Qualitative Data Analysis

2 In This Section, We Will Discuss:  How to load data files.  How to use Signal Options for data display.  How to apply annotation to your chromatogram.  Ways to identify components in your sample.  How to check the purity of a chromatographic peak.

3 Preferences – Signal Options

4 Display Files in Navigation Table Double-click on sequence or Single Runs to display associated data files.

5 Load Signals using Navigation Table Right click the mouse anywhere on the row to activate menu Click on the ‘+’ to activate signal details Turn on/off Navigation Table Grouping can be customized Data review tools

6 Load Signals using Menu

7 Signal Options Use Signal Options... to define how you want a chromatographic signal to look when it is loaded or reproduced.

8 Signal Options Tools Graphics Task Tool Same Scale and each in Full Scale Compound Names Retention Times Object Titles Axis Separate and Overlay of Signals Baseline Print Window Copy to Clipboard

9 Use the Signal Tool set to graphically work with your chromatogram, then select one of the following: Align the x-axis of multiple signals Align the y-axis of multiple signals Reset the alignment of your signals Create a 3D overlay of signals Mirror signals Subtract signals Integrate all chromatograms Signal Manipulation

10 Annotation Options: l Font and Style l Font Size l Color, Justification, Rotation 1.Select New Annotation. 2.Click where you want the annotation to appear. 3.Select Options. 4.Type in text. 5.Press OK.

11 Edit Annotation Add Annotation Draw Line in Window Move Annotation Delete Annotation Annotation with Tools

12 Peak Identification Compare k’ values of the unknown with standards. Spike the sample with a standard. Use a fraction collector to obtain the unknown. Use a hyphenated technique such as LC-MS. Use a diode array to compare spectra of unknowns with standards.

13 Spike Sample

14 Displaying Spectra Display Spectra

15 Displaying Spectra Selected Spectrum and Reference Spectra Resulting Background Subtracted Spectrum

16 Spectral Task Tools Select Spectrum at any time position Select Spectrum at Peak Apex Position Average a selected set of spectra Select a set of Spectra of a peak Select Spectrum to set as first reference Select Peak to display Purity Select Spectrum to set as second reference

17 Take peak spectrum Compare with library Print match factor Match: 998 Chlortoluron ? W a v e l e n g t h (nm) W a v e l e n g t h (nm) *Library Searching may be performed in an automated fashion. Spectral Libraries: Principle

18 Match Factor: Definition

19 UV spectra not very characteristic UV spectra dependent on mobile phase (pH) Non-separated compounds give wrong results Overlay of sample matrix gives wrong results No spectral libraries available UV/VIS Spectral Libraries: Current Limitations

20 Creating a Library Create a new Library in Data Analysis

21 Search Results Unknown and Library Spectrum Overlaid

22 Peak Purity Assess chromatographic purity by comparing spectra across the peak.

23 Analysis Indicates Peak Purity Threshold Curve Similarity Curve Iso-Plot of Spectra for Selected Peak 3-D Display for Selected Peak Purity Information Spectral Overlay Print Exit

24 Purity Information - Peak Pure

25 Analysis Indicates Impurity Three or more diamonds in red band Similarity Curve crosses Threshold Curve

26 Purity Information - Peak Impure

27 l Compound Specificity l Spectral Absorption of Matrix Compounds l Spectral Noise Level l Spectra Chosen for Comparison Match factor influenced by: For best results: l Use an intense lamp and clean flow cell l Select the appropriate flow cell and slit. l Collect an adequate number of spectra. l Set the peakwidth on the diode array detector screen to the width of the narrowest peak of interest. l Use a sample concentration within the linear range of the detector. Peak Purity Performance