Optimizing DAD Aquisition Method Settings

This Information Applies To: Agilent LC Systems with DAD (diode array detector)

Issue

The detector settings may change the quality of the data results. This article discusses the influence of DAD acquisition method settings for Agilent diode array detectors. 

Resolution

The following settings can be adjusted depending on the analytical method needs. 

• Data acquisition
• Bandwidth
• Reference wavelength and bandwidth
• Wavelength
• Step setting
• Peak suppression

Data acquisition rate:

The data acquisition frequency determines the number of data points collected and is expressed in Hertz. Higher frequency will result in more data points, increased peak resolution, increased background noise and large data file sizes. Figure 1 shows the influence of data acquisition frequency on peak aspect. Data acquisitions at high frequency give sharper peak shape, while peak height remains approximately the same. Figure 2 shows the influence of data acquisition frequency on background noise. Data acquisitions at high frequency have less filtering, increasing the baseline noise. 

Figure 1: Influence of data acquisition rate on peak aspect
Blue signal = 80 Hz, highest data rate, Green signal = 5 Hz, Red signal = 0.31 Hz, lowest data rate 

Figure 2: Influence of data acquisition data on baseline (zoom-in)
Blue signal = 80 Hz, highest data rate, Green signal = 5 Hz, Red signal = 0.31 Hz, lowest data rate

Bandwidth:

The bandwidth is range of wavelengths detected either side of the target wavelength. For example, a bandwidth setting of 4 nm on a 250 nm wavelength setting will detect wavelengths 248, 249, 250, 251 and 252 and averages the results. Narrow bandwidth increases selectivity, and uses a unique wavelength for the target analyte. Large bandwidth results in lower peak and background noise responses, which change the signal-to-noise ratio, and can improve sensitivity. Figure 3 shows the effect of bandwidth on peak aspect. The ideal bandwidth is determined as the range of wavelength at 50% of the spectral feature being used for the determination.

Figure 3: Influence of bandwidth on peak aspect
Bandwidth splits set at 2, 10, 30, 60, 100 nm.

Reference wavelength and bandwidth:

The reference wavelength is used to compensate for fluctuations in lamp intensity. Causes include lamp fluctuations, background absorbance changes during gradient elution (i.e. mobile phase changes) and negative peaks where the sample absorbance is greater than the reference absorbance. 

Use the Isoabsorbance plot feature to optimization reference wavelength selection. 

Wavelength:

The wavelength used for each analyte affects sensitivity according to the extinction coefficient of the substance; it varies with the wavelength and impacts the measured intensity (Lambert-Beer's law). The chosen wavelength is one where the compound absorbs strongly. If the sample has several compounds with different absorbance maximum, then choose one where there will be reasonable absorbances for each component. Alternatively, use several signals with a wavelength maximum for each component. The drawback is that multiple signals will need to be analyzed.

Figure 4 shows the difference in response with different spectral wavelengths for the same compound. Some wavelengths can saturate and cause signal overload and distort the peak shape and interfere with quantitative results. To avoid this problem, either decrease concentration of the sample, or use a different wavelength.

Figure 4: Influence of wavelength on peak intensity

Step setting:

The step setting is jump between wavelengths when measuring a range of wavelengths. Lower step settings mean smoother peaks due to the higher number of data points. Figure 5 and Figure 6 show the effect of step setting on wavelength data acquired between 190 – 400 nm. Figure 5 shows a step setting of 1 nm, which makes a smooth peak shape. Data is measured at every wavelength, and it provides the best resolution, but the largest file size. This spectra setting is especially useful if doing investigative work or to extract chromatograms from an isoabsorbance plot. Figure 6 shows a step setting of 8 nm, which is not enough data points to draw the curve.

Figure 5: 1 nm step size

Figure 6: 8 nm step size

 Peak suppression:

Peak suppression allows a parasite peak signal from a known compound to be suppressed from the chromatogram. It requires a reference wavelength with the same wavelength of this known compound. Figure 7 shows the influence of the reference wavelength when it is used for peak suppression. 

Figure 7. Peak suppression example. Unresolved hydrochlorothiazide and caffeine chromatograms
1. No signal suppression. Wavelength 204 nm, Reference wavelength: None.
2. No signal suppression. Wavelength 222 nm, Reference wavelength: None. 
3. Hydrochlorothiazide signal suppressed. Wavelength 204 nm, Reference wavelength: 260 nm
4. Caffeine signal suppressed. Wavelength 222 nm, Reference wavelength: 282 nm

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