[RASMB] Strange AUC noise

Tue Oct 20 18:03:29 PDT 2015

Hi Tom and all,
The practice in our lab is to use Spin50 cells always for absorbance scanning since even at best it seems that Spin60 cells always have a higher RMSD for absorbance scanning (maybe a bit less signal to noise due to being narrower, or because of the timing issues, don't know, just observed).  Since we had been averaging about 600 samples a year, we have other practices not found in the best academic labs.  I instruct my analysts to load 400 microLiters on the sample side and 405 - 410 on the reference side.  We always use gel loading pipettes for loading to avoid scratching the walls with needles.  We use Beckman supplied thin tubing or pipette tips to remove the sample rather than blunt needles, also to avoid wear and tear on the centerpieces.  Given that some samples are precious and we are always given only enough for one filling, and some samples are irreplaceable, trying to fill up closer to the top risks over filling spills both by getting the bubble too close to the top and by dipping the pipette tip in three times since most of our pipettes get a bit non-linear over 200 micro liters.   And since the main data we want is percent aggregate, and we want to avoid as much as possible any possible artifacts, we use absorbance data only and are very conservative in choosing the area to fit fit.  Empirically I am of the opinion that there are plenty of data points, and another mm of data is not going to be very important to the SEDFIT final answer.   Jack has mentioned to me before, and stated in this email chain, that his practice is to insist on analysts optimizing the column height in the cells to near the max possible, which makes sense theoretically, but practically I don't think the extra data points change the final answers enough to justify the extra stress and time taken by the analysts to try to achieve that optimization.  I get much more bang for the nagging by telling them to be more careful washing their cells:  I have observed noticeable differences in final answers between dirty and clean cells when doing replicates, and also when the lamp is cleaned, I sometimes resolve peaks better in certain samples.

At first the Beckman repair person did not see anything wrong with the slit assembly, but later after I wrote the email to RASMB the instrument failed a radial calibration and he did replace the slit assembly.  Subsequently, the instrument is now running OK again.
However, the last time we saw noise like this, replacing the slit assembly helped for only a few weeks and then the noise came back, so I am not completely convinced there is nothing else going on  (The last time we saw this kind of noise, quite a few parts were changed for reasons that are a bit complex and can't be put in the email).  The noise is never seen in the scan of a blank hole without a cell, so maybe it is both the slit assembly and something with the timing and cell light beam geometry.  We also had a slit assembly become unglued in our oldest centrifuge and give the "jumping pattern" noise John Philo described (as well as finding the bottom of the cell to be outside the normal 7.15 to 7.2 cm range) but none of our older machines have made this exact strange pattern.
Thanks for the discussion.  If there are any new developments, I will update the conversation.  For now, changing the slit assembly has fixed the problem and prevents further investigation:  an unexpected good news, since last time changing out the assembly provided only limited help.
Kind Regards,
David
      From: "Laue, Thomas" <Tom.Laue at unh.edu>
 To: "jphilo at mailway.com" <jphilo at mailway.com>; 'David Hayes' <drdavidbhayes at yahoo.com>; 'RASMB' <rasmb at rasmb.org> 
 Sent: Tuesday, October 20, 2015 8:54 AM
 Subject: RE: [RASMB] Strange AUC noise

#yiv7929042017 #yiv7929042017 -- _filtered #yiv7929042017 {font-family:Helvetica;panose-1:2 11 6 4 2 2 2 2 2 4;} _filtered #yiv7929042017 {font-family:Helvetica;panose-1:2 11 6 4 2 2 2 2 2 4;} _filtered #yiv7929042017 {font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;} _filtered #yiv7929042017 {font-family:Tahoma;panose-1:2 11 6 4 3 5 4 4 2 4;}#yiv7929042017 #yiv7929042017 p.yiv7929042017MsoNormal, #yiv7929042017 li.yiv7929042017MsoNormal, #yiv7929042017 div.yiv7929042017MsoNormal {margin:0in;margin-bottom:.0001pt;font-size:12.0pt;}#yiv7929042017 a:link, #yiv7929042017 span.yiv7929042017MsoHyperlink {color:#0563C1;text-decoration:underline;}#yiv7929042017 a:visited, #yiv7929042017 span.yiv7929042017MsoHyperlinkFollowed {color:#954F72;text-decoration:underline;}#yiv7929042017 p.yiv7929042017MsoAcetate, #yiv7929042017 li.yiv7929042017MsoAcetate, #yiv7929042017 div.yiv7929042017MsoAcetate {margin:0in;margin-bottom:.0001pt;font-size:8.0pt;}#yiv7929042017 span.yiv7929042017EmailStyle17 {color:blue;font-weight:normal;font-style:normal;text-decoration:none none;}#yiv7929042017 span.yiv7929042017EmailStyle18 {color:#1F497D;}#yiv7929042017 span.yiv7929042017BalloonTextChar {}#yiv7929042017 .yiv7929042017MsoChpDefault {font-size:10.0pt;} _filtered #yiv7929042017 {margin:1.0in 1.0in 1.0in 1.0in;}#yiv7929042017 div.yiv7929042017WordSection1 {}#yiv7929042017 Hi- Were these Spin60 or Spin50 cells? If they were Spin60, it might be that the lamp timing on the ‘noisy’ machine is causing the light pulse to partially hit the centerpiece wall. This is a machine-to-machine problem, consistent with what you are seeing. However, the telltale for the timing problem is increased noise in the scans, often at the top of the cell, not a wave.  Consequently, I vote for the slit assembly lifting (John’s suggestion). Tom       

From: RASMB [mailto:rasmb-bounces at list.rasmb.org]On Behalf Of John Philo
Sent: Monday, October 19, 2015 5:00 PM
To: 'David Hayes'; 'RASMB'
Subject: Re: [RASMB] Strange AUC noise    David, I will speculate that you have a problem with the slit assembly. You might see something like that if the foil on the bottom side has come partially loose. Or perhaps there is some dirt or a burr causing the whole assembly to lift up at certain radial positions. I once had the foil come loose in a way that produced wild outlier scans, but only every once in while (a few per run). Intermittently the foil would actually catch on something (probably the ring on top of the PMT) and fold completely back on itself. And by the way, you should remind your people to fill the cellsmuch more. You want the meniscus to be up near 5.9 cm, not at ~6.25 cm. John From: RASMB [mailto:rasmb-bounces at list.rasmb.org]On Behalf Of David Hayes
Sent: Monday, October 19, 2015 11:14 AM
To: RASMB <rasmb at rasmb.org>
Subject: [RASMB] Strange AUC noise    Hi all,    We had some strange AUC noise in a recent experiment.  In the pictures below from SEDFIT, the first two runs produced data as in the picture NNBekman1 (the IT guys name our instruments and they pruposely mispelled Beckman as Bekman years ago). A strange noise pattern showed up in the scans that were produced on day one.  Then the sample was loaded into a new cell and run again on the same centrifuge producing the strange noise pattern in the picture called NNBekman1.   the filled cell was taken and gently mixed and then run on another centrifuge Bekman17 and made a perfectly fine patter shown in picture Beckman17.    We have seen the strange noise before on our NNBekman1 centrifuge, but this is the first time on NNBekman2.    Has anyone seen noise like this?   Any theories on the cause of the noise?    It certainly looks like an optical artifact of some kind, but the time dependence is hard to explain.    Kind Regards,    David Hayes          

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