How Instrument Parameters influence Spectral absorbance?

How Instrument Parameters influence Spectral absorbance?

It is well established that instrument parameters and settings influence the absorbance measurements and appearance of absorbance spectra. Such settings have influence on the following absorbance features

  • Resolution
  • Signal intensity
  • Accuracy

In this article we shall discuss such parameters

Spectral Resolution

Spectral resolution is the ability of a spectrometer to differentiate between two closely spaced absorption peaks. In practice resolution verification is done by recording absorbance of a 0.02% toluene solution in hexane at 266 and 269 nm. The ratio of λ max at 269 and λ min at 266 nm should be greater than 1.5

Spectral resolution depends on physical slit width of instrument and the dispersion efficiency of the monochromator. Lower physical slit width will result in decrease of spectral bandwidth and consequent improvement of resolution but will result in decrease in sensitivity due to reduction of light throughput.

Spectral Bandwidth

Peak Spectral Bandwidth
Peak Spectral Bandwidth

Spectral bandwidth is peak width at half height of light leaving the monochromator. Spectral bandwidth will always be less than the physical slit width. Most instruments provide a choice of slit widths ranging from 0.5, 1.0, 1.5, 2.0 and 4.0 nm. Wide slit width should be selected for quantitation work at particularly low concentrations.

Stray Light

Non Linear Behavior of Beer Lambart Plot with Increase in Stray Light
Non Linear Behavior of Beer-Lambart Plot with Increase in Stray Light

Stray light is any light reaching the detector which lies outside the selected wavelength bandwidth. It causes decrease in absorbance and affects the linearity of the absorbance plot thereby affecting accuracy of measurement at high absorbance values

Noise

Noise is random and varies continuously due to light source or electronic component output variations. Higher the noise the lower is the S/N ratio which reduces sensitivity of measurements.

Baseline Flatness

The intensity of the light source (tungsten – halogen or deuterium lamp) is not constant over the selected wavelength. On this account the detector may not give stable output. Absorbance measurements should only be made once flat baseline is achieved.

Instrument should be capable of maintaining a stable baseline over long time periods with minimal drift.

Photometric Accuracy

Photometric accuracy refers to the closeness of measured absorbance with specified values using standard absorption filters or solutions such as Potassium dichromate and Holmium oxide.

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