OpenLAB and EZChrom PDA Analysis

Agilent OpenLAB and
Agilent EZChrom Elite
PDA Analysis
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Contents
1 Using This Guide..............................................................................................................................1
Introduction ............................................................................................................................................................. 1
Who Should Read This Guide?............................................................................................................................. 1
2 About the PDA Software.................................................................................................................1
3 About PDA Views.............................................................................................................................1

About the PDA Toolbar.......................................................................................................................................... 2
About the 3D View ................................................................................................................................................. 2
Set the 3D Properties .................................................................................................................................... 4
Set the 3D Axis Properties ........................................................................................................................... 6
Rotate the 3D plot .......................................................................................................................................... 8
About the Contour View........................................................................................................................................ 8
Set the Contour Properties........................................................................................................................... 9
Set up the Contour Axis.............................................................................................................................. 11
About the Mixed View......................................................................................................................................... 13

Mixed View Options Button ....................................................................................................................... 13
Mixed View Actions Button ....................................................................................................................... 14
About Mixed View w/ 3D........................................................................................................................... 14
About Chromatogram View ................................................................................................................................ 14
About the Max Plot .............................................................................................................................................. 15
About the Multi-Chromatogram View .............................................................................................................. 15
About the Spectrum View................................................................................................................................... 16
Interpolate Spectrum ................................................................................................................................... 17
Export a Spectrum........................................................................................................................................ 17
Overlay Spectra............................................................................................................................................. 17
About the Peak Purity Plot.................................................................................................................................. 18
Purity View Properties................................................................................................................................. 18
Select Peak for Purity Similarity Display.................................................................................................. 19

Similarity View Properties .......................................................................................................................... 20
PDA Analysis iii

Select Peak for Purity Similarity Display.................................................................................................. 21
About the Ratio View........................................................................................................................................... 21
PDA Utilities .......................................................................................................................................................... 22
About the Spectrum Similarity Table........................................................................................................ 23
Set the Similarity Table Properties ........................................................................................................... 24
Extract a Chromatogram ............................................................................................................................. 24
4 About PDA Method Options..........................................................................................................24

Set Library Options............................................................................................................................................... 25
PDA Options Purity............................................................................................................................................... 26
PDA Options Spectrum........................................................................................................................................ 28
Set Multi-Chromatogram Options...................................................................................................................... 30
PDA Options Ratio................................................................................................................................................ 32
5 Spectral Library Search ................................................................................................................33

Define a Spectral Library ..................................................................................................................................... 33
Search the Spectral Library................................................................................................................................. 34
Choose a Spectrum to Search ................................................................................................................... 36
6 How to Perform a Manual Library Search .................................................................................37
7 How to Collect Spectra for a Library...........................................................................................37

Reports and Peak Table....................................................................................................................................... 38
8 About PDA Report Items ...............................................................................................................38

Custom Report Graph Items for PDA................................................................................................................ 38
3D Data Graph............................................................................................................................................... 38
Insert Contour Graph ................................................................................................................................... 38
9 Custom Report PDA Parameters..................................................................................................38

Create a Library Search Report .......................................................................................................................... 38
Create a Library Definition Report ..................................................................................................................... 40
Create a Purity Report.......................................................................................................................................... 40
Create a Spectrum Report................................................................................................................................... 41
10 Changes to the Peak Table for PDA Option................................................................................44
PDA Analysis iv
11 PDA Analysis and Calculations ...................................................................................................44
General ................................................................................................................................................................... 44
About Chromatograms Extracted from the Contour View ............................................................................ 45
About Multi-Chromatogram Channels.............................................................................................................. 45
About the Working Chromatogram ................................................................................................................... 46

About Spectra Extracted from the Contour View ........................................................................................... 46
About Analysis Spectra ....................................................................................................................................... 47
About Working Spectrum.................................................................................................................................... 47
Background Correction........................................................................................................................................ 47
Spectrum Interpolation........................................................................................................................................ 47
Upslope and Downslope Spectra....................................................................................................................... 48
Spectrum Operations ........................................................................................................................................... 48
Spectrum Smoothing ........................................................................................................................................... 48
Spectrum Derivatives........................................................................................................................................... 48
Library Search Calculations ................................................................................................................................ 49
General ................................................................................................................................................................... 49
Pre-Filters............................................................................................................................................................... 49
Ratio Chromatogram Calculation....................................................................................................................... 50

Similarity Calculations ......................................................................................................................................... 50
Lambda Max/Min Calculations ......................................................................................................................... 50
Peak Purity Calculations...................................................................................................................................... 51
Calculating Total Purity ....................................................................................................................................... 51
Three Point Purity................................................................................................................................................. 51
12 Index................................................................................................................................................53
PDA Analysis v
1 Using This Guide
Introduction
The PDA software provides functionality to extract and analyze spectral data from
Diode Array Detectors.
Who Should Read This Guide?

This document is designed for users of the PDA analysis software.
2 About the PDA Software

The PDA software enables you to view and analyze spectra generated with a photo
diode array detector on your LC. In order to use the PDA analysis software, you must
have control software installed for your LC that contains PDA detector capabilities.
To turn on the PDA analysis option,
1. Open an instrument configuration.
2. In the Instrument Configuration window, click Configure.
3. In the available/configured modules configuration window, click Options.
4. Select PDA and then click OK.
5. Configure a PDA detector for your LC instrument. For details on
configuration of your particular instrument, see the control help for that
instrument.
6. In the Instrument Window choose PDA Options from the Method menu to
set post-run PDA analysis parameters. Note that data acquisition
parameters for the method may need to be set in Instrument Setup
according to the requirements of the control software for your PDA
detector.
Once the PDA analysis software has been turned on, you can
• View spectra and chromatograms in a variety of formats.
• Create and search spectral libraries.
• Perform on-demand peak purity calculations and program peak purity
calculations to occur as part of analysis.
• Set up spectral filtering and extract spectra from 3D data.
• Set Multi-Chromatogram Options
• Set up chromatogram ratios.
• Create custom PDA reports.
3 About PDA Views

The PDA views provide a variety of ways to view PDA data. You can choose to display
one view at a time, or a combination of views, such as 3D and Contour or Mixed with
3D.
PDA Analysis 1
To select the view, choose PDA View from the View menu or by selecting Views from
the PDA Toolbar. A right mouse click within any of the view windows provides a
unique menu of options for that view. You must have a data file open in order to
access PDA Views.
Mixed View
3D Views
Contour View
Max Plot View
Chromatogram View
Multi-Chromatogram View
Spectrum View
Ratio View
About the PDA Toolbar

Whenever one of the PDA Views is selected, a toolbar will appear with one or more
menus and buttons that enable you to manipulate the view or perform various actions.
Click Views to select or change the current PDA View.

Click Synchronize to synchronize the axis and zoom limits with the other two plots.
When the contour plot is zoomed, the time range of the chromatogram and the
wavelength range of the spectrum plot are automatically adjusted to the same range as
the contour. When synchronized zooming has been performed, the x axis plot limits for
the chromatogram and spectrum plot are changed. To unzoom these plots to the limits
of the data, you must select Full Unzoom from the contour plot context menu.
Click Arrange to restore the view windows to default positions. Use this after you
have resized the windows by clicking and dragging the window borders.
The Overlay button appears when a Multi-chromatogram view containing multiple
chromatograms is displayed. This toggles between chromatogram overlay view and
separate chromatogram view.
Click Actions to open the Actions menu.
Click Options to open the Options menu.
About the 3D View

The 3D View provides a three-dimensional view of absorbance versus time and
wavelength. Wavelengths of appreciable absorbance and interference, which may be
invisible in a single wavelength plot, are easy to locate with the 3D View. The plot can
be elevated and rotated around its axis for display from any angle.
PDA Analysis 2
Note: During a run, the user must manually refresh this display. It does not update automatically.
Right-click inside the 3D view to display the pop-up menu that gives you access to plot
rotation and axis setup features.
3D Plot movement selections

Click on the type of plot movement you wish to use (elevate, roll, rotate, rotate
XYZ, or spin). A checkmark will appear next to the selected movement option.
Once you have selected one of the options, you will be returned to the plot and
the cursor will indicate the type of movement selected.
Hold down the left mouse button and move the cursor in the direction you wish
to move the plot. The plot will move as you move the cursor. The movement
option will remain in effect until you turn it off. When finished, click the right
mouse button, and de-select the movement option. If you wish to return to the
original view, select the Reset command.
Note: These values can be viewed or changed using the 3DPlot/Properties dialog.
PDA Analysis 3
Set the 3D Properties
To set or change the properties of the 3D data display,
7. From View menu, select PDA View.
8. Select Mixed View or one of the other views that includes 3D.
9. Do a right mouse click on the 3D view and select Properties.
10. Click the General tab to enter or change the properties for the 3D plot.
Click the Axis Setup tab to set or change the axis parameters.
Style
Select Color or Grayscale for the plot. Checking the Wire Mesh box will cause
the data to be rendered as a wire frame plot, rather than as a solid fill plot.
Colors
Range
Select how you want the coloration on the plot to appear. When Light & Dark
Range is selected, alternating light and dark bands are used. When Full
Spectrum is selected, then a continuous color spectrum is used.
PDA Analysis 4
Background
Select the color for the background of the plot.
Axes
Select the color for the plot axes.
Display
This specifies the relative quality of the displayed contour plot. Lower Display
Detail will result in faster drawing of the plot.
Print
This specifies the relative quality of the printed contour plot. Lower Print
Quality will result in faster printing.
Viewing
Use these fields to view current rotation settings, or to set them manually.
Light source
Selecting this option causes the plot to be shaded as if an external light source
were shining upon it.
Rotation
This reports the current level of rotation (front to back) in the aspect position
of the plot. When a new value is entered, the plot is redrawn to reflect the new
value upon exiting the dialog.
Elevation
This reports the current level of tilt (forward or backward) in the aspect
position of the plot. When a new value is entered, the plot is redrawn to reflect
the new value upon exiting the dialog.
Roll
This reports the current level of roll (side to side) in the aspect position of the
plot. When a new value is entered, the plot is redrawn to reflect the new value
upon exiting the dialog.
Zoom
This reports the current level of magnification in the plot. When a new value is
entered, the plot is redrawn to reflect the new value upon exiting the dialog.
Performance - Use Zoom/Rotate Bounding Box

