Integration
The UV Spectra Plot

Overview

The Spectra Plot enables the display of UV spectra. The prerequisite for the spectra plot is that the corresponding raw data is available. Raw data is generated by recording a 3D field, using a Photodiode Array Detector. Open the spectra plot from the Integration method.

• To open the spectra plot window, select Show Spectra on the View menu or click the following icon: .

The representation of a spectrum in the Spectra Plot is usually (height) normalized: The height of the spectrum is represented in percent. Thus, it is independent of the concentration (also, refer to Normalization). As a default, normalization is by the greatest relative maximum within the spectrum.

Normalization allows objectively comparing two spectra of different concentrations. If spectra of the same peak but different peak heights are overlaid, these will generally coincide despite the differences in concentration.

• Select Decorations on the View or context menu. On the Frame & Axes tab page, select which type of normalization is applied to the spectrum.

Normalized spectra representation allows you to perform the following tasks:

• Compare two spectra such as a standard and a sample

• Determine the number and position of minima and maxima, even with less distinct spectra at low concentrations

• Select an appropriate type of normalization

• Enable or disable baseline correction

• Determine exact integration limits by checking peak purity at various wavelengths and peak heights

• Identify components

Displaying Spectra of one Peak

• In the chromatogram plot, select the peak for which to display the peak spectrum (= spectrum in the peak maximum).

• If several peak heights were enabled on the Peak Spectra tab page in the Decoration dialog box of the spectra plot, the spectra of different peak heights are simultaneously displayed when you click the peak.

Displaying any Spectra of a Chromatogram

To extract any spectra of a chromatogram via mouse-click, follow the steps below:

• Select Tools on the context menu and select Spectra/I-t Plots Tool or click the corresponding icon on the Integration Toolbar. A spectra symbol is added to the mouse pointer, indicating that the mode was changed.

• Click anywhere in the chromatogram to display the corresponding spectrum.

• Repeat the operation while pressing the SHIFT key to overlay several spectra.

Displaying Spectra of Different Samples

To objectively compare spectra of different samples:

• Select Decoration on the context menu of the spectrum to open the Decoration dialog box and select the Peak Spectra tab page.

• Select the Retention time spectrum of a fixed sample option and click Browse to navigate to the sample of interest.

• Alternatively, you may also use the retention time spectrum of the last standard (= Retention time spectrum of recent standard), the reference spectrum of the peak table (= Reference spectrum in corresponding peak table), or any spectra that was found during library screening (Spectra library screening result).

Match Factor, Difference Spectra, and 1st and 2nd Derivative of Spectra

As soon as two or more spectra are represented on the spectra plot, a frequent question is the similarity between the various spectra.

The similarity is indicated by the Match Factor, the formation of difference spectra or by representing the first or second derivative of a spectrum.

• Select Decorations on the View or context menu of the spectrum, and then select the Show match check box on the Label tab page. Chromeleon returns a value for each represented spectrum specifying the match degree relative to the main spectrum (0 = no match; 1000 = perfect match).

• On Analysis tab page, select whether the difference spectrum or the first or second derivative of a spectrum shall be displayed in a second window in addition to the actual spectra.

In the case of the match factor and the difference spectrum, the question which spectrum is considered a main spectrum is especially important, as this is the basis for the comparison and for all calculations.

The main spectrum is usually the peak spectrum extracted at the retention time. If there is no peak spectrum, distinguish the following cases: If you used the Spectra/I-t Plots Tool to extract the single spectra from the chromatogram, the spectrum that was extracted first is the main spectrum. If the spectra were automatically extracted at different peak heights (see Displaying Spectra of one Peak), the spectrum with the "oldest" retention time is considered the main spectrum. When representing difference spectra, the Difference to entry indicates the basis for calculation.

Comparing a Spectrum with Spectra of an Existing Spectra Library

To clearly identify a spectrum, compare it to a reference spectrum stored in a Spectra Library.

• On the context menu, select Library Search to start the comparison. The  Spectra Library window lists all library spectra with a minimum similarity to the (normalized) spectrum. For more information about how to perform library screening, refer to Displaying and Using UV Spectra  Searching Single Reference Spectra.

If the spectra plot contains more than one starting spectrum, Chromeleon always uses the spectrum displayed first and then compares it to library spectra.