Peak Purity and Peak Identification

Introduction

Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity.

Peak Purity

• 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:

Peak Identification

• The absorbance ratio can be used for peak identification. Different compounds have a specific ratio between absorbances at different wavelengths.
• The illustration below shows a chromatogram of two components with differences in their absorbance spectra:

ALSO READ: Chromatographic Calculations

Divide the Curves

• 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. TIP: You can also subtract corresponding blank runs if there are blank runs available.
4	Repeat steps 2 and 3 for the second UV curve.
5	Select Operations: Divide. Result: The Divide dialog box opens.
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. Note: Default Threshold values are entered by UNICORN. The values can be changed.
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.

Filter the Result curve

• 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. TIP: The Median filter is recommended to remove noise that appears as spikes or occurs in a small area of the curve. Click OK. Result: A new curve is added to the chromatogram in the first available position. By default, the curve name will have an SMTH ending.

ALSO READ: SOP for Verification of System Suitability Test

Ratios between UV curves measured at different wavelengths give useful information about peak purity or peak identity.

Peak Purity

• 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:

Peak Identification

• The absorbance ratio can be used for peak identification. Different compounds have a specific ratio between absorbances at different wavelengths.
• The illustration below shows a chromatogram of two components with differences in their absorbance spectra:

ALSO READ: What is Peak Tailing?

Divide the Curves

• 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. TIP: You can also subtract corresponding blank runs if there are blank runs available.
4	Repeat steps 2 and 3 for the second UV curve.
5	Select Operations: Divide. Result: The Divide dialog box opens.
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. Note: Default Threshold values are entered by UNICORN. The values can be changed.
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.

Filter the Result curve

• 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. TIP: The Median filter is recommended to remove noise that appears as spikes or occurs in a small area of the curve. Click OK. Result: A new curve is added to the chromatogram in the first available position. By default, the curve name will have an SMTH ending.

ALSO READ: Do's and Don'ts: HPLC Analysis