiments (the
stretching<br>
function will predict the offsets at each speedi). See the recent posts
on<br>
this topic.<br><br>
Regards, -borries<br><br>
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<br>
Glen Ramsay, Ph.D.<br>
Chief Scientist<br>
Aviv Biomedical, Inc.<br>
750 Vassar Avenue, Suite #2<br>
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(732) 370-1300, Ext. 25<br>
(732) 370-1303, FAX<br>
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www.avivbiomedical.com<br>
</a> Skype: GlenAtAviv<br><br>
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