When this box is checked, the plot will be temporarily replaced by a box during
zoom and rotation operation. When the operation is completed, the plot will be
redrawn. Checking this box will increase performance on computers with
slower graphic subsystems.
PDA Analysis 5
Set the 3D Axis Properties
To set the axis properties for the 3D plot,
1. From View menu, select PDA View.
2. Select Mixed View or one of the other views that includes 3D.
3. Do a right mouse click on the 3D view and select Properties.
4. Click the Axis Setup tab to enter or change the axis properties for the 3D plot.
PDA Analysis 6
Limits
Enter the limits for the 3D plot.
Time
Click Autoscale if you want to have the software automatically scale the time
axis to the maximum values. To enter a manual range, de-select the autoscale
box, and add your own limits, or click the Get Limits button to enter the limits
displayed on the current 3D graph.
Wavelength
Click Autoscale if you want to have the software automatically scale the
wavelength axis to the maximum values. To enter a manual range, de-select the
autoscale box, and add your own limits, or click the Get Limits button to enter
the limits displayed on the current 3D graph.
Absorbance
absorbance axis to the maximum values. To enter a manual range, de-select the
autoscale box, and add your own limits, or click the Get Limits button to enter
the limits displayed on the current 3D graph.
PDA Analysis 7
Labels
You can customize the labeling of the 3D plot using the parameters in this area.
You can select font, size, color and style using the selections provided.
Rotate the 3D plot

A right mouse click on the 3d plot provides a menu containing various rotation
options. When you select one of these rotation options, a checkmark will
appear next to the option and that rotation option will become active on the
plot.
The cursor will change to indicate the option is in effect.
While the option is active, you can move the plot in the designated way by
holding down the left mouse button and moving the mouse in the desired
direction on the plot. When you have the plot in the desired position, click the
right mouse button and turn off the option by selecting it again (checkmark
removed). If you want to return to the original plot view, click the right mouse
button and select Reset.
About the Contour View

The Contour Plot (also referred to as an Isoabsorbance Plot) provides an aerial view of
the absorbance of the sample at each wavelength versus time. The contour view
supplies quick and easy-to-assimilate information about those wavelengths at which
the sample exhibits appreciable absorbance. With contour view, it is also possible to
generate a Chromatogram View for an individual wavelength and a Spectrum View for
a given point in time.
PDA Analysis 8
Right-click inside the window to display the pop-up menu. Select Properties to display
the Contour Properties dialog box.
To generate a chromatogram view from contour view of Mixed View or Mixed View w/
3D:
1. Select PDA Views from the View menu, and then select Mixed View to
display the Contour Map, Chromatogram, and Spectrum.
11. Move the cursor to the triangle-shaped handle located on the left- hand
wavelength axis of the Contour Map and press the left mouse button.
12. Drag the cursor up or down to the desired wavelength and release the
mouse button.
13. The chromatogram associated with the specified wavelength is displayed in
the Chromatogram View.
To generate a spectrum view from contour view of Mixed View or Mixed View w/ 3D:
15. Move the cursor to the triangle-shaped handle located on the time axis of
the Contour Map and press the left mouse button.
16. Drag the cursor to the desired peak and release the mouse button
17. The Spectrum associated with the specified retention time value is
displayed in the Spectrum View.
Set the Contour Properties

To set the properties of the Contour View,
18. Do a right mouse click anywhere on the contour plot and then select
Properties.
19. Click the General tab and set or change the properties for the Contour plot.
PDA Analysis 9
Style
Select how you want the plot to appear, Grayscale or Color.
Colors
Range: Use this to select how the coloration of the plot is to be displayed. When
Light & Dark Range is selected, alternating light and dark bands are used.
When Full Spectrum is selected, then a continuous color spectrum is used.
Background: Select the color to be used for the background of the plot.
Display
This specifies the relative quality of the displayed contour plot. Less Display
Detail will result in faster rendering of the plot.
Print Quality
This specifies the relative quality of the printed contour plot. Coarse Print
Quality will result in faster printing.
Cursors
The values in these boxes reflect the current contour cursor positions. When
you change the values, the cursors on the plot will be changed when you exit
the dialog or when you click the Apply button.
PDA Analysis 10
Time
This specifies the time position (X Value) of the cursor. When a value is
entered, the X cursor is updated to the new position upon exiting the dialog.
Wavelength
This specifies the wavelength position (Y Value) of the cursor. When a value is
entered, the Y cursor is updated to the new position upon exiting the dialog.
Bandwidth
This specifies the wavelength band that will be averaged when a chromatogram
is extracted from the contour plot. The extracted chromatogram is an average
of the absorbances at each wavelength in the wavelength band. The wavelength
band is equal to the selected wavelength (see above) +/- one half of the
bandwidth.
Set up the Contour Axis

To set or change the Axis Setup of the Contour Plot,
1. Do a right mouse click anywhere on the contour plot and then select
Properties.
2. Click the Axis Setup tab and set or change the axis properties for the
Contour plot.
If the Autoscale box is selected, the software will automatically set the axis parameter.
If you want to enter the parameter manually, clear the Autoscale box, then enter the
range values, or click Get Limits to bring in the limits from the current view.
PDA Analysis 11
Time
Click Autoscale if you want to have the software automatically scale the time
axis to the maximum values. To enter a manual range, de-select the autoscale
box, and add your own limits, or click Get Limits to enter the limits displayed
on the current contour graph.
Wavelength
wavelength axis to the maximum values. To enter a manual range, de-select the
autoscale box, and add your own limits, or click Get Limits to enter the limits
displayed on the current contour graph.
Absorbance
absorbance axis to the maximum values. To enter a manual range, de-select the
autoscale box, and add your own limits, or click Get Limits to enter the limits
displayed on the current contour graph.
Labels
You can customize the labeling of the contour plot using the parameters in this
area. You can select font, size, color and style using the selections provided.
PDA Analysis 12
About the Mixed View
This view displays the contour, chromatogram and spectrum views along with the
fourth pane displaying similarity, purity and peak profile.
To zoom in on a portion of either the contour plot, chromatogram or spectrum, hold the
left mouse button down, move the mouse over the plot until the area of interest is
highlighted and release the mouse button.
To quickly move to the previous level of zoom, double-click on the plot.
To Unzoom to the full plot after multiple zooming operations, use Ctrl+Z, or use Shift +
double-click in the window, or click the right-mouse button anywhere in the window
and select Full Unzoom from the pop-up menu.
Note: You can synchronize the axis and zoom limits with the other two plots using the
Synchronize button on the PDA toolbar. When the contour plot is zoomed, the time
range of the chromatogram and the wavelength range of the spectrum plot are
automatically adjusted to the same range as the contour. When synchronized zooming
has been performed, the x axis plot limits for the chromatogram and spectrum plot are
changed. To unzoom these plots to the limits of the data, you must select Full Unzoom
from the contour plot context menu.
Mixed View Options Button
This button presents options that enable you to toggle the view for the lower right pane
of the mixed view:
• Show 3D Plot
• Show Peak Similarity and Threshold Plot
• Show Peak Purity Plot
• Show Spectrum Similarity Table
PDA Analysis 13
Mixed View Actions Button
This button presents the following actions:

Search Library…
Opens the Spectral Library Search window.
Overlay Spectra…
Allows you to select a spectrum to overlay in the Spectrum view pane.
Select Peak for Similarity/Purity Display…
Enables you to select a peak for display in the Purity view pane.
Add to Multi-Chromatogram Table…
Enables you to select a chromatogram to add to the Multi-Chromatogram table
in the PDA options.
Add Spectrum to Spectral Library

Selecting this menu item allows you to create a new library entry from the
spectrum currently displayed in the spectrum pane. The spectrum will be
added to the currently open library (opened by selecting File > Spectrum
Library > Open). A name of the form "Spectrum at time xx.xx” will be used as
the Compound Name for the new library entry.
About Mixed View w/ 3D

Select Mixed View w/ 3D from the PDA Views menu to display the contour,
chromatogram and spectrum views along with a fourth pane containing a three
dimensional plot of PDA data.
About Chromatogram View

The Chromatogram View (also called the Working Chromatogram) may be used alone
or as part of the Mixed View or Mixed View with 3D displays.
To generate a working chromatogram from the Contour plot, click and drag the Y-Axis
arrow to the wavelength of the desired chromatogram.
To generate a working chromatogram from the Spectrum plot, click and drag the X-Axis
arrow to the wavelength of the desired chromatogram.
To add a chromatogram from the Contour or Spectrum plot, click and drag the arrow
as described above to select the wavelength, and then select Actions followed by Add
to multi-chromatogram table and then click Add followed by Close.
The following operations can be performed using the working chromatogram:
To select a peak for the Similarity/Purity display, hold down the CTRL key and click
the mouse on the apex of the desired peak. This function requires that the
chromatogram has been analyzed first by clicking the Analyze button or selecting
Analyze from the Analysis menu.
Right-click within the chromatogram window to display the pop-up menu. This menu
contains the same options as the basic chromatogram window for all detector types,
and in addition enables you to overlay chromatograms from different wavelengths and
change the Gallery view.
PDA Analysis 14
About the Max Plot
To view a Max Plot
1. From the Channel Selection box on the toolbar, click the arrow and then
select Spectrum Max Plot.
A Max Plot is a chromatogram with each point plotted at its maximum absorbance. This
plot gives an indication of the appearance of the chromatogram when the wavelengths
are optimized for each peak. This plot is useful in selecting an appropriate Analysis
Channel for each peak. Note: The Max Plot is not an analysis channel and is not used
to analyze chromatographic data.
About the Multi-Chromatogram View

