Fw: [RASMB] Bad scans from XLA
James
james.falabella at nist.gov
Fri Oct 3 10:54:51 PDT 2008
Hi Sam,
Thank you so much for your message. I just corresponded with John Philo and
he said that the noise to the left of the reference cell meniscus looks
excessive so it probably will be necessary to clean the system to see if
this improves the signal smoothness. It would be great if you can stop by on
Monday to clean the optics the timing will work well with my experiment
schedule. With the improved technique and clean optics the results should be
much better.
James
-----Original Message-----
From: sefishpaw at Beckman.com [mailto:sefishpaw at Beckman.com]
Sent: Friday, October 03, 2008 9:08 AM
To: 'James'; rasmb at server1.bbri.org
Cc: Edward Eisenstein
Subject: Re: Fw: [RASMB] Bad scans from XLA
Hi James,
Following the email traffic on the RASMB, in reading this it sounds
like
you had another cell leak after I was there on Monday. You can confirm
this.
If so I may need to come out and clean the system again. I can come back out
on
Monday October 6. Let me know if I need to return, thanks.
Take Care, Sam.
Beckman Coulter, Inc.
District Manager, Chesapeake Team
LSR, Eastern Zone
To
sefishpaw at beckman.com
cc
Subject
Fw: [RASMB] Bad scans from XLA
----- Forwarded by Erby Perdue/Remote/BII on 10/02/2008 07:59 PM -----
"John Philo"
<jphilo at mailway.com
>
To
Sent by: "'James'"
<james.falabella at nist.gov>,
rasmb-bounces at rasmb <rasmb at server1.bbri.org>
.bbri.org
cc
Subject
10/02/2008 04:35 PM RE: [RASMB] Bad scans from XLA
Please respond to
jphilo at mailway.com
James, after seeing the 2nd, correct PDF I notice one other symptom: in that
last experiment the data all the way at the left in the air-air space are
actually changing with time, which obviously should never happen, and seem
to be
extraordinarily noisy too. So that does suggest there may be a problem with
the
optics or slit assembly movement.
Sorry, if I'd paid attention to the sedimentation coefficient scale it
should
have been obvious you are running at low speeds so I strongly doubt the
leaks
caused bending of the centerpiece rib.
John
From:, James [mailto:james.falabella at nist.gov]
Sent: Thursday, October 02, 2008 12:58 PM
To: jphilo at mailway.com; rasmb at server1.bbri.org
Subject: RE: [RASMB] Bad scans from XLA
John,
Thank you for your insight into the problem, I will make certain that the
reference meniscus does not move throughout the run. I am using 15,000 rpm
for
the 10 nm gold particles and 5,000 rpm for the 20 nm particles I will apply
the
other corrections and hopefully the data will not show a bent center rib. I
will
run another radial calibration at 3000 rpm to ensure that this is not the
issue.
I will also check the masks in the centerpiece to ensure that they are not
inserted upside down. I think also my fit of the data may be a problem too
with
the right boundary too far over and I will correct this in the future data
analysis.
If all of this does not work, I will call Beckman to clean the slit assembly
since I am a rather new user with about a year of experience using the XLA.
Thank you so much for your advice on rectifying this problem that I am
experiencing. I will follow up with results when I try these things.
James
From: John Philo [mailto:jphilo at mailway.com]
Sent: Thursday, October 02, 2008 3:27 PM
To: 'James'; rasmb at server1.bbri.org
Subject: RE: [RASMB] Bad scans from XLA
James,
I am wondering if you are possibly getting intermittent leakage between the
channels as the rotor accelerates. This can sometimes make it seem like
sedimentation has started somewhere far away from the true meniscus
position, as
seems to be the case in the second 'bad' run. In that data your reference
meniscus seems to be at ~5.96 cm, i.e. not right on top of the sample
meniscus
as it will be if there is extensive cross-channel leaking, but possibly the
reference started out significantly further to the left and moved as the
rotor
accelerated.
You also want to be very careful about aligning the cell in the rotor or you
will get convection (and very screwy data).
Another possibility is that your leaked gold particles are getting into the
slit
assembly and causing problems with the radial scanning.
I'm not clear what rotor speed you are using but when you leak out half a
sample
and the speed is high enough the center rib of the centerpiece can get
permanently bent, after which you will always get strange data with that
centerpiece.
I am not clear why you keep doing radial calibration and in general I would
not
recommend this, but I doubt that is the fundamental problem here. The
calibration should not change during normal operation---it will only change
if
someone works on the optical components. Also you should not turn off the
delay
calibration. Definitely never ask for radial calibration at speeds above
3000
rpm.
Having said that I do wonder whether your radial calibration is correct. It
looks to me like you are getting good data (flat plateaus) well beyond 7.14
cm,
which is typically not true for the standard epon centerpieces and window
holders, which suggests the radial calibration may be faulty. The true base
of
the channel should be very close to 7.2 cm (and seems not to be positioned
correctly in your fits). If the radial cal is bad it might be the
counterbalance
itself. The masks that slip into the sides of the counterbalance to create
the
precision reference points can fall out and hence can be put back in upside
down.
John
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of James
Sent: Thursday, October 02, 2008 10:38 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] Bad scans from XLA
Dear RASMB members,
I have recently been struggling with a recurring problem when I centrifuge
10
and 20nm gold particles that causes my XLA to generate poorly formed OD data
scans that cannot be fit by Sedfit. I had initially been getting well formed
scans that were easily fit by Sedfit using the continuous c(s) model however
after half of the sample side in one cell (in a 4 cell rotor) leaked out
during
a run I was no longer able to get the nice sigmoidal scans that I originally
had
even though I tightened and refilled the leaking cell. I asked the Beckman
service rep to investigate and he cleaned the optics and told me to have the
XLA
perform a radial calibration and the scans were back to a sigmoidal shape
using
the same sample of gold nanoparticles and Sedfit could fit the data well
again.
However after another cell leaked in the centrifuge, the scans could not be
fit
with Sedfit even though the cell was tightened and refilled and no longer
leaked. I have been performing a radial calibration before each run and have
not
turned off the delay calibration hoping the instrument will recalibrate and
improve the scans however, this has not happened. I have attached screen
shots
showing the quality of the data before and after the cell leak. It looks
like it
could be the optics but I am not convinced this is the whole story since it
seems extreme that one cell leak can alter the XLA performance this much
until
the optics are cleaned.
James B. Falabella
Process Sensing Group
National Institute of Standards and Technology Gaithersburg, MD
Tel: 301-975-5041
Fax: 301-975-2643
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