153 Eu peak shape
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Please be polite and civil. We know that troubleshooting is vexing...
Please be polite and civil. We know that troubleshooting is vexing...
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I recently have trouble with the CV of the 153 Eu beads test. I ran both beads test before and after clean the neutralizer, spray chamber, injector, skimmer/sampler. The Tb counts increased from 500K to 900K. But the CV of the Eu153 increased from 11% to 28%. Could someone kindly give me some hint? I used to change the sample line and sample capillary, this worked to decrease the CV number. Could something major goes wrong which I am not aware of? I should not be able to get the Tb counts around 900K if there is the alignment issue. Am I correct?
Re: 153 Eu peak shape
Hi Shelly,
1. Did you realign the XY, etc after cleaning?
2. Did you *replace* any components, or just clean them?
3. Did you save screenshots or FCS files from your Eu bead acquisitions, so we could look at the data?
Mike
1. Did you realign the XY, etc after cleaning?
2. Did you *replace* any components, or just clean them?
3. Did you save screenshots or FCS files from your Eu bead acquisitions, so we could look at the data?
Mike
Re: 153 Eu peak shape
I did autotuning on XY without changing any parts for cleaning. I have the tuning files and the 153 Eu Massspec files. Please see the attachment. Usually, the CV were little high, it will fine if I changed the sample line and capillary. But this time, it did not help much.
Re: 153 Eu peak shape
Hi Shelly,
In addition to Mike's comments, one of the big factors that I find influences the bead cvs is the oxide level (higher oxidation = wider cvs; poorer resolution). The autotuning will typically maximize your Tb159 dual counts, but I will sometimes follow that with a manual tuning and deliberately reduce either the makeup gas or current a little bit, sacrificing signal intensity for lower oxide ratio, which gives better cvs.
Hope that helps,
Adeeb
In addition to Mike's comments, one of the big factors that I find influences the bead cvs is the oxide level (higher oxidation = wider cvs; poorer resolution). The autotuning will typically maximize your Tb159 dual counts, but I will sometimes follow that with a manual tuning and deliberately reduce either the makeup gas or current a little bit, sacrificing signal intensity for lower oxide ratio, which gives better cvs.
Hope that helps,
Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
Icahn School of Medicine at Mount Sinai, NYC
Re: 153 Eu peak shape
Hi Shelly,
1. Did you acquire for the same amount of time for the two Eu plots? I see that the tighter CV one has half the events of the broader CV one.
2. I think there are a couple things at issue here. Based on the profile of your single-axis histograms (above and to the right of the axes in the bivariate plots), I think you have a broad CV because you actually have two overlapping peaks. Particularly in the Eu151 (top) plot, the peak isn't symmetric: there's a distinct shoulder on the left (low signal) side of the peak.
It's important to remember that there's a functional difference between large CV from a single, symmetric, but broad peak; and a large CV from a combination of more than one poorly resolved peak. The single-but-broad-peak is a problem: you would be losing mass resolution. Broad-because-multiple peaks, on the other hand, can often result from running your sample too concentrated and therefore causing too many overlapping Event clouds.
In short, you can get a large output CV from two separate and distinct problems. The fact that the broader CV sample is from a larger number of bead Events would be consistent with the too-concentrated-sample idea (assuming you acquired for the same length of time, of course).
Mike
1. Did you acquire for the same amount of time for the two Eu plots? I see that the tighter CV one has half the events of the broader CV one.
2. I think there are a couple things at issue here. Based on the profile of your single-axis histograms (above and to the right of the axes in the bivariate plots), I think you have a broad CV because you actually have two overlapping peaks. Particularly in the Eu151 (top) plot, the peak isn't symmetric: there's a distinct shoulder on the left (low signal) side of the peak.
It's important to remember that there's a functional difference between large CV from a single, symmetric, but broad peak; and a large CV from a combination of more than one poorly resolved peak. The single-but-broad-peak is a problem: you would be losing mass resolution. Broad-because-multiple peaks, on the other hand, can often result from running your sample too concentrated and therefore causing too many overlapping Event clouds.
In short, you can get a large output CV from two separate and distinct problems. The fact that the broader CV sample is from a larger number of bead Events would be consistent with the too-concentrated-sample idea (assuming you acquired for the same length of time, of course).
Mike
Re: 153 Eu peak shape
Hi Mike,
Thank you for your note. The answer to the first question is yes. I always use the same acquisition method for beads tests. I cleaned the system thoroughly before I ran the beads test, I do not quite understand what you mean that the concentrated samples were injected before I clean the system. The sample with narrow CV was ran after I finished all samples. The sample with large CV was ran later after I cleaned the front end parts.
Warmly,
Shelly
Thank you for your note. The answer to the first question is yes. I always use the same acquisition method for beads tests. I cleaned the system thoroughly before I ran the beads test, I do not quite understand what you mean that the concentrated samples were injected before I clean the system. The sample with narrow CV was ran after I finished all samples. The sample with large CV was ran later after I cleaned the front end parts.
Warmly,
Shelly
Re: 153 Eu peak shape
Hi Shelly,
Sorry, I may have been unclear.
Beadsbeforecleaning_23OCT2015.png
Cluster(s) Events: 9927
(Eu151Di)[m] = 922.7
(Eu151Di)[cv] = 11.8%
- single, symmetric peak
Beadsaftercleaning_23OCT2015.png
Cluster(s) Events: 19413
(Eu151Di)[m] = 1663.7
(Eu151Di)[cv] = 28.3%
- broader peak; likely to be two partially merged peaks, with one being a shoulder at lower signal
I think that "Beadsaftercleaning_23OCT2015.png" represents a bead sample that either was actually at a higher concentration, or the cleaning reduced or eliminated a blockage present before the cleaning (thereby increasing the bead transmission efficiency).
Try running more beads, at half the concentration from that used for "Beadsaftercleaning_23OCT2015.png"; if it's simply a concentration effect merging your populations, a more dilute sample should avoid that, and restore your tighter CV. If that doesn't happen, then there may be more of a hardware issue.
Mike
Sorry, I may have been unclear.
Beadsbeforecleaning_23OCT2015.png
Cluster(s) Events: 9927
(Eu151Di)[m] = 922.7
(Eu151Di)[cv] = 11.8%
- single, symmetric peak
Beadsaftercleaning_23OCT2015.png
Cluster(s) Events: 19413
(Eu151Di)[m] = 1663.7
(Eu151Di)[cv] = 28.3%
- broader peak; likely to be two partially merged peaks, with one being a shoulder at lower signal
I think that "Beadsaftercleaning_23OCT2015.png" represents a bead sample that either was actually at a higher concentration, or the cleaning reduced or eliminated a blockage present before the cleaning (thereby increasing the bead transmission efficiency).
Try running more beads, at half the concentration from that used for "Beadsaftercleaning_23OCT2015.png"; if it's simply a concentration effect merging your populations, a more dilute sample should avoid that, and restore your tighter CV. If that doesn't happen, then there may be more of a hardware issue.
Mike
Re: 153 Eu peak shape
Hi Mike,
I forgot to attach the tuning file before I cleaned the instrument. Basically, the Tb counts before the cleaning was half of the Tb counts of the tuning after cleaning. I am not sure if this explains the difference of the events number before and after the cleaning. If the problem is due to the hardware, what should I do to track down?
Thanks,
Shelly
I forgot to attach the tuning file before I cleaned the instrument. Basically, the Tb counts before the cleaning was half of the Tb counts of the tuning after cleaning. I am not sure if this explains the difference of the events number before and after the cleaning. If the problem is due to the hardware, what should I do to track down?
Thanks,
Shelly
Re: 153 Eu peak shape
Hi Mike,
As you suggested that it might be the hardware issue. I just realigned the whole front end. A magic just came out. Tb counts reached over 1000K, 153 Eu beads reaches almost 2000 with perfect peak shape and the RSD are within 10%.
Thanks,
Shelly
As you suggested that it might be the hardware issue. I just realigned the whole front end. A magic just came out. Tb counts reached over 1000K, 153 Eu beads reaches almost 2000 with perfect peak shape and the RSD are within 10%.
Thanks,
Shelly
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