This view displays multiple chromatographic plots of absorbance versus time, each at a
different wavelength. This displays all of the chromatograms that were specified in the
Multi-Chromatogram tab of the PDA Options.
Click Overlay on the PDA Toolbar to toggle between separate chromatograms and
chromatograms overlaid on a single axis.
Right-click within the window to display the data system menu for chromatogram
window options.
PDA Analysis 15
About the Spectrum View
This view displays the spectrum associated with a time on the chromatogram.
To change the displayed spectrum,
1. From the View menu, select PDA View and then choose Mixed View, or
select Views from the PDA Toolbar, and then select Mixed View.
2. Drag the X-Axis arrow on the Contour plot to a time where the desired
spectrum is displayed in the Spectrum pane.
To change the working chromatogram,
1. From the Mixed View click and drag the arrow in the Spectrum pane to
the desired wavelength. The chromatogram associated with this
wavelength will be displayed in the Chromatogram pane.
Change the Spectrum Properties

Do a right mouse click in the Spectrum pane, and then select Properties to
access the standard data system trace properties dialog. This enables you to
add another trace to the view, or change scaling. It also allows you to
selectively remove overlaid traces from the view.
Spectrum Background Correction
A background correction can be performed if the current spectrum has been
extracted from an integrated peak of the working chromatogram.
To perform a spectrum background correction,
1. Do a right mouse click in the Spectrum window and then select Operations
followed by Background Correction.
2. Click the spectrum on which you wish to perform the calculation. A
background correction for the displayed spectrum will be performed based
on the working chromatogram. The background-corrected spectrum is then
displayed along with the original spectrum.
Refer to Background Correction in the PDA Analysis and Calculations
section for details on how this calculation is performed.
PDA Analysis 16
Notes:
Background Correction may only be performed once on a spectrum. If a second
background correction is attempted on an already corrected spectrum, a message box
is displayed and the operation ignored.
If the current spectrum has not been extracted from an integrated peak of the working
chromatogram, selecting this menu item will have no effect on the spectrum.
Background subtraction must always be the first operation performed on a spectrum. If
a background correction is attempted on a spectrum after another operation has
already been performed (including operation specified in the Spectrum tab of PDA
Options), a message box is displayed and the background correction request ignored.
Interpolate Spectrum
A spectrum interpolation can be performed if the current spectrum has been
extracted from an integrated peak of the working chromatogram.
To perform a spectrum background correction,
1. Do a right mouse click in the Spectrum window and then select Operations
followed by Interpolate.
2. Click the spectrum on which you wish to perform the calculation. A 10:1
interpolation will be performed on the spectrum. The new interpolated
spectrum is then displayed along with the original spectrum.
Refer to Spectrum Interpolation in the PDA Analysis and Calculations
section for details on how this calculation is performed.
Export a Spectrum
To export a spectrum,
1. In the Spectrum pane, do a right mouse click. Select Utilities followed by
Export. A check-mark will appear next to the Export command.
2. Click on the spectrum to be exported as an ASCII file.
3. The Export Spectrum As dialog box will appear. Browse to the folder
where you want to save the exported spectrum and then type a name for
the file in File Name and then click Save.
Overlay Spectra
To overlay more than one spectra in the Spectrum pane,
1. Click Actions from the PDA toolbar, followed by Overlay Spectra...
2.In the dialog box presented, enter a time for which you want the spectrum
displayed and then click Overlay, or drag the cursor on the Contour or
Chromatogram View to the time for which you want the spectrum
displayed.
To remove the spectra you have added to the view,
1. Do a right mouse click in the Spectrum pane, and then select Clear
overlays.
You can also clear selected spectra using the Trace Setup dialog accessed by doing a
right mouse click in the view and then selecting Properties.
PDA Analysis 17
About the Peak Purity Plot
To show the Peak Purity Plot in the 4th pane of the Mixed View
1. From the View menu select PDA View followed by Mixed View.
2. Click Options followed by Show Peak Purity Plot.
3. Click the Action button, and choose Select Peak for Purity/Similarity
Display to select a peak for purity display.
The Total Peak Purity view displays the purity profile for a chromatogram extracted
from the 3D data. The view displays purity information for the Max Plot chromatogram
that is displayed in the Chromatogram pane of the Mixed View. The pane will be blank
until the data has been analyzed, and a peak has been selected for the purity
calculation.
Purity View Properties

To set up the properties for the Purity View, do a right mouse click in the Purity View,
and select Properties.
PDA Analysis 18
By default, the Similarity View will use the current method settings from the Purity tab
of PDA Options. To override the method settings, click the Override method settings
box, then enter the values you wish to use.
Wavelength range
Specify the wavelength range over which the purity calculations will be
performed, for example, from 200nm to 400nm.
Wavelength Step
Specify the wavelength spacing (in nm) to be used when purity calculations are
performed.
Purity threshold
The comparison of two spectra gives a Similarity Index (SI), the closer to 1.00
number is the more similar the spectra are. The purity threshold is used to
eliminate spectra that do not match. If the SI is greater than the purity
threshold, the spectra will be considered pure. In general, a spectra with an SI
greater than 0.9900 would have a high probability of being the same as the apex
spectra. A SI greater than 0.9000 but less than 0.9900 shows some similarity
but would need to be evaluated with care and caution. SI less then 0.9000
should be considered non-similar.
Absorbance threshold
This value represents the percentage of peak height that spectra will included
in purity calculation. Spectra in sections of the peak that do not exceed this
threshold will not be included in the purity calculation. This provides a
method of eliminating spectra where the concentration of the compound is so
low that the solvent spectrum interferes.
Background compensation
Checking this box will cause spectra to be corrected for background using the
peak baseline prior to being used in the calculation of purity.
Select Peak for Purity Similarity Display

To select the reference spectrum for the purity calculation,
PDA Analysis 19
1. Make sure the data has been analyzed. (If you are not sure, click the
analyze button on the toolbar.)
2. Click Actions followed by Select Peak for Purity/Similarity Display.
3. Select a peak by holding down the CTRL button and then clicking on a peak
from the Chromatogram or Contour pane. The retention time of the
reference spectrum is displayed along with a value indicating peak purity.
4. Continue to select or change the peak. When you are finished, click Close.
Similarity View Properties

To set up the properties for the Similarity View,
1. In the Peak Similarity and Threshold Plot, do a right mouse click, and
then select Properties.
By default, the Similarity View will use the current method settings. To override the
method settings, click the Override method settings box, then enter the values you wish
to use.
Wavelength range
PDA Analysis 20
Wavelength Step
performed.
Purity threshold
but would need to be evaluated with care and caution. SI less then 0.9000
should be considered non-similar.
threshold will not be included in the purity calculation. This provides a
method of eliminating spectra where the concentration of the compound is so
low that the solvent spectrum interferes.
Select Peak for Purity Similarity Display

To select the reference spectrum for the purity calculation,
1. Make sure the data has been analyzed. (If you are not sure, click the
analyze button on the toolbar.)
2. Click Actions followed by Select Peak for Purity/Similarity Display.
3. Select a peak by holding down the CTRL button and then clicking on a peak
from the Chromatogram or Contour pane. The retention time of the
reference spectrum is displayed along with a value indicating peak purity.
4. Continue to select or change the peak. When you are finished, click Close.
About the Ratio View

To show the Ratio Plot
1. From the View menu select PDA View followed by Ratio Plot.
PDA Analysis 21
The Ratio view displays two PDA wavelength channels and the ratio of those two
channels. These may be viewed during real-time acquisition as well as during post-run
analysis. The flat tops on the ratio peaks are a preliminary indication of peak purity.
The ratio wavelengths and parameters are set in the Ratio tab of PDA Options.
The Y-axis of the ratio chromatogram is auto-scaled to 1.
Right-click within the window to display the pop-up context menu. The context menu is
the same as for a standard chromatogram graph window.
PDA Utilities
To access the Utilities menu,
1. Do a right mouse click from any PDA View and then select Utilities.
The Utilities menu allows displayed spectra to be printed, copied, saved or exported.
Some or all of the following options will be presented.
Print
Select Print to automatically print the currently displayed spectra.
Copy to Clipboard
Select Copy to Clipboard to copy the displayed spectra to the Clipboard. The
contents of the Clipboard may then be pasted into other software.
Save trace
Select Save trace to save the spectrum to a file with an .spc extension for later
inclusion in a library or report.
Export
Select Export to enable export of the item in the method.
PDA Analysis 22
About the Spectrum Similarity Table
The Spectrum Similarity table is displayed in the fourth pane of the PDA Mixed View
when the Options Mode is set to Show Spectrum Similarity Table.
To view the Spectrum Similarity Table
1. In the Mixed View, click Options.
2. Select Show Spectrum Similarity Table.
Spectra are automatically added to and deleted from this table whenever a spectrum is
added to and deleted from the Spectrum pane of the Mixed View.
Spectrum Name
This column displays identifications for each of the spectra added to the table.
For spectra extracted from the 3D data, the identification includes the time.
For spectra extracted from a chromatogram based on a peak name, the
identification includes the peak name. For spectra loaded from a file, the
identification includes the retention time.
A check mark next to the colored line for a spectrum indicates that the
spectrum will serve as the Reference Spectrum for the similarity calculation.
Spectra may be added to the table in the Mixed View by selecting the Add to
Similarity Table from the Actions button menu.
Similarity
This column displays the similarity of the spectrum on that row relative to the
Reference Spectrum. The Reference Spectrum is determined by double-clicking
on a row of the table or by highlighting a row and pressing the ‘Set Reference’
button.
Until a reference spectrum has been selected the similarity for all spectra in the
table will be zero.
Refer to the PDA Analysis and Calculations section for details on how this
calculation is performed.
Source
This column displays the source of the spectrum. (Current Data) refers to
working spectra extracted form the current 3D data. When spectra come form a
stored data file, this column will display the source filename.
PDA Analysis 23
Set Reference
Click this button will designate the spectrum in the currently highlighted row
of the table as the new reference spectrum. The similarities for all of the rows
will be recalculated based on the new reference spectrum.
Print
Click this button will output a simple text report representation of the table to
the default printer.
Properties
Click this button will display a dialog with parameters related to the
calculation of similarity.
Set the Similarity Table Properties

