Qualitative Data Analysis
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.
2
Preferences – Signal Options
3
Display Files in Navigation Table
Double-click on sequence or Single Runs
to display associated data files.
4
Load Signals using Navigation Table
Turn on/off
Navigation
Table
Right click
the mouse
anywhere on
the row to
activate
menu
Click on the
‘+’ to activate
signal details
Data review tools
Grouping can be customized
5
Load Signals using Menu
6
Signal Options
Use Signal Options... to define how you want a chromatographic
signal to look when it is loaded or reproduced.
7
Signal Options Tools
Graphics Task Tool
Separate and
Overlay of Signals
Compound Names
Same Scale and
each in Full
Scale
Retention Times
Baseline
Object Titles
Axis
Print Window
Copy to
Clipboard
8
Signal Manipulation
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
9
Annotation
1. Select New Annotation.
2. Click where you want
the annotation to appear.
3. Select Options.
4. Type in text.
5. Press OK.
Options:
Font and Style
Font Size
Color, Justification, Rotation
10
Annotation with Tools
Edit Annotation
Add Annotation
Draw Line in Window
Move Annotation
Delete Annotation
11
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.
12
Spike Sample
* Spiked with Pure Standard of Insulin
* Insulin?
Identification
0
7
10
15
20
25
30 min.
0
0
7
10
*
Not Insulin
7
10
15
15
20
25
30 min.
20
25
30 min.
13
Displaying Spectra
Display Spectra
14
Displaying Spectra
Resulting Background
Subtracted Spectrum
Selected Spectrum
and Reference Spectra
15
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 Peak to display Purity
Select Spectrum to set as first
reference
Select Spectrum to set as second reference
16
Spectral Libraries: Principle
Chlortoluron ?
Take peak spectrum
Print match factor
Match: 998
Compare with library
250
300
W a v e l e n g t h (nm)
250
300
W a v e l e n g t h (nm)
*Library Searching may be performed in an automated fashion.
17
Match Factor: Definition
1
0
0
1
2
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8
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l
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t
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a
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8
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6
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4
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Standard(mAu)
Ndormalize
4
0
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n
k
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2
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(
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18
UV/VIS Spectral Libraries: Current Limitations
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
19
Creating a Library
Create a new
Library in
Data Analysis
20
Search Results
Unknown and
Library Spectrum
Overlaid
21
Peak Purity
Assess chromatographic purity by comparing spectra across the peak.
22
Analysis Indicates Peak Purity
Threshold Curve
Spectral
Overlay
Similarity Curve
Print
Purity
Information
Iso-Plot of Spectra
for Selected Peak
Exit
3-D Display for
Selected Peak
23
Purity Information - Peak Pure
24
Analysis Indicates Impurity
Similarity
Curve crosses
Threshold Curve
Three or
more diamonds
in red band
25
Purity Information - Peak Impure
26
Peak Purity Performance
Match factor influenced by:




Compound Specificity
Spectral Absorption of Matrix Compounds
Spectral Noise Level
Spectra Chosen for Comparison
For best results:





Use an intense lamp and clean flow cell
Select the appropriate flow cell and slit.
Collect an adequate number of spectra.
Set the peakwidth on the diode array detector screen to the
width of the narrowest peak of interest.
Use a sample concentration within the linear range of the
detector.
27