All the elements of the optical system, shown in Optical path, including Hg-Xe lamp, excitation condenser lens, ellipsoid excitation mirror, excitation grating, flow cell, emission condenser mirror, cut-off filter, emission slit, ellipsoid emission mirror, emission grating and photo-multiplier tube are housed in the metal casting inside the detector compartment. The fluorescence detector has grating/grating optics, enabling the selection of both excitation and emission wavelengths. The flow cell can be accessed from the front of the fluorescence detector.
The mercury-doped Hg-Xe lamp of the G7123B produces high radiant energy, especially in the UV region. In addition, there are several strongly pronounced Hg lines across the spectrum of the lamp, which make it easy to excite a fluorophore on a molecule. Distinct Hg-lines can be found at 254, 313, 365, 407, 436, 546 and 579 nm.
The Hg-Xe lamp produces a spectrum of light from 200 nm to 900 nm. The light output distribution can be expressed as a percentage in 100 nm intervals, see Lamp Energy Distribution (vendor data). The lamp can be used for 1400 hours (on-time) and needs to be replaced then, due to safety considerations.
UV light is powerful and can cause degradation of optical components over time, especially below 250 nm is significantly higher compared to visible wavelength range.
The radiation emitted by the lamp is dispersed and reflected by the excitation monochromator grating onto the cell entrance slit. The holographic concave grating is the main part of the monochromator, dispersing and reflecting the incident light. The surface contains many minute grooves, 1125 on the excitation side and 1500 on the emission side. The grating carries a blaze to show improved performance in the visible range.
The geometry of the grooves is optimized to reflect almost all of the incident light, in the 1st order and disperse it with about 70 % efficiency in the ultra-violet range. Most of the remaining 30 % of the light is reflected at zero order, with no dispersion. Dispersion of Light by a Grating illustrates the light path at the surface of the grating.
The grating is turned using a precise stepper motor, the position of the grating determining the wavelength or wavelength range of the light falling onto the flow cell. The grating can be programmed to change its position and therefore the wavelength during a run. For spectra acquisition and multi-wavelength detection, the grating performs continuous position changes.
The flow cell is a solid fused silica body with a maximum back pressure of 60 bar. Excessive back pressure will result in destruction of the cell. Operating the detector close to waste with low back pressure is recommended. In case another device (e.g. another detector, MS, fraction collector...) is installed after the Fluorescence Detector, the use of a pressure relief valve can help preventing overpressure from the Fluorescence Detector flow cell.
On the photocathode, Photo-multiplier Tube, incident photons generate electrons. These electrons are accelerated by an electrical field between several arc-shaped dynodes. Depending on the voltage difference between any pair of dynodes, an incident electron may spark-off further electrons which accelerate onto the next dynode. An avalanche effect results: finally so many electrons are generated that a current can be measured. The amplification is a function of the voltage at the dynodes and is microprocessor controlled. Amplification can be set to three levels (LOW/STANDARD/HIGH), but the majority of applications will work just fine with the STANDARD setting. With the digitized photo-multiplier signal, a very broad dynamic range can be achieved.
PMTs are designed for specific wavelength ranges. The standard PMT offers optimum sensitivity from 200 to 600 nm. In the higher wavelength range, a red-sensitive PMT can improve performance, but at the cost of a deterioration in I-to-I variation, because the factory-made EM calibration is only valid with the default PMT. Contact your Agilent representative for more details.
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