To set the properties for the Similarity Table
1. From the Spectrum Similarity Table, click Properties to display the table
properties dialog box.
2. Enter the wavelength range and data point spacing (in nm) for similarity
calculations.
Note: During analysis, it is possible that a portion of this wavelength range will be outside the
range of the acquired data. In that case, the wavelength range will be truncated to the limits of
the acquired data.
Extract a Chromatogram
To generate a chromatogram view (extract a working chromatogram) from contour
view of Mixed View or Mixed View w/ 3D:
1. From the View menu, select PDA View followed by Mixed View, or click
Views from the PDA Toolbar and then select Mixed View. This will
mouse button.
4. The chromatogram associated with the specified wavelength will be
displayed in the Chromatogram View.
4 About PDA Method Options

The PDA Options enable you to set up a variety of analysis options for use with a
configured photo diode array detector. To access the PDA Options for your method,
5. From the Method menu, select PDA Options...
PDA Analysis 24
6. Click the desired tab to set the parameters for data analysis.
Tab Used for
Library Enabling spectral libraries and specifying search
parameters.
Purity Entering parameters for purity calculations.
Spectrum Defining filtering and per-peak spectrum
parameters
Multi-chromatogram Enabling multi-wavelength chromatograms
Ratio Setting up ratio plots
Set Library Options

To set the PDA Library options,
1.From the Method menu, select PDA Options and then click the Library
tab.
Using this tab, you can enter library parameters for spectral library searches that will
be saved as part of your method. When you do a spectral library search "using method
parameters", these parameters will be used.
Library/Enabled
Enter the spectral library to be searched or select from available libraries by
clicking the Library field followed by clicking the file button. You can select
more than one library. To enable the library for searching, click the Enabled
box. If this list is empty, or no library is enabled, no hits will be returned when
a search is performed.
PDA Analysis 25
Search parameters
Enter the parameters to use for the search.
Wavelength range
Enter the wavelength range to search
Wavelength step
Enter a step number for the search. Larger numbers will make the search
faster, but if you use too large of a step, spectral details may not be picked up.
Max hits
Specify the number of hits that will be reported in the results of a library
search. Note that this works in conjunction with the Similarity Threshold
parameter to limit the number of hits reported.
Similarity threshold
Enter a number for threshold of similarity. The library search results will only
display matches whose similarity to the unknown exceeds this value.
Pre-filters
The options in this group allow you to specify search pre-filters that will be
performed on library spectra prior to the test for similarity. All pre-filters are
optional.
Retention time range
When values are entered in these fields, PDA limits its search to library spectra
obtained from peaks whose apex are within the specified retention time range.
This pre-filter is optional and may be left blank.
Lambda max
When one or more of these values is specified, library search will be restricted
to those library entries containing lambda max value(s) within 5nm of all of the
specified values. Entries without matching lambda max value(s) are
automatically excluded from the search (no similarity calculation is made).
Entering values for lambda max is optional.
Compound name filter
If you are searching only for spectra that contain a certain name, enter the
name here. Only spectra containing that name will be searched.
PDA Options Purity

To set the PDA Purity options,
1. From the Method menu, select PDA Options and then click the Purity tab.
The Purity tab is used to set the parameters necessary to perform on-demand peak
purity calculations and peak purity calculations that occur as part of analysis.
PDA Analysis 26
Purity Calculations
From/To – wavelength range
Step
performed.
Purity threshold
but would need to be evaluated with care and caution. SI less than 0.9000
should be considered dissimilar.
threshold will not be included in the purity calculation. This provides a method
of eliminating spectra where the concentration of the compound is so low that
the solvent spectrum interferes.
PDA Analysis 27
Per-peak spectrum calculations
Checking any of these boxes indicates that the indicated value will be
calculated on a per-peak basis during analysis. The result of this calculation
will then be available in reports and as chromatogram annotations.
Unchecking values that are not of interest will speed up analysis. If a box is unchecked
and the field appears in a run report, it will be reported as zero
Total Purity
Checking this box will cause total peak purity to be calculated on a per-peak
basis during analysis. The result of this calculation will then be available in
reports and as chromatogram annotations. If this box is unchecked and a
report contains the field, a zero will be reported.
3 Point Purity
Checking this box will cause 3-point peak purity to be calculated on a per-peak
basis during analysis. The result of this calculation will then be available in
reports and as chromatogram annotations. If this box is unchecked and a
report contains the field, a zero will be reported.
PDA Options Spectrum

To set the PDA Spectrum options,
1. From the Method menu, select PDA Options and then click the Spectrum
tab.
The Spectrum tab is used to specify the types of filtering and processing to be
performed on spectra that are extracted from the 3D data during analysis as well as to
spectra displayed in the PDA Spectrum View.
Note: The processing specified on this page is performed prior to any use of the spectra in
the software, including display, searching and reporting.
PDA Analysis 28
Spectral filtering
In this area you designate how spectral filtering (if any) will be performed.
Filtering type
Choose the type of filtering for the spectral plot. Choices are None, Smooth, 1st
Derivative, and 2nd Derivative. Selecting one of these smoothing algorithms can
remove noise from the spectrum.
Background correction
If this box is checked, then, prior to display, a correction for spectral
background is made, as follows:
1. The spectra from the baseline start and baseline stop times for the peak are
extracted from the data.
2. For each spectrum in the peak, a corresponding background spectrum is
generated by linear interpolation between the baseline start and baseline stop
spectra.
3. This background spectrum is subtracted from the original spectrum.
Note: The baseline start and stop times that are used in calculating background
compensation are based on the detected peaks for the channel currently selected.
Interpolate spectra
Select this check box to automatically perform 10:1 interpolation of spectra
displayed in the spectrum window using a cubic spline curve fit. This
interpolation is performed after the applying any spectral filtering option (1st
derivative, 2nd derivative or smooth) to the display spectrum.
Note: Interpolated spectra may not be stored or added to a library.
The peak spectrum is defined as

This specifies how the spectra extracted from the 3D data are averaged before
being exported or used in the Spectrum Report.
Spectrum Averaging is the calculation of a spectrum whose absorbance values
are the average of the absorbances.
The peak apex spectrum
When this is selected, the spectrum at the apex of the peak will be used,
without averaging.
The average of upslope/apex/downslope spectra
When this is selected, three spectra (the spectra at the upslope inflection point,
the apex and the downslope inflection point) are averaged.
The average of every n spectra across the peak
When this is selected, enter a value for n. Every nth spectrum from the peak is
extracted, beginning at the start of the peak and continuing to the end of the
peak. These extracted spectra are averaged and become the spectrum that is
used.
PDA Analysis 29
Per-peak spectrum calculations
Checking any of these boxes indicates that the indicated value will be
calculated on a per-peak basis during analysis. The result of this calculation
will then be available in reports and as chromatogram annotations.
Unchecking values that are not of interest will speed up analysis. If a box is
unchecked and the field appears in a run report, it will be reported as zero
Similarity
Checking this box causes the Peak Apex Similarity to the reference spectrum to
be calculated on a per-peak basis during analysis. The result of this calculation
will then be available in reports and as chromatogram annotations. If this box
is unchecked and a report contains the field, a zero will be reported.
Peak Apex Similarity applies only to named peaks and its use requires that a
reference spectrum be included in the peak table.
Upslope Similarity
Checking this box causes upslope similarity to be calculated on a per-peak
basis during analysis. Upslope similarity compares the peak apex spectrum to
the spectrum at the peak inflection point on the upslope side (to the left of the
apex). The result of this calculation will then be available in reports and as
chromatogram annotations. If this box is unchecked and a report contains the
field, a zero will be reported.
Downslope Similarity
Checking this box causes downslope similarity to be calculated on a per-peak
basis during analysis. Downslope similarity compares the peak apex spectrum
to the spectrum at the peak inflection point on the downslope side (to the right
of the apex). The result of this calculation will then be available in reports and
as chromatogram annotations. If this box is unchecked and a report contains
the field, a zero will be reported.
Lambda Max
Checking this box indicates causes the lambda max (wavelength at which the
highest absorbance occurs) to be computed for each peak of each multi-
chromatogram extracted from the 3D data during analysis. The result of this
calculation will then be available in reports and as chromatogram annotations.
If this box is unchecked and a report contains the field, a zero will be reported.
Lambda Max Calculation Range

This allows the user to restrict the wavelength range over which the Spectrum
Max Plot will be calculated. Default is the detector acquisition range.
Set Multi-Chromatogram Options

