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5 Diode Array Detector Settings That Should be Optimized

Don’t just ‘set and forget’ your detector parameters.  Let us help you make the most of your Diode Array Detector with our guide to the 5 settings that should be optimized, and our Quick Optimization Tips. 

 

1. Bandwidth

The Bandwidth parameter in Diode Array detection is related to the number of diode responses which are averaged in order to obtain a signal at a particular wavelength.  A wide bandwidth has the advantage of reducing noise by averaging over a greater diode range.  Noise is random; therefore, averaging the response over a large range of diodes will reduce noise.  As the bandwidth in increased, the signal intensity (detector sensitivity) increases as some diodes will result in a lower absorbance compared to a reading using only the single most intensive wavelength (λmax).  A wide bandwidth results in a larger range of wavelengths being averaged when producing a spectral data point, which results in a loss of spectral resolution.  A bandwidth of 10 nm is often a good starting point and will give good sensitivity and selectivity

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The effect of changing bandwidth is summarized in Table 1.

Table 1: Summary of the effect of changing bandwidth on S/N noise and spectral resolution.

 

 
 

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2. Slit Width

A narrow slit width provides improved spectral resolution for analytes which give UV spectra with enough fine detail to be useful for qualitative analysis.  For example, improved spectral resolution will increase the confidence of library matching search results when attempting to identify unknown peaks within a chromatogram.  A wide slit width allows more of the light passing through the flow cell to reach the photodiode array, hence, the signal intensity and detector sensitivity will increase.  Baseline noise will also be reduced leading to an increase in signal to noise ratio.  However, with a wider slit width the optical resolution of the spectrophotometer (its ability to distinguish between different wavelengths) diminishes.  The wavelength of light falling on each diode becomes less specific as the light becomes more diffuse.  Any photodiode receives light within a range of wavelengths determined by the slit width, and so spectral resolution decreases.

The effect of changing slit width is summarized in Table 2

Table 2: Summary of the effect of changing slit width on baseline noise and spectral resolution.

 

 

 

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3. Reference Wavelength

The reference wavelength compensates for fluctuations in lamp intensity as well as changes in the absorbance/refractive index of the background (i.e. mobile phase) during gradient elution.  During gradient elution the composition of the eluent will change and, hence, so will its refractive index.  To compensate for the change in refractive index properties a reference wavelength should always be set otherwise drifting baselines will occur.  Noise will also be reduced as the reference wavelength is moved closer to the sample signal.  Without any reference measurement all noise and variability in lamp intensity is recorded within the signal.  When using a reference signal all lamp intensity and background (mobile phase) variability is subtracted out of the signal being measured.  The closer the reference wavelength is to the sample wavelength the more effectively these background deviations are catered for and the better the detector sensitivity.  However, the reference wavelength should not be selected too close to the analyte wavelength or the signal intensity may be seriously reduced.  Choice of a proper reference wavelength can reduce variability and drift in the chromatographic baseline resulting in better signal to noise performance.

 

 

 

 

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4. Sample and Reference Settings

A diode array detector can compute and store several signals simultaneously and also manipulate the signals together in order to yield a composite or deconvoluted chromatogram.  The following signals are usually collected using diode array detectors:

  • Sample wavelength – the center of a wavelength band with the width of the sample bandwidth
  • Reference wavelength – the center of a wavelength band with the width of the reference bandwidth

The signals comprise a series of data points over time with the average absorbance in the sample wavelength band minus the average absorbance of the reference wavelength band.  An empirical method is detailed below for setting typical sample and reference wavelengths.

 

 

 

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5. Response Time

Response time describes how fast the detector signal follows a sudden change of absorbance in the flow cell.  The detector uses digital filters to adapt response time to the width of the peaks in a chromatogram.  These filters do not affect peak area or symmetry.

Decreasing the response time allows the detector to take more measurements per unit time, resulting in a larger data file and a better defined chromatogram.  Low response times are characterized by and increases in peak height (or area) as well as a noisier baseline.

Increasing response time results in averaging more data points and so reduces the noise by the square root of the number of data points.  The drawback to increasing response time is a slight loss in the peak height or area. 

Altering response time results in a compromise between how well the chromatographic signal and UV spectrum are defined (i.e. spectral resolution) and the sensitivity (as defined by the signal to noise ratio). 

Generally 20 – 25 data points across a chromatographic peak are required for accurate quantification.

 

 

 

 

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Quick Optimization Tips

Wavelength and Bandwidth
Sample Wavelength

  • A browser wavelength can be used to detect all peaks.  For example use a wavelength of 250 nm with 100 nm bandwidth.
  • For increased selectivity select a specific wavelength with a narrower bandwidth for sample and reference wavelengths.
  • For samples with high concentrations use a peak or valley in the spectrum to give the optimum linearity.

Reference Wavelength

  • A reference wavelength should be in a range where analytes show little or no absorbance.
  • A broad bandwidth should be used.  Note:  Ensure that the reference bandwidth does not obscure and analyte peaks which will affect quantitation.

Peak Width

  • Use a narrow peak of interest from the chromatogram and set the peak width close to its width.

Slit Width

  • Set the slit width to 4 nm for normal applications.
  • Use a narrow slit (1 or 2 nm) for the identification of analytes with fine spectral structures or for high concentrations.
  • To detect very low concentration analytes use a wide slit (e.g. 16 nm).  The signal used should have a bandwidth at least as wide as the slit width.

Response Time/Data Collection Rate

  • Set the response time to 1/3 of the peak width at half height of the narrowest peak of interest i.e. 0.02 min peak, 0.5 s response time.
  • Each peak should ideally be defined by at least 20 data points.  If peaks co-elute or when there is a low signal to noise ratio 40 data points per peak should be used.
  • Lower data collection rates can be used for wide peaks, and conversely, with peaks of interest that are less than a few seconds wide faster data collection rates can be used.
 
 

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