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Use of UV and Diode-Array (PDA) Detectors in (U)HPLC

  • Liquid chromatography systems feature detectors chosen according to the chemistry of the analytes of interest to the user. The vast majority of detectors for (U)HPLC are light absorbing detectors which focus on ultraviolent (UV) and visible (Vis) regions of the spectrum in the 190 - 900 nanometer (nm) wavelength range and are often abbreviated UV-Vis or UV/Vis. 
  • Most analyses of organic analytes are in the ultraviolet range 190 - 350 nm. The HPLC/UV generally only measures a couple of user-selectable specific wavelengths simultaneously. For natural products analyses, it is common to monitor 220 nm and 274 nm to determine analytes of interest. So, confirmation of the specific analyte is based on its retention time in the chromatography.

What is the use of PDA detector in HPLC?
Diode-Array Detection (DAD) or Photodiode-Array Detection (PDA) is an analytical technique that can be used to determine the purity of an analyte or related impurity peak eluting during an HPLC separation.

What is the difference between diode array detector and UV detector?

UV/UV-VIS detectors
  • UV/UV-VIS detectors are most frequently used to measure components showing an absorption spectrum in the ultraviolet or visible region.
  • A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm.
  • If components are to be detected at wavelength longer than this, a UV-VIS detector is used, which employs an additional tungsten lamp (W lamp).

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  • Above Figure shows the optical system. Light from the lamp is shone onto the diffraction grating, and dispersed according to wavelength. For example, when the measurement is performed with a wavelength of 280 nm, the angle of the diffraction grating is adjusted so that 280 nm light can shine on the flow cell.  
  • By monitoring the reference light divided from the light in front of the flow cell, the difference in light intensity can be determined between the back and front of the flow cell, and this is output as absorbance.
  • A many components have an absorption in the ultraviolet or visible region. However, attention should be given to the fact that different components have a different spectrum. Components with a large molar extinction coefficient can show a large peak even in small amounts. Thus, the concentration cannot be determined from peak size. Typically, the measurement is performed at a certain fixed wavelength.
  • If all of the components of a sample are to be detected with high sensitivity, the time program function can be used to measure each component along with its maximum absorption wavelength during the analysis.

Diode array detector (DAD, PDA: Photodiode Array Detector)
  • Photodiode arrays (semiconductor devices) are used in the detection unit. A DAD detects the absorption in UV to VIS region. While a UV-VIS detector has only one sample-side light-receiving section, a DAD has multiple (1024 for L-2455/2455U) photodiode arrays to obtain information over a wide range of wavelengths at one time, which is a merit of the DAD.
  • The idea is that spectra are measured at intervals of 1 second or less during separation by HPLC with continuous eluate delivery. If the measurement is performed at a fixed wavelength, components are identified from only their retention time; thus, a minor deviation in retention time can make identification of components difficult. In such a case, the DAD can be used to identify components by a comparison of the spectrum.
  • DADs differ from UV-VIS detectors in that light from the lamps is shone directly onto the flow cell, light that passes through the flow cell is dispersed by the diffraction grating, and the amount of the dispersed light is estimated for each wavelength in the photodiode arrays.
  • Compared with a UV-VIS detector, the DAD has the following disadvantages: 
  1. Noise is large because the amount of light is small; 
  2. the DAD is also susceptible to various changes, such as lamp fluctuations, because the reference light cannot be received. 
However, the DAD has recently been improved to reduce its difference in performance from UV-VIS detectors.

  • PDA has other advantages in that the spectral profile may assist in determining an unknown peak in the chromatograms. For full confirmation, analyses should be performed by mass spectrometry. 
  • In pharmaceutical industry the PDA is often used to determine peak purity of the target compound. The absorbance spectra are compared at multiple points across the peak for similarities and differences. 

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