To set the PDA Multi-Chromatogram options,
1. From the Method menu, select PDA Options and then click the Multi-
Chromatogram tab.
The Multi-Chromatogram tab is used to specify data channels within the 3D data for
integration and quantitation. It allows you to select wavelengths to display on the
Multi-Chromatogram plot. When you select an Enabled box, enter a wavelength and
bandwidth to be used for one plot. After you have set up several wavelengths, you can
disable them temporarily by clicking the Enabled box such that the check mark is not
displayed. The Multi-Chromatogram display is selected from the View Gallery. The
PDA Analysis 30
Multi-Chromatogram view is not functional unless one or more valid wavelengths are
enabled in the Multi-Chromatogram Definition tab.
Enabled
To enable a wavelength, click the checkbox. To disable a wavelength, click the
checkbox again to remove the checkmark. Only enabled wavelengths will
appear in the multi-chromatogram view.
Wavelength
Enter the wavelength you want to view.
Bandwidth
Specify the nm range to be averaged in generating the analog signal for each
channel.
The multi-chromatogram data is calculate by averaging the data points of
chromatograms across the wavelength range of "wavelength - 1/2*bandwidth"
to "wavelength + 1/2*bandwidth".
For example, given a wavelength of 600 nm and a bandwidth of 4 nm, for each
sample time, the data points from the chromatograms at 598, 599, 600, 601 and
602 nm are added, and the sum divided by 5.
If, in the example above, the detector's range is 190 - 600 nm (inclusive), the
software only averages the points across chromatograms at 598, 599 and 600
nm (dividing by 3). The range would not be exceeded.
Subtract baseline chromatogram

Click this to enable baseline subtraction of a designated chromatogram to be
extracted from the 3D data.
PDA Analysis 31
Wavelength
Enter the wavelength at which you wish to extract a chromatogram to be used
for baseline subtraction.
Bandwidth
Specify a bandwidth range to be averaged in generating the extracted
chromatogram.
PDA Options Ratio

To set the PDA Ratio options,
1. From the Method menu, select PDA Options and then click the Ratio tab.
The Ratio tab is used to set the displays of the Ratio Plot View. The Ratio View displays
two PDA wavelength channels and the ratio of those two channels. These may be
viewed during real-time acquisition as well as during post-run analysis. The flat tops on
the ratio peaks are a preliminary indication of peak purity.
Ratio plot
These controls specify the wavelength of the ratio multi-chromatogram
channels. The extracted chromatograms will be centered about the specified
wavelength.
Channel 1, Channel 2
Wavelength
Enter the wavelength of the chromatogram channel.
Bandwidth
Enter the nm range to be averaged in generating the chromatogram channel.
Threshold
Enter the ratio threshold. The threshold is the minimum absorbance value
required in the chromatograms of both channels for calculation of the ratio. It
is a method of eliminating ratio calculation at minor absorbance values, such as
those that occur with noise. If the threshold value is not met, the ratio plot will
be zero.
PDA Analysis 32
5 Spectral Library Search
Define a Spectral Library
In order to define a library, you must have the spectra to be added saved on disk as
spectra files (.spc) or you can create a library from the spectra obtained during the
current run. To define a spectral library
1. From the File menu, select Spectral Library followed by New to create a
new spectral library. Select Open to browse for and open an existing
library to modify.
2. Click Spectrum Data Source and select Current Data, Spectrum File or
Named Peak.
If you select Current Data, a dialog box will appear where you enter the retention
time for the spectra to be added to the library.
If you select Spectrum File, browse to the spectrum file to be added to the library
and then click OK.
If you select Named Peak, chose a peak whose spectrum is to be added to the
spectral library from the dialog box presented. (The current chromatogram must be
Analyzed first in order to use this option.)
Spectrum Data Source

Click on the arrow in this field to select either Current Data, Spectrum File,
or Named Peak to be used to select the spectrum to be added to the library.
To add a spectrum from a spectrum file, the spectrum must have been
previously saved as a file with the .spc extension, for example, using the Save
Trace function accessed by doing a right mouse click in the Spectrum pane
followed by Utilities.
If you chose to enter a spectrum from the current data file, a dialog box will
appear where you must select the retention time at which you wish to select
the spectrum.
PDA Analysis 33
If you chose to enter a spectrum from a named peak, a dialog box will appear
where you must select the named peak from which the spectrum will be
extracted.
Component Name
Enter the chemical name or description of the spectrum. This field is used for
display and search pre-filter purposes.
Lambda Max From ... To

The wavelength range of the spectrum over which the software will calculate
the lambda (absorbance) max value is displayed in these columns.
Retention time
When the spectrum was extracted from a chromatographic run, the software
automatically identifies the retention time of the spectrum and enters the value
in this field. When desired, the value in this field may be edited. This field can
also be used as a search pre-filter.
Comment
Enter any descriptive information desired related to the spectrum. This field is
used only for display and documentation purposes.
Library Notes
This field is used to document relevant information about the library as a
whole, e.g., documentation about run conditions or general sample information.
Note: Fields in existing library entries may be edited by selecting the fields with the cursor.
Right-click on a row in the library table to display a pop-up menu used to cut, paste,
copy, and insert and delete lines from the library as necessary.
Search the Spectral Library

To open Spectral Library Search,
1. From Mixed View, click Actions followed by Search Library.
From here you can perform a library search on a spectrum you have selected in the
contour map, and you can also select a spectrum from the current data or stored
spectrum file to search.
PDA Analysis 34
Hits Displayed
Select the number of hits you wish to display. Hits will be displayed in order of
similarity.
Search
Select Method if you want to use the parameters from the current method for
your search. Select Quick if you want to enter or change the parameters
yourself before the search.
Display Params
Click this button if you want to display the parameters. It toggles between
display and no display. If you always use the method parameters, you may not
wish to display them when you search.
Search Now
When you click this button, a library search will be performed on the selected
spectra using the parameters, and the number of hits requested will be
displayed.
Search Spectrum
Click the arrow to select a new spectrum to search on. You can choose from the
current data, or from a stored spectrum file. The current spectra source will be
displayed.
Params
This tab contains search parameters. If you have elected to use the Method
search parameters, the parameters displayed will be those in your current
method in the Library tab of the PDA Options. If you have elected to do a
Quick search, the parameters will be default parameters.
Wavelength Range
These specify the wavelength range over which the library search will be
performed.
PDA Analysis 35
Wavelength Step
This specifies the data point spacing to be used when a library search is
performed.
Max Hits
This specifies the number of hits that will be reported in the results of a library
search. Note that this works in conjunction with the Similarity Threshold
parameter to limit the number of hits reported.
Similarity Threshold
Specifying a value for this control will cause the library search results to only
display matches whose similarity to the unknown exceeds this value. Note that
this control works in conjunction with the Max Hits parameter to limit the
number of hits reported.
Library
Displays the library from the method, or for Quick enables you to select a
library to be used.
Pre-Filters
When Quick is selected as the Search Mode, the items on this tab allow the
user to specify search pre-filters that will be performed on library spectra prior
to the test for similarity. All pre-filters are optional. If Method is selected as the
Search Mode, the items on this tab are read-only and reflect the parameters
values on the Library tab of PDA Options.
Retention Time Range
When a Retention Time Range is specified, library search will be restricted to
those library entries whose retention time is within the specified range. Entries
outside this range are automatically excluded from the search (no similarity
calculation is made). Entering a value for this is optional.
Lambda Max
When one or more of these values is specified, library search will be restricted
to those library entries containing a lambda max within 5 nm of one of the
specified values. Entries without a matching lambda max are automatically
excluded from the search (no similarity calculation is made). Entering values
for this is optional.
Compound Name Filter
When a Compound Name Filter is specified, library search will be restricted to
those library entries whose name contains the specified string as a case-
insensitive sub-string. Entries without a matching sub-string are automatically
excluded from the search (no similarity calculation is made). Entering a value
for this is optional.
Choose a Spectrum to Search
To chose a spectrum to use for the library search,

1. From the Mixed View, click Actions and then select Search Library.
2. Click Search Spectrum.
3. Select Current Data, Spectrum File, or Named Peak.
PDA Analysis 36
4. Enter the Retention Time, Spectrum File, or Named Peak to be used for
the search.
6 How to Perform a Manual Library Search

1. In the Contour pane of the Mixed View window, drag the vertical cursor to the
apex of the peak of interest to display the corresponding spectrum in the
spectrum pane.
2. Click on Actions and then click Search library… to open the Spectral Library
Search dialog box. On the toolbar Search selection, click either Method to use
the library parameters from the method, or Quick to enable you to modify the
search parameters. Before you do the search, make sure you have either
selected a library in your method, or have opened a library to do a quick
search.
3. Click Search Now to display the Library Search Results window, showing the
three closest matches in the specified library. When appropriate, click on the
>> or << button to display additional matches.
7 How to Collect Spectra for a Library

5. Perform an acquisition using a sample with known components or
standards to be placed in the library.
6. Perform an analysis using the appropriate integration parameters. (Ideally,
Spectral Background Correction would be checked in the Spectrum tab of
PDA Options.)
7. Using the Contour plot in the Mixed View window, drag the vertical cursor
to the apex of a peak to display the corresponding spectrum in the
spectrum view. See also About Spectra Extracted from the Contour View.
8. Right-click within the spectrum view to display the Spectrum pop-up menu,
select Utilities followed by Save Trace. A check-mark will appear next to
Save Trace. Click on the spectrum you wish to save. Browse to the
location where you want to save the spectrum file, and enter a file name in
the dialog box and then click Save. Repeat this procedure for each
PDA Analysis 37
spectrum to be added to the library. Files are saved automatically with the
.spc extension.
Note: To add the 1st or 2nd Derivative of a spectrum to the library, select the appropriate filter in the
Spectrum tab of PDA Options and repeat steps 3 and 4.
Reports and Peak Table
8 About PDA Report Items

When the PDA option is enabled, the following report items will be available to insert
into a custom report: Library Search Report, Library Definition Report, Purity Report,
and Spectrum Report.
Custom Report Graph Items for PDA

The Insert Graph menu has the following additional items available when the PDA
Option is enabled: 3D Data Graph, and Contour Graph.
3D Data Graph
The 3D View provides a three-dimensional view of absorbance versus time and
wavelength. Wavelengths of appreciable absorbance and interferences, which may be
invisible in a single wavelength plot, are easy to locate with the 3D View. Select this
function to automatically enter the 3D map into the report. Click the right-mouse
button within the 3D map and select Properties to display the 3D Properties dialog box
and enter appropriate changes
The 3D map can be elevated and rotated around its axis for display from any angle.
These functions work the same as in the 3D view window.
Insert Contour Graph

A data file containing PDA data may include a Contour Plot in a custom report. The
contour view (also referred to as Isoabsorbance plot) provides an aerial view of the
absorbance of the sample at each wavelength vs. time. The contour view supplies quick
and easy–to-assimilate information about those wavelengths at which the sample
exhibits appreciable absorbance.
Changing parameters for the contour graph in a custom report works exactly as in the
Contour view.
9 Custom Report PDA Parameters

A variety of PDA information can be placed in a custom report. These items are
inserted in the report by placing the cursor at the location where you want to insert the
item, then do a right mouse click to access the custom report menus.
Create a Library Search Report

To create a Library Search Report, or add a Library Search Report to your custom
report,
PDA Analysis 38
1. Click Edit Custom Report to open the custom report.
2. On the custom report, do a right mouse click and select Insert Report
followed by Library Search Report. The Library Search Report template
will be inserted at the location of the cursor in the custom report.
To modify the parameters of the report, do a right mouse click in the report table, then
select Properties.
Search Libraries for

This specifies what peaks should be searched for the report. When Apex
Spectra of Peaks is selected, the Specific Spectrum button is disabled. When
A Specific Spectrum is selected, the other controls of this group are disabled.
Apex spectra of peaks
Choose this option to enable search based on the apex of peaks detected by
criteria you select.
Detected on
Select the detection criteria for the peaks to be searched. Choices include All
Multi-Chroms, All PDA Traces, Max Spectrum Plot, or the available
wavelength channels.
Limit to named peaks only
This allows the user to restrict the peak selection (made above) to a time range.
When checked, only peaks within the specified time range will be searched
When this box is unchecked, the Time Range edit fields are disabled.
Limit Peaks to time range
This allows you to restrict the peak selection (made above) to a time range.
When checked, only peaks within the specified time range will be searched.
PDA Analysis 39
A Specific Spectrum
Select this to specify the spectrum to be searched. When this is selected, click
the button to select the spectrum source. The menu contains the following
choices: Current Data, Named File, or Spectrum Peak.
Spectral Display
The in this group specify the layout of the search results graphs.
Graph Height
This specifies the relative height of a search results graph. A value of 100%
corresponds to a standard sized graph. A larger value may be selected to
provide more a detailed plot.
Graph Properties
Pressing this button displays the graph properties dialog as found on the
standard Spectrum graph.
Create a Library Definition Report

To create a Library Definition Report, or add a Library Definition Report to your
custom report,

followed by Library Definition Report. The Library Definition Report
template will be inserted at the location of the cursor in the custom report.
To specify parameters for the report, do a right mouse click in the report table, then
select Properties. The Library Definition Report Properties dialog will appear.
Enter or select the library you wish to include in the report, then click OK.
Create a Purity Report

To create a Purity Report, or add a Purity Report to your custom report,

followed by Purity Report. The Purity Report template will be inserted at
the location of the cursor in the custom report.
Selecting this option will insert a Purity Report into your custom report. To specify
parameters for the report, do a right mouse click in the report table, then select
Properties.
PDA Analysis 40
Detected on
This allows the user to select the channel or channels from which peaks will be
detected.
This allows the user to restrict the peak selection (made above) to named
peaks.
Limit to time range
Graph
Select the type of curve you want to graph - Purity Curve or
Similarity/Threshold Curve.
Height %
This value will enable you to "zoom" the graph. A value of 100% will maximize
the curve to the page.
Graph Properties...
Pressing this button displays the graph properties dialog as found on the
standard graph.
Create a Spectrum Report

To create a Spectrum Report, or add a Spectrum Report to your custom report,

followed by Spectrum Report. The Spectrum Report template will be
inserted at the location of the cursor in the custom report.
The report will display a table showing spectra extracted from peaks of the
chromatogram. Based on the selection on the Spectrum tab of PDA Options, the report
PDA Analysis 41
will contain either the apex spectrum, an average of the upslope, apex and downslope
spectra, or several averaged spectra.
To set parameters for this report, do a right mouse click on the table, and then select
Properties.
Report peak spectra

Detected on
This allows the user to select the channel or channels from which peaks will be
detected.
When checked, only peaks within the specified time range will be searched
Limit Peaks to time range
Time Range
When the Limit to time range box is unchecked, these fields are disabled.
Report information
Checking any of these indicates that the indicated value will be calculated and
printed to the right of the graph. Unchecking values that are not of interest will
speed up analysis.
Peak Area
Checking this indicates that the peak area should be printed to the right of the
spectrum graph.
PDA Analysis 42
Lambda Max
Checking this indicates that the three largest lambda max values should be
printed to the right of the spectrum graph.
Lambda Min
Checking this indicates that the three largest lambda min values should be
printed to the right of the spectrum graph.
Total Purity
Checking this indicates that total peak purity should be printed to the right of
the spectrum graph.
3 Point Purity
Checking this indicates that 3 point peak purity should be printed to the right
of the spectrum graph.
Similarity
Checking this indicates that peak apex similarity to the reference spectrum
should be printed to the right of the spectrum graph. The use of this value
requires that a reference spectrum be included in the peak table. See the
section on Analysis and Calculations for details.
Spectral Display
The selections in this group specify the content and labeling of the spectrum
graphs.
Show Lambda Maxima
Checking this box will annotate each spectrum graph with the 3 largest lambda
max absorbance.
Show Lambda Minima
Checking this box will annotate each spectrum graph with the 3 largest lambda
minima absorbance.
Background Correction
Checking this will cause each apex spectrum to be corrected for background
using the chromatographic baseline prior to being used elsewhere in analysis.
Refer to the PDA Analysis and Calculations section for details on the formula
used.
Filtering Type
This allows the user to specify a mathematical filtering function to be
performed on all spectra extracted from the 3D data during analysis. Refer to
the Analysis and Calculations section for details on the formula used for each
filter.
Graph Height
This specifies the relative height of the spectrum graph. A value of 100%
corresponds to a standard sized graph. A larger value may be selected to
provide more a detailed plot.
Graph Properties
Click this button to display the graph properties dialog as found on the
standard Spectrum graph.
PDA Analysis 43
10 Changes to the Peak Table for PDA Option
When using the PDA option, certain columns will be added to the peak table that
enable you to analyze peaks from the PDA detector.
Detection
Select the basis for the identification of the peak. If you choose Ret Time, only
the retention time will be used for identification of the peak. If you choose Ret
Time with Spectral Confirm, the Similarity of the peak’s spectrum to that of a
designated reference spectrum will be used in addition to the retention time as
the basis of peak identification.
Spectrum
If you want Similarity to be used as a basis for peak identification, then click
on the arrow to the right of this field to specify the stored reference spectrum
to be used for comparison. During identification, this reference spectrum is
compared to the peak apex spectrum and a similarity index is computed. A
peak is considered identified if this calculated similarity index is at least the
value specified in the Similarity column of the peak table.
If Similarity is not specified as a basis for peak identification, then this field is
ignored.
Similarity
If Similarity is specified as a basis for peak identification, then this field
specifies required minimum similarity index for a peak to be considered
identified. During identification, the reference spectrum (see previous section)
is compared to the peak apex spectrum and a similarity index is computed. A
peak is considered identified if the calculated similarity index is at least the
value in this field.
If Similarity is not specified as a basis for peak identification, then this field is
ignored.
Analysis Channel
Specify which Diode Array wavelength channel is to be used for analysis of the
peak. The choices will be those specified in Instrument Setup/DAD.
11 PDA Analysis and Calculations
This section describes the various types of calculations that are related to PDA data
and how the data is analyzed.
General
In order to maintain accuracy during the application of multiple operations, all
calculations are performed using double-precision floating-point numbers.
Analysis spectra
Background correction
PDA Analysis 44
Calculating total purity
Chromatograms extracted from the 3D data
Lambda max/min calculations
Library search calculations
Multi-chromatogram channels
Peak purity calculations
Pre-filters
Ratio chromatogram calculation
Similarity calculations
Spectra extracted from the 3D data
Spectrum derivatives
Spectrum interpolation
Spectrum operations
Spectrum smoothing
Three-point purity calculations
Upslope and downslope spectra
Working chromatogram
Working spectrum
About Chromatograms Extracted from the Contour View

Two types of chromatograms may be extracted from the 3D data:
• Multi-Chromatogram Channels: One or more chromatograms defined on the
Multi-Chromatogram tab.
• Working Chromatogram: A single chromatogram extracted from the Contour
plot on the Mixed View display. (This chromatogram is displayed in the
Chromatogram View)
To generate a chromatogram view (extract a chromatogram) from contour view of
Mixed View or Mixed View w/ 3D:
mouse button.
6. The chromatogram associated with the specified wavelength is displayed in
the Chromatogram View.
About Multi-Chromatogram Channels

The following apply to multi-chromatogram channels:
PDA Analysis 45
• In the Channel Selection drop down list, each multi-chromatogram channel
have a unique identifier that includes the wavelength and bandwidth of the
channel.
• Each Multi-Chromatogram Channel have a separate Integration Events Table,
Manual Integration Table, Export Table and Performance Table associated with
it.
• All Multi-Chromatogram Channels share a common peak and group table.
Within the peak and group tables, any channels may be selected as the analysis
channel for quantitative information.
• When an analysis is performed, all multi-chromatogram channels are
automatically analyzed.
• Each multi-chromatogram channel is an average of the absorbances monitored
at each wavelength in the wavelength range. The wavelength range is equal to
the selected wavelength +/- one half of the bandwidth.
About the Working Chromatogram

The following apply to the working chromatogram:
• In the Channel Selection drop down list, a single entry is added that identifies
the channel as applying to the working chromatogram.
• This channel has a separate Integration Events Table, Manual Integration
Table, Export Table and Performance Table associated with it. These are
separate from the tables of the Multi-Chromatogram Channels (above).
• When an analysis is performed, the working chromatogram will automatically
be analyzed.
• The working chromatogram is an average of the absorbances monitored at each
wavelength in the wavelength range. The wavelength range is equal to the
selected wavelength +/- one half of the bandwidth.
About Spectra Extracted from the Contour View

Two types of spectra may be extracted from the 3D data:
• Analysis Spectra: One or more spectra automatically extracted from the 3D
data during analysis and used for peak identification, similarity and/or library
search.
• Working Spectrum: A spectrum extracted from the Contour plot on the Mixed
View display. (This chromatogram is displayed in the Spectrum View)
To generate a spectrum view (working spectrum) from the contour view of Mixed View
or Mixed View w/ 3D:
7. Select PDA Views from the View menu, then select Mixed View to
8. Move the cursor to the triangle-shaped handle located on the time axis
of the Contour Map and click the left mouse button.
9. Drag the cursor to the desired peak and release the mouse button
10. The spectrum associated with the specified retention time value is
displayed in the Spectrum View.
PDA Analysis 46
About Analysis Spectra
The following applies to the analysis spectra:
• Prior to being used elsewhere, all analysis spectra extracted from the data are
filtered according to the settings on the Spectrum tab of PDA Options.
About Working Spectrum

The following apply to the working spectrum:
• Working spectra extracted from the 3D data are never filtered according to the
settings on the Spectrum tab of PDA Options.
• The working Spectrum is not available as a trace in Custom Reports.
Background Correction
Background Correction may only be performed on a spectrum as a result of the settings
on the Spectrum tab of PDA Options, the settings on the Purity tab of PDA Options,
or by setting the properties of the Peak Purity Pane.
Background subtraction is always the first operation performed on a spectrum. If a
background correction is attempted on a spectrum after another operation has already
been performed (including an operation specified in the Spectrum tab of PDA
Options), a message box is displayed and the background correction request ignored.
Background subtraction may only be performed once on a spectrum. If a second
background correction is attempted on a spectrum after the operation has already been
performed (including the operation specified in the Spectrum tab of PDA Options), a
message box is displayed and the background correction request ignored.
A background correction is performed as follows:
1. The spectra from the baseline start and baseline stop times for the peak are
extracted from the 3D data. The Max Plot is used to determine the peak that is used.
2. For each spectrum in the peak, a corresponding background spectrum is generated
by linear interpolation between the baseline start and baseline stop spectra.
3. These background spectra are subtracted from the original spectra.
Note: While compensation for background will provide more precise purity calculations, it can slow
down re-analysis of large data files.
Spectrum Interpolation
Spectrum interpolation may be performed on a spectrum as a result of the settings on
the Spectrum tab of PDA Options or by doing a right mouse click in the Spectrum
window and then selecting Operations followed by Interpolate.
Interpolated spectra may not be stored in spectrum libraries.
The calculation is performed by doing a 10:1 interpolation of the spectrum data points
using a cubic spline curve fit. This interpolation is performed after the applying any
spectral filtering option (1st derivative, 2nd derivative or smooth) to the spectrum.
PDA Analysis 47
Upslope and Downslope Spectra
The upslope and downslope spectra of a peak are identified by calculating the second
derivative of the portion of the chromatogram containing the peak. The two times at
which the second derivative plot crosses zero are known as the inflection points. For
normal peaks (i.e. non-Negative peaks), the Upslope Spectrum is the spectrum at the
time represented by the first inflection point, while the Downslope Spectrum is the
spectrum at the time represented by the second inflection point.
Spectrum Operations
The following table describes what operations can be done on what type of spectra
Raw Spectra (no filtering Smoothed, 1st Derivative and Interpolated Spectra
or operations) 2nd Derivative Spectra
Search against Yes
Libraries
Similarity Yes
Peak Identification Yes, but generate an error if the operations that were performed on the reference
using spectral spectrum do not match the current method.
confirmation
Appear in Reports Yes Yes, but label must include what operations were
performed
Purity Yes No.
Add to Spectrum Yes No
Library
Spectrum Smoothing
Spectrum smoothing may be performed on a spectrum as a result of the settings on the
Spectrum tab of PDA Options or by doing a right mouse click in the Spectrum
window, and then selecting Operations followed by Smooth.
Smoothing may be performed repeatedly on a spectrum.
The calculation is performed by doing a 9 point Savitsky-Golay smooth on the spectrum
data points.
Spectrum Derivatives
Calculation of the 1st and 2nd derivatives of a spectrum may be performed on a
spectrum as a result of the settings on the Spectrum tab of PDA Options or by doing a
right mouse click in the Spectrum window, and then selecting Operations followed by
1st or 2nd Derivative .
Derivatives may be computed repeatedly on a spectrum.
The absorbance values of the 1st derivative of a spectrum are computed by calculating
the differences between adjacent absorbance values to create a new spectrum. The 2nd
PDA Analysis 48
derivative of a spectrum is defined as the 1st derivative of the 1st derivative of the
spectrum.
Library Search Calculations
General
During analysis, if one or more search libraries are defined on the Library tab of PDA
Options, then an automated library search is performed on every integrated peak of
every PDA analysis channel.
Note: Unless a Library Search Results object is part of the method custom report, no
automated library searching will done when analysis is performed.
In this section, a Query Spectrum is defined the unknown spectrum that is being
searched. A Reference Spectrum is defined as a spectrum from a spectrum library file.
During a search, the apex spectrum of the peak (the query spectrum) is compared to
each spectrum contained in the libraries (reference spectra) to determine the similarity
of the query spectrum to the reference spectrum. The similarity is quantified through
the calculation of a Similarity Index for each query/reference pair. The Similarity
Indices are used to generate a hit list of the best matching entries. A perfect match will
have a Similarity Index of 1.0000. Similarity indices less than 1 indicate differences in
the spectral patterns.
If the query spectrum and the reference spectrum have different wavelength ranges,
then the intersection of the two ranges is used in the similarity calculation.
If the query spectrum and the reference spectrum have different wavelength steps, then
the higher resolution spectrum is de-resolved to match the other spectrum before being
used in the similarity calculation.
Pre-Filters
A pre-filter is a criterion on a reference spectrum that must be met before that
spectrum is used in similarity calculations. One or more pre-filters may be specified in
the search parameters. When multiple pre-filters are specified, a reference spectrum
must meet all of the individual pre-filters in order to be considered for similarity
calculations.
Reference spectra that do not meet all of the pre-filter criteria are automatically
excluded from calculations and from being a candidate for a hit list. No similarity
calculation is performed on these spectra.
The following pre-filters are supported:
Retention Time Range: When a Retention Time Range is specified, a reference
spectrum is excluded unless the retention time for the
spectrum in the library is within the specified range.
Lambda Max: Up to three Lambda Max values may be specified. When one or
more Lambda Max values are specified, a reference spectrum is
excluded unless all of the specified maxima are within 5 nm of
a maxima for the spectrum in the library.
Compound Name: When a Compound Name is specified, a reference spectrum is
excluded unless the specified string is a case-insensitive sub-
string of the compound name for the spectrum in the library.
PDA Analysis 49
Ratio Chromatogram Calculation
The ratio chromatogram plot consists of data points calculated as follows:
Ratio Pt. = abs1 / sqrt(abs1 * abs1 + abs2 * abs2)
where: abs1 = the absorbance in chromatogram 1 at this wavelength
abs2 = the absorbance in chromatogram 2 at this wavelength
If the absorbance of any point in chromatogram 1or chromatogram 2 is less than the
threshold, the Ratio Pt. for that wavelength is set to 0.
Similarity Calculations
The Similarity Index (SI) compares two spectra across the wavelength range defined in
Method > PDA Setup > Library. A Library Search is performed using the Similarity
Index, determined as follows:
A spectrum is considered as an array of absorbances at each wavelength.
(a (λ1), a (λ2), ...., (a (λn))
Where a(λi) is the absorbance at λi
The spectrum can also be represented as an n-dimensional vector, with each
absorbance corresponding to one dimension of the vector, as follows:
S = (a (λ1), a (λ2), ...., (a (λn))
When two spectra are obtained from the same compound, the ratio between
corresponding elements in S1 and S2 is constant; consequently, the vectors have the
same direction. In such a case, the angle between the two vectors would be 0. As a
general rule, the greater the angle between the two vectors, the less similarity they
have. This relationship is calculated as follows:
The closer the SI value is to 1, the more similar two spectra are considered to be. Two
spectra are considered to be identical when the SI value is close to 1.
Lambda Max/Min Calculations

A lambda max (min) is defined as a local maximum (minimum) of the absorbance
values of a spectrum. The user must define the wavelength range over which the
calculation should be performed.
To compute n lambda max (min) values for a spectrum, the software will find the n
local maxima (minima) with the largest (smallest) absorbance values.
The calculation range of Lambda Min/Max is based on the X-axis settings for the graph
in the Axis Setup… context menu item of the graph.
PDA Analysis 50
Peak Purity Calculations
The purity of a peak is calculated by comparing each spectrum in the peak to the apex
spectrum using the calculation given in the Similarity Calculation section. To derive a
purity number, the peak area under the spectra whose similarity meets the purity
threshold will be divided by the total area of the peak.
Calculating Total Purity

Total purity for an integrated peak is calculated as follows:
1. The purity calculation range of the peak is determined by starting from the
peak apex and working toward peak start and peak end. Each chromatographic
point in the peak is compared to the absorbance threshold. The start and stop
values of the range are defined as the point at which the absorbance drops
below the absorbance threshold.
2. For each point in the calculation range, a similarity index is calculated by
comparing the spectrum for that point to the peak apex spectrum.
3. All spectra whose similarity index meets or exceeds the Purity Threshold will
be considered similar to the apex spectra.
4. A sum is calculated of the chromatographic area represented spectrally similar
points.
5. The Purity Index is calculated by dividing this sum by the total area over the
calculation range of the peak.
6. The purity index will have a range of between 0.000000 and 1.000000.
Three Point Purity

Three Point Purity is calculated by comparing the spectrum at the apex of a peak (Point
1) with the spectra at the up-slope (Point 2) and down-slope (Point 3) of the peak. The
up-slope spectrum is defined as the spectrum located 20% of the distance from the peak
start to the peak apex. The down-slope spectrum is defined as the spectrum located
20% of the distance from the peak end to the peak apex. These two similarity indices
are then averaged to get the 3-point purity value.
Time up = Time start + (Time apex – Time start) * 1/5
Time down = Time apex + (Time end – Time apex) * 4/5
Similarity indices are generated for the up-slope and down-slope spectra, relative to
the apex. Since spectra taken at different points along a pure peak will look identical,
they will have high similarity indices. The closer the Similarity Index is to 1.0000, the
more similar or more pure the peak is deemed to be.
While it is easy to decide that a peak with a very low Purity value (0.0000-0.8900) is
indeed not pure, it is more difficult to decide whether peaks with purity values
between 0.9000 and 0.9500 are actually contaminated. The user's judgment is required
to make this determination and may necessitate using other methods to determine
purity, such as overlaying the spectra or comparing first and second derivatives.
PDA Analysis 51
12 Index
3 Changes to the Peak Table for PDA Option. 44
3D Plot Rotation Options................................... 8 Choose a spectrum for library search........... 36
3D Properties....................................................... 4 Chromatogram View ........................................ 14
3D Properties Axis.............................................. 6 Contour Axis Setup .......................................... 11

Contour Properties ............................................. 9
A
Contour View....................................................... 8
About Analysis Spectra ................................... 47
Create a Library Definition Report ................. 40
About Chromatogram View ............................ 14
Create a Library Search Report ...................... 38
About Chromatograms Extracted from the
Contour View ................................................ 45 Create a Purity Report...................................... 40
About Mixed View w/3D ................................ 14 Custom Report Graph Items for PDA............ 38
About Multi-Chromatogram Channels.......... 45 Custom Report PDA Parameters ................... 38
About PDA Method Options........................... 24 Custom Reports................................................. 40
About Spectra Extracted from the Contour D
View................................................................ 46
Define a Spectral Library ................................. 33
About the 3D View ............................................. 2
E
About the Contour View.................................... 8
Export.................................................................. 17
About the Max Plot .......................................... 15
Extract a Chromatogram.................................. 24
About the Mixed View..................................... 13
About the PDA Option ....................................... 1 H
About the PDA Toolbar...................................... 2 How to Add Spectra to a Library.................... 33
About the Ratio View....................................... 21 How to add spectra to an existing library .... 33
About the Spectrum Similarity Table............ 23 How to Collect Spectra for a Library............. 37
About the Working Chromatogram ............... 46 How to Perform a Manual Library Search ... 37
About Working Spectrum................................ 47 How to rotate the 3D plot ................................. 8
Actions Button .................................................. 14 How to set 3D Properties.................................. 4
B I
Background Correction..............................16, 47 Interpolate Spectrum ....................................... 17
C L
Calculating Total Purity ................................... 51 Lambda Max/Min Calculations ..................... 50
Change the Spectrum Properties................... 16 Library Definition Report ................................. 40
PDA Analysis 53
Library Search.................................................... 37 Purity View...................................................19, 21
Library Search Calculations ............................ 49 Purity View Properties ..................................... 18
Library Search Report ...................................... 38 R
M Ratio Chromatogram Calculation................... 50
Max Plot ............................................................. 15 Ratio View.......................................................... 21
Mixed View ........................................................ 13 S

Mixed View Actions Button ........................... 14 Search Spectrum .............................................. 36
Mixed View Options Button ........................... 13 Search the Spectral Library............................. 34
Mixed View w/ 3D ........................................... 14 Select Peak for Purity/Similarity Display.... 19,
Multi-Chromatogram View.............................. 15 21
Set Library Options........................................... 25
O
Set Multi-chromatogram Options .................. 30
Options Button .................................................. 13
Set the 3D Axis Properties................................ 6
Overlay Spectra ................................................. 17
Set the Contour Properties ............................... 9
P
Similarity Calculations ..................................... 50
PDA Analysis and Calculations...................... 44
Similarity Table Properties.............................. 24
PDA Method Options ....................................... 24
Similarity View ............................................19, 21
PDA Option Overview ........................................ 1
Similarity View Properties............................... 20
PDA Options Library......................................... 25
Spectral Library Definition .............................. 33
PDA Options Multi-Chromatogram ............... 30
Spectral Library Search.................................... 34
PDA Options Purity........................................... 26
Spectrum ............................................................ 28
PDA Options Spectrum.................................... 28
Spectrum background correction .................. 16
PDA Options X Y Ratio..................................... 32
Spectrum Derivatives....................................... 48
PDA Options X Y Ratio and Max Plot ........... 32
Spectrum Information ...................................... 36
PDA Report Items ............................................. 38
Spectrum Interpolation.................................... 47
PDA Utilities ...................................................... 22
Spectrum Operations ....................................... 48
PDA Views ........................................................... 1
Spectrum Properties ........................................ 16
Peak Purity Calculations.................................. 51
Spectrum Report ............................................... 41
Peak Purity Plot................................................. 18
Spectrum Similarity Table ............................... 23
Pre-Filters........................................................... 49
Spectrum Smoothing ....................................... 48
Purity................................................................... 26
Spectrum View.................................................. 16
Purity Report...................................................... 40
Purity Report Properties .................................. 40
PDA Analysis 54
T U
Three Point Purity............................................. 51 Upslope and Downslope Spectra................... 48

Total Purity......................................................... 51 X
XY Ratio .............................................................. 32
PDA Analysis 55

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Who Should Read This Guide?............................................................................................................................. 1

2 About the PDA Software.................................................................................................................1

3 About PDA Views.............................................................................................................................1

About the 3D View ................................................................................................................................................. 2

Set the 3D Properties .................................................................................................................................... 4

Set the 3D Axis Properties ........................................................................................................................... 6

Rotate the 3D plot .......................................................................................................................................... 8

About the Contour View........................................................................................................................................ 8

Set the Contour Properties........................................................................................................................... 9

Set up the Contour Axis.............................................................................................................................. 11

About the Mixed View......................................................................................................................................... 13

Mixed View Actions Button ....................................................................................................................... 14

About Mixed View w/ 3D........................................................................................................................... 14

About Chromatogram View ................................................................................................................................ 14

About the Max Plot .............................................................................................................................................. 15

About the Multi-Chromatogram View .............................................................................................................. 15

About the Spectrum View................................................................................................................................... 16

Interpolate Spectrum ................................................................................................................................... 17

About the Peak Purity Plot.................................................................................................................................. 18

Purity View Properties................................................................................................................................. 18

Select Peak for Purity Similarity Display.................................................................................................. 19

PDA Analysis iii

About the Ratio View........................................................................................................................................... 21

PDA Utilities .......................................................................................................................................................... 22

About the Spectrum Similarity Table........................................................................................................ 23

Set the Similarity Table Properties ........................................................................................................... 24

Extract a Chromatogram ............................................................................................................................. 24

4 About PDA Method Options..........................................................................................................24

PDA Options Purity............................................................................................................................................... 26

PDA Options Spectrum........................................................................................................................................ 28

Set Multi-Chromatogram Options...................................................................................................................... 30

PDA Options Ratio................................................................................................................................................ 32

5 Spectral Library Search ................................................................................................................33

Choose a Spectrum to Search ................................................................................................................... 36

6 How to Perform a Manual Library Search .................................................................................37

7 How to Collect Spectra for a Library...........................................................................................37

8 About PDA Report Items ...............................................................................................................38

Insert Contour Graph ................................................................................................................................... 38

9 Custom Report PDA Parameters..................................................................................................38

Create a Purity Report.......................................................................................................................................... 40

Create a Spectrum Report................................................................................................................................... 41

10 Changes to the Peak Table for PDA Option................................................................................44

About Multi-Chromatogram Channels.............................................................................................................. 45

About the Working Chromatogram ................................................................................................................... 46

About Analysis Spectra ....................................................................................................................................... 47

About Working Spectrum.................................................................................................................................... 47

Upslope and Downslope Spectra....................................................................................................................... 48

Spectrum Operations ........................................................................................................................................... 48

Spectrum Smoothing ........................................................................................................................................... 48

Library Search Calculations ................................................................................................................................ 49

Ratio Chromatogram Calculation....................................................................................................................... 50

Lambda Max/Min Calculations ......................................................................................................................... 50

Peak Purity Calculations...................................................................................................................................... 51

Calculating Total Purity ....................................................................................................................................... 51

Three Point Purity................................................................................................................................................. 51

Who Should Read This Guide?

2 About the PDA Software

3 About PDA Views