- The absorbance ratio can be used to check peak purity. If the peak is pure, the absorbance spectra are the same over the whole peak and the ratios should therefore remain constant. The peak is probably not pure if the absorbance ratio is not the same over the whole peak.
- The illustration below shows a chromatogram of two co-eluting components with differing absorbance spectra and a small difference in retention time:
- The absorbance ratio can be used for peak identification. Different compounds have a specific ratio between absorbancies at different wavelengths.
- The illustration below shows a chromatogram of two components with differences in their absorbance spectra:
- Both curves must have a baseline close to zero AU before they can be divided. This is achieved with baseline subtraction. The table below describes how to subtract the baseline from an earlier integration and divide the curves:
Step |
Action |
||
1 |
Perform
peak integrations to create a baseline for each UV curve, see "Perform
a peak integration". |
||
2 |
Select
Operations: Subtract. Result:
The Subtract dialog opens. |
||
3 |
Select
the UV curve in the first Source chromatogram and curve list. Select
its baseline in the second Source chromatogram and curve list. Click
OK. Result: A
curve with the subtracted baseline is added to the chromatogram in the first
available curve position. By default, the curve name will have a SUB ending.
|
||
4 |
Repeat
steps 2 and 3 for the second UV curve. |
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5 |
Select
Operations:Divide. Result:
The Divide dialog box opens. |
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6 |
Select
the first result curve from the subtractions in the first Source chromatogram
and curve list. Select
the second result curve from the subtractions in the second Source
chromatogram and curve list. |
||
7 |
Click
the checkbox for Threshold and type values for each curve. This results in
the following: The
quotient is set to 1.0 if either of the sample values is closer to zero than
the threshold value. Very high quotient values are prevented if division is
performed with values close to zero. Very low quotient values are also
prevented.
|
||
8 |
Click
OK. Result:
A new curve is added to the chromatogram in the first available position. By
default, the curve name will have a DIV ending. |
- The resulting curve can be filtered to reduce noise and to remove ghost peaks. The table below describes how to filter the curve.
Step |
Action |
||
1 |
Select
Operations: Smooth. Result:
The Smooth dialog opens. |
||
2 |
Select
the Source Chromatogram. Select a
Filter Type.
Click OK. Result:
A new curve is added to the chromatogram in the first available position. By
default, the curve name will have a SMTH ending. |
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