ESTD Quantitation

OpenChrom enables users to quantify chromatograms using internal standards (ISTD). It’s also possible to use external standards (ESTD). First of all, load the external calibration chromatograms into OpenChrom.

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Please select the “Overlay Perspective”. It easily allows to review the peaks designated for calibration or, in case they haven’t been determined yet, to detect candidate peaks that may be suitable for quantitation purposes.

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Via the preferences button, it’s also possible to disable the display of the chromatogram area. This is helpful in some cases.

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The peaks at position ~10.7 minutes and ~16.4 minutes seem to be suitable to be used as an external calibration.

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After suitable peaks have been detected, please apply the peak detector on each chromatogram.

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After detecting the peaks, apply the peak integrator.

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Then delete all peaks except both at position ~10.7 minutes and ~16.4 minutes.

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Two peaks remain in each calibration file.

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Then identify both peaks manually.

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The first peak gets the name STD-1 and the second gets the name STD-2. You can choose whatever name you want, but it must be the same for all calibration chromatograms. This makes it easier to setup the quantitation table later.

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The identification is shown in the “Peak Targets” view.

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Do the same for the seconds peak: STD-2.

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After setting the targets for all peaks in each calibration chromatogram, select the “Quantitation Support” perspective.

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If no database has been created yet, empty views are displayed.

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Create a new database in the “Databases View“.

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The new database is shown in the database list then.

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Please select the database, so that it will be used for all further steps.

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Then add the peaks of the selected chromatogram to the selected quantitation table.

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Please add the quantitation details of the file Calibration1.ocb.

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Do the same for all other calibration files. The peaks are automatically matched by the peak name “STD-1” and “STD-2“.

Calibration1.ocb    3.0    mg/g
Calibration2.ocb    6.0    mg/g
Calibration3.ocb    1.0    mg/g
Calibration4.ocb    4.0    mg/g
Calibration5.ocb    10.0    mg/g
Calibration6.ocb    2.0    mg/g

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Each quantitation peak can be edited afterwards. Edit STD-1:

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Edit STD-2:

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Select the first entry “STD-1“, select the view “Quantitation Peak List” and create the response table.

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The response entries are listed then in the view “Concentration Response Entries“.

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You can also edit the quantitation signals. In this case, the TIC is used as we already recorded the data in SIM modus.

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All views can be drag & dropped within in the application. For a better overview, please move “Quantitation Peaks” view and select the “Concentration Response Entries Chart“. The peak with a concentration of 4 mg/g seems to be out of range.

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It’s easy to delete the response entry from the list.

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The calibration looks better after deleting the response entry.

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The second peak looks better and needs no further adjustments. Then load a sample which needs to be quantified. Detect and integrate peaks. Then apply the quantifier.

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The results for each peak are shown in the “Peak Quantitation Entries (MSD)” view. The report option exports the data to a text format *.txt.


PEAK QUANTITATION RESULTS
——————————
[#]    Name    ChemicalClass    Concentration    ConcentrationUnit    Area    Description    Ion

[5]    STD-1    SB    3.15636    mg/g    456697.11942        TIC

[12]    STD-2    SB    4.01875    mg/g    334451.11274        TIC

We expect that the sample chromatogram contains 4 mg/g. Hence, STD-2 seems to be suitable for quantitation.

 

 

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