integration parameters and calibration

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sindibad
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Joined: Fri Feb 08, 2013 4:11 pm

integration parameters and calibration

Post by sindibad »

Dear Sir
can you please explain how clarity is able to calculate amounts after calibration because when changing integration parameters and copy them in template and running samples, the retention times of compounds can vary little in left and right windows and standards also can vary little regarding their retention times

My last question, if we want to calibrate with only one standard, how the result table indicate the amount of other pic than the unique standard we have, are there indicate by errors in result table?....
many thanks
Last edited by sindibad on Fri May 10, 2019 2:10 am, edited 1 time in total.
samira

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Daniel Mentlik
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Re: integration parameters and calibration

Post by Daniel Mentlik »

Dear Samira,
let me answer the questions one by one:
1) For Clarity to correctly identify the peak with a name and to be able to quantify it correctly then, the peak itself must fall to the certain range on the time axis. This time interval is defined in the calibration file by using the Retention Time parameter there, as well as Left Window and Right Window. The peak is considered to be the compound, if its retention time is higher than retention time as set in the calibration table minus the left window, and lower then the time plus the right window - you are defining the range of times where the peak may appear. This holds true for both calibration standards and unknown samples.
In case there are more peaks in the given time interval found in the chromatogram, only one of those is considered to be the compound of interest. It is the peak which retention time is closest to the retention time set in the calibration table by default, but you can change this behavior for particular compounds to use the first peak in the interval, last peak in the interval or the biggest peak in the interval using the Peak Selection column, which is hidden by default.
2) There are two columns for response factor in the calibration window, you are right about that. As you may note, one of those is common for the compound as it is, the other one is specific to the calibration level. As you may notice, the one specific for each calibration level is automatically calculated for any level and compound which has non-zero response and non-zero amount and cannot be manually modified. The other one, common across the different calibration levels, is only used if the Curve Fit Type is set to Free Calibration - no calibration curve is computed, but the compound uses the entered Response Factor instead.
3) I possibly do not understand this question correctly - you mean you are using just one-level calibration, or that you want to use just one compound for calibration and use its response factor to calculate the amounts for other compounds not present in the standard mixture, but present in the unknown samples? Please specify.
Daniel Mentlí­k
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sindibad
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Joined: Fri Feb 08, 2013 4:11 pm

Re: integration parameters and calibration

Post by sindibad »

Many thanks dear Daniel
for my last question if we want to quantitate a lignan in plant, I have only one standards lariciresinol for example and should quantite it among other present in my plant extract
so I have to create project, then method, then open calibration file which I must create a new one for that analyse
after my sequence I will have for example tree chromatogram if I use only two levels in calibration, from unknown and for a tw0 level standard of lariresinol
in calibration file I have to add peak of lariresinol alone (is that correct?) or add all peaks and remove the other peaks from peak table and leave lariresinol , suppose everything in integration is ok
my question how about the result table appear if I have quantited only lariresinol
should we observe the other peaks in that result table

maybe it is more clear
many thanks daniel
samira

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Daniel Mentlik
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Re: integration parameters and calibration

Post by Daniel Mentlik »

Dear Samira,
I understand now.
Actually, for this you have two options of display - selecting the one you want is up to your choice.
Suppose I create a calibration with just one calibrated peak and the rest of them are uncalibrated. When I return to Chromatogram window, the result table would like similar to this:
web-all-peaks.png
Please note the file has multiple peaks, just one of them is in the calibration and all others are displayed without the name and amounts.
The other option is to switch the display of the result table only for peaks present in the calibration - you can switch the radio button in the pane right of the result table to see this result:
web-calibration.png
The printed reports will look exactly like on the screen, nothing changes with the graph - just the result table is influenced.
Daniel Mentlí­k
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sindibad
Posts: 20
Joined: Fri Feb 08, 2013 4:11 pm

Re: integration parameters and calibration

Post by sindibad »

Dear daniel
when creating calibration file ( a new one), I have some problems when opening standards and and adding peaks in table
so when adding existing peaks in each level, I couldnt see calibration curve for each standard, I see instead in response columns ##### instead values, can you please explain when that problem can comes or maybe mistake that I had made
please assistance
samira

sindibad
Posts: 20
Joined: Fri Feb 08, 2013 4:11 pm

Re: integration parameters and calibration

Post by sindibad »

Dear daniel
even when creating new method my chromatograms are linked to another method when I try to lionk them into the new method,should I work in a new project each time I have a new method? maybe you can advise me please
also can you sow me how to calculate performance parameters (capacity factor, symmetry, theoretical plates, please explain how to get them.
many thanks,
samira

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Daniel Mentlik
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Re: integration parameters and calibration

Post by Daniel Mentlik »

Dear Samira,
once again, problems one by one:
so when adding existing peaks in each level, I couldnt see calibration curve for each standard - there might be two things to note regarding this. The ##### in the response columns (or any other, for that matter) is shown when the value in the cell has more digits that can fit into the displayed cell - so you wouldn't see just a part of the number. The remedy to this is to widen the response column by dragging its right border in the column header until the whole value is shown. The second thing - a calibration curve not appearing - would be most probably caused by the missing amounts for those levels. The level which has a response but not amount filled in will not be shown in the calibration graph of the compound. However, the causes might be more and I could only find the real one when inspecting the calibration file - if the above will not help, please send the calibration file (*.cal) causing problems to support@dataapex.com .

even when creating new method my chromatograms are linked to another method when I try to lionk them into the new method,should I work in a new project each time I have a new method? - again, I do not understand what is causing you the troubles, can you explain in more details, please? Generally, I wouldn't use separate project for each method unless the method would be widely used - by that I mean you would be measuring the same 5 types of analyses, using the same 5 methods, for next year or so. Then, having results organized by methods might be useful.

can you sow me how to calculate performance parameters (capacity factor, symmetry, theoretical plates, please explain how to get them. - these can be displayed on the Performance tab or in the Result table. To display the performance tab in the Chromatogram window, use the Results - Performance command. To display these columns in the result table, right-click on the result table to bring its context menu up and use the Setup Columns... command. In the dialog, move the columns you are interested in from the left list (hidden ones) to the right list (displayed ones). Please note that for the calculations to be effective, you will have to set the Unretained Peak Time and Column Length parameters on the right side of the Performance tab of the given chromatogram, or on the Method Setup - Advanced tab of the method you are using. All chromatograms measured according to that method will have the fields on the Performance tab already filled.
Daniel Mentlí­k
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sindibad
Posts: 20
Joined: Fri Feb 08, 2013 4:11 pm

Re: integration parameters and calibration

Post by sindibad »

Dear daniel
thank you for everythings
Now, Im willing to know how to make results and quantification with statistics, means and rsd in the results table
how to make sequence and set with calibration and then to obtain means and sem or rsd in the result table for the compounds
many thanks by avdvance
samira

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Daniel Mentlik
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Re: integration parameters and calibration

Post by Daniel Mentlik »

Dear Samira,
please check the manuals available on the DataApex website, context help invoked by pressing the F1 button from any Clarity window and/or the Online Clarity Guide containing suggested workflow with the station.

If anything will still be unclear, feel free to ask, but make the questions as specific as possible - I'm really not sure what exactly is unclear from your last post as it mentions functions throughout Clarity and analysis process itself.

Best Regards,
Daniel
Daniel Mentlí­k
DataApex

sindibad
Posts: 20
Joined: Fri Feb 08, 2013 4:11 pm

Re: integration parameters and calibration

Post by sindibad »

thank you daniel I will see the manuals
I have also another quuestion, if I want to quantitate some metabolites and see that one has a maximum of absorbANCE different to the others, should I create 2 calibration file for each wavelenghts and quantitate
second question what is the number of hits, taked in software as 3
the last quetion what is the utility of isoplot, means why we have to look for it, is it to localize the max of absorbance for after quatitate?
sorry for my several questions
best regards
samira

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