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The 2487 Detector operates in single or dual wavelength mode, allows spectrum scanning using a flow cell or a cuvette, and provides RatioPlot, difference plot, and MaxPlot functions.

1.3.1 Single Wavelength Mode

Single wavelength is the default mode of operation for the 2487 Detector. The 2487 Detector supports monitoring of a single wavelength from 190 nm to 700 nm, settable in 1-nm increments on Channel A. You can configure the analog outputs for Channel B while the detector is operating in single wavelength mode, so you can use Channel B to obtain additional information about the wavelength selected on Channel A.

In single wavelength mode, the 2487 Detector automatically engages the second order filter for wavelengths 370 nm and above and removes it for wavelengths under 370 nm. The second order filter is an optical filter that blocks unwanted ultraviolet (UV) light produced by the diffraction grating that can interfere with absorbance detection above 370 nm.

You can configure several additional parameters when using the 2487 Detector in single wavelength mode.

Primary Parameters

The following are the major parameters you can use in single wavelength mode:

• Wavelength in nm – You can specify a wavelength for Channel A from 190 to 700 nm settable in 1-nm increments.

• Chart type selection – In single wavelength mode, you are limited to the following selections:

– Absorbance: The detector scales absorbance in real time and sends it out

over the analog output. Scaling depends on the AUFS setting and the optional chart zero parameter.

– Log absorbance: The detector can plot the Log (base 10) of absorbance

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– Reference energy: You can select reference energy for charting out the

analog output. Scaling for reference energy is fixed at 150 nA/V.

• Sensitivity in AUFS – This value specifies the scaling factor for the analog output channels and corresponds to the AU value where the analog outputs saturate at full scale values. AUFS varies from 0.0001 to 4.000 AU. Sensitivity is ignored if you select Log absorbance.

• Chart polarity (+ or –) – You can use the polarity to reverse the polarity of the charted chromatogram. Select + for a normal chromatogram, or – for an inverted chromatogram. This function changes the direction of the plot on both the 2 V and 10 mV outputs, similar to reversing the leads to an external chart recorder.

• Filter time constant in seconds – You can program a filter time in seconds. Selecting 0.0 disables all filtering.

Secondary Parameters

When on the Absorbance (or HOME) screen in single wavelength mode, pressing Next

brings you to several pages of secondary, or less frequently used, parameters. They are: • Absorbance offset

• Autozero on inject • Autozero on λ changes

• Chart mark on keypad/panel active • Autozero on keypad/panel active

Section 3.2.4, Primary and Secondary Functions, and Table 3-3 explain the functions, ranges, and defaults of these parameters.

1.3.2 Dual Wavelength Mode

In dual wavelength mode, the detector can monitor two wavelengths, one on Channel A and one on Channel B. The sampling frequency is reduced from 10 to 1 Hz, limiting use of this mode to more standard chromatography where peaks span at least 20 seconds to enable full characterization of a peak. You can use dual wavelength mode to obtain additional information about an analyte by running a RatioPlot or a MaxPlot.

The 2487 Detector allows you to select any two wavelengths from 190 to 700 nm.

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Attention: Changing the sensitivity (AUFS) setting affects both the 2 V and 10 mV outputs.

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Chart Out Selection Modes

When operating in dual wavelength mode, the 2487 Detector offers these choices for analog output in addition to the selections offered in single wavelength mode and explained in Section 1.3.1, Single Wavelength Mode. The default selection for dual wavelength mode is Absorbance.

• Absorbance (A and B) – This is the standard LC mode where the current absorption is scaled and sent directly out the analog output. The scaling is dependent upon the AUFS setting and the absorbance offset. The absorbance value is scaled for both the 2 V and 10 mV analog outputs. If a setting of 1 AU/V is desired, you can set an AUFS of 2.0000 for either the A or B output channels which can be controlled independently, even in single wavelength mode.

• Log Absorbance (A and B) – This mode allows the Log (base 10) of absorbance to be plotted allowing both small and large peaks to be viewed on a single

chromatographic output. See Section 1.3.1, Single Wavelength Mode.

• MaxPlot – This mode results in the output of the larger of the two absorbance values, scaled to the selected AUFS setting. This mode is useful when looking at multiple compounds with absorbancies at two separate wavelengths with one data channel.

• RatioPlot (A/B or B/A) – This mode produces the ratio of absorbance from two wavelengths. Theoretically, the ratio is constant for a pure chromatographic peak and variable for an impure peak. This results in a nonsquared response. Instead of a programmable AUFS, the 2487 Detector provides minimum and maximum ratio

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Attention: In dual wavelength mode:

• If both selected wavelengths are greater than 370 nm, the detector applies the second order filter to block unwanted UV light.

• If both selected wavelengths are less than or equal to 370 nm, the detector removes the second order filter.

• If the selected wavelengths bracket the 370 nm threshold, the detector does not apply the second order filter and issues a warning message that any data collected for the wavelength above 370 nm may contain inaccuracies because of possible UV light interference (second order effects).

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Attention: To prevent acquisition of invalid data when operating the 2487 Detector in dual wavelength mode under the control of the Millennium³² software or a PowerLine system, you must select a data sampling rate of 1 point per second.

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• Difference Plot (A-B or B-A) – This mode plots the arithmetic difference in absorbance for the two monitored wavelengths.

1.3.3 Spectrum Scanning

You can use the 2487 Detector as a spectrophotometer to acquire spectra from either the flow cell or the cuvette. You may scan and store up to five spectra (five reference or zero scans, and five sample scans) in memory for playback or to compare with other spectra. The major difference between the 2487 Detector and a double-beam spectrophotometer is that the 2487 Detector employs only one flow cell or cuvette, rather than a simultaneous sample and a reference pair.

Note: To prevent contamination when scanning using the cuvette, you should use a matched pair of cuvettes for the zero and sample scans.

The 2487 Detector obtains an absorbance spectrum by performing two types of scan on either the flow cell or using the cuvette (see Section 1.3.4, Cuvette Operations):

• Zero scan – A zero scan characterizes the baseline absorbance spectrum of a solvent.

• Sample scan – A sample scan subtracts the zero scan, so the results displayed or charted are of the sample only.

To obtain a spectrum of a sample with the 2487 Detector, you need to run a zero scan first, followed by a sample scan. Typically, the zero scan is run with pure solvent and the sample scan is of the analyte dissolved in that solvent.

Spectra can be simultaneously charted on the Channel A output, or acquired and stored in memory for later playback.

See Section 3.4.7, Scanning Using the Cuvette, and Section 3.4.8, Scanning Using a Static Flow Cell, for detailed procedures.

1.3.4 Cuvette Operations

The 2487 Detector cuvette option is used to measure the absorbance spectrum of a sample in a cuvette. To generate and store a spectrum, you need to acquire:

1. A zero scan, which measures the absorbance of the contents of the cuvette and flow cell over the desired wavelength range.

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Attention: When the 2487 Detector is operating under the control of the MIllennium³² software, the scanning function is disabled.

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2. A sample (absorbance) scan, which measures the absorbance of the analyte dissolved in mobile phase.

The 2487 Detector subtracts the zero scan from the sample scan to create a sample spectrum.

Since the cuvette scan is acquired by measuring the absorbance from a light path that includes both the flow cell and the cuvette, the solvent conditions in the flow cell should be identical for both scans. For a detailed explanation of cuvette scanning, see Section 3.4.7, Scanning Using the Cuvette.

1.3.5 RatioPlot

The 2487 Detector allows ratio plotting, that is, comparing the absorbances of a

compound or analyte at two different wavelengths. The RatioPlot divides absorbances at two selected wavelengths, and plots the resulting ratio on a chart recorder or data system over one output channel (Channel A). The RatioPlot is useful in detecting hidden

components present within individual peaks.

The RatioPlot of a spectrally homogeneous peak appears as a rectangular wave. The RatioPlot of an impure peak appears as a distorted wave. When obtaining a RatioPlot, you must be operating the 2487 Detector in dual wavelength mode; the RatioPlot is output on the selected channel.

For an explanation of the RatioPlot procedure, see the discussion “Obtaining a RatioPlot”

in Section 3.3.5, Operating the Detector in Dual Wavelength Mode.

1.3.6 MaxPlot

The 2487 Detector allows you to obtain a MaxPlot while in dual wavelength mode. The MaxPlot function monitors absorbance at two selected wavelengths and plots the maximum absorbance value for each sample component.

When obtaining a MaxPlot, you must be operating the 2487 Detector in dual wavelength mode. The MaxPlot outputs the greater of the two absorbance values on the selected channel.

For an explanation of the maxplot procedure, see the discussion “Obtaining a MaxPlot” in

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Installing the 2487 Detector