The Multiwavelength Fluorescence Detector

One form of multi-wavelength fluorescence detector consists of two monochromators: the first selects the wavelength of the excitation light, and the second disperses the fluorescent light, and provides a fluorescence spectrum, or allows the separation to be monitored at a selected fluorescence wavelength. The multi-wavelength fluorescence detector is shown in Figure 3. The detector comprises a fluorescent spectrometer fitted with a suitable absorption cell that can be used with high efficiency LC columns without degrading the resolution of the column. The spectrometer involves two distinctly different light systems. The function of the detector is easier to understand if the different light systems and the respective light paths are considered separately. The detector

Figure 3 The fluorescence spectrometer detector.

excitation light;

fluorescent light.

Figure 3 The fluorescence spectrometer detector.

excitation light;

fluorescent light.

comprises an excitation light system and fluorescent light system.

The excitation source (emitting light over a wide wavelength range, such as a deuterium lamp) is situated at the focal point of an ellipsoidal mirror, shown at the top left-hand corner of the diagram. The parallel beam of light is collimated to fall on to a toroidal mirror, which then focuses it on to the grating, shown on the left-hand side of the diagram. This grating is used to select the wavelength of the excitation light or it can be used to scan the complete range of excitation wavelengths and provide a corresponding excitation spectrum that is monitored at a specific fluorescent wavelength. The selected wavelength then passes to a spherical mirror and then to a ellipsoidal mirror, shown at the base of the diagram, which focuses it on to the sample. The excitation light path is mostly depicted on the left-hand side of the diagram.

In the centre of the diagram, between the spherical mirror and the ellipsoidal mirror, is a beam splitter that diverts a portion of the incident light on to another toroidal mirror. This mirror focuses the light on to the reference photo cell. The reference photo cell provides an output that is proportional to the intensity of the excitation light. The path of the fluor escent light is depicted on the right-hand side of the diagram. Fluorescent light, emitted from the cell, is focused by an ellipsoidal mirror on to a spherical mirror at the top right-hand side of the diagram. This mirror focuses the light on to a grating which is situated at about centre right of the diagram. This grating selects a specific wavelength of the fluorescent light to monitor, or can scan the fluorescent light produced by excitation light of a given and selected wavelength, and provide a fluorescent spectrum. Fluorescent light from the grating passes to a photoelectric cell which monitors the intensity. The instrument is complex and relatively expensive; however, for measuring fluorescence, it is extