The Single Wavelength Excitation Fluorescence Detector

With the exception of the electrochemical detector, the single wavelength excitation fluorescence detector is probably the most sensitive detector generally available to LC but, as already stated, it is so at the cost of limited versatility. A simple form of the fluorescence detector excited by light from a single wavelength UV source is shown in Figure 1.

The UV excitation source is usually a low pressure mercury lamp which is comparatively inexpensive and provides relatively high intensity UV light at 253.7nm. Many substances that fluoresce will, to a lesser or greater extent, be excited by light at this wavelength. The excitation light is focused by a quartz lens, through the cell. Another lens situated normal to the incident light focuses the fluorescent light through a circular mask on to a photocell. Typically, a fixed wavelength fluorescence detector will have a minimum detectable concentration at an excitation wavelength of 254 nm of c. 1 x 109 g mL-1 and a linear dynamic range of 1 x 10~9-5 x 10~6 g mL-1. One of the disadvantages of the fluorescence detector is this rather limited linear dynamic range.

Detectors have been designed as a compromise between the expensive fluorescence spectrometer and

Figure 1 The single wavelength fluorescent detector.

Figure 2 The seperation of some amino acids by monitoring their o-phthalaldehyde derivatives with a fluorescence detector. Column: Supelcosil LC-18, 5cmx4.6mm, 5 ^m particles. Mobile phase: methanol-tetrahydrofuran-0.02mol L~1 sodium acetate (pH 5.9 with acetic acid) A, 22.5:2.5:77.5, B, 80:2.5: 17.5. 2 min at 100% (A) to 100% (B) in20min. Flow rate: 2 mLmin"1. Sample: 50-100 pmol of each derivative in solvent A. Peak identification: 1, Aspartic acid; 2, glutamic acid; 3, asparagine; 4, serine; 5, glutamine; 6, glycine; 7, threonine; 8, arginine; 9, alanine; 10, tyrosine; 11, methionine; 12, valine, 13, phenylalanine; 14, isoleucine; 15, leucine; 16, lysine. (Courtesy of Supelco Inc.)

Time (min)

Figure 2 The seperation of some amino acids by monitoring their o-phthalaldehyde derivatives with a fluorescence detector. Column: Supelcosil LC-18, 5cmx4.6mm, 5 ^m particles. Mobile phase: methanol-tetrahydrofuran-0.02mol L~1 sodium acetate (pH 5.9 with acetic acid) A, 22.5:2.5:77.5, B, 80:2.5: 17.5. 2 min at 100% (A) to 100% (B) in20min. Flow rate: 2 mLmin"1. Sample: 50-100 pmol of each derivative in solvent A. Peak identification: 1, Aspartic acid; 2, glutamic acid; 3, asparagine; 4, serine; 5, glutamine; 6, glycine; 7, threonine; 8, arginine; 9, alanine; 10, tyrosine; 11, methionine; 12, valine, 13, phenylalanine; 14, isoleucine; 15, leucine; 16, lysine. (Courtesy of Supelco Inc.)

the fixed wavelength detector. A typical example of this compromise is the fluorescence detector that utilizes the monochromator of a dispersive UV spectrometer in conjuction with light filters. It consists of a UV dispersion spectrometer fitted with a special absorption cell having reduced dimensions.

The small sensor cell ensures that the narrow peaks produced by high efficiency LC columns can be monitored without loss of chromatographic resolution. The wavelength of the excitation light is selected by the monochromator which will be within the normal UV range of the spectrometer (c. 200-360 nm). The excitation light passes through the cell and the fluorescent light, emitted at right angles to the path of the excitation light, is focused on to a photocell. Up to this point, the sensor system is very similar to that of the fixed wavelength fluorescence.

In most of these types of compromise detectors, appropriate light filters can be inserted between the sensor cell and the lens that focuses the emitted fluorescent light on to the photocell. In this way, the wavelength of the light monitored by the sensor can be selected by the choice of an appropriate filter. This, in fact, is a rather primitive way of selecting the emission wavelength. Nevertheless, the arrangement can be quite effective, and certainly eliminates the need for a second monochromator and the added cost. The use of this type of detector in monitoring the separation of the o-phthalaldehyde derivatives of some amino acids is shown in Figure 2. It is seen that a very high sensitivity is realized and the integrity of the chromatographic resolution is well maintained.

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