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 simulated 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 absorbancies at different wavelengths.

The illustration below shows a simulated chromatogram of two components with differences in their absorbance spectra:

How to 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

Create a baseline for each UV curve.

2

Select Operations:Subtract.

Result: The Subtract dialog box opens.

3

  • Select the UV curve in the first list of curves.

  • Select its baseline in the second list of curves.

  • Click OK.

Note: 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 list of curves.

  • Select the second result curve from the subtractions in the second list of curves.

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.

How to 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 box opens.

2

  • Select the Source Chromatogram.

  • Select a Filter Type.

Note: The Median filter is recommended to remove noise that appears as spikes or occurs in a small area of the curve.

  • Click OK.

2005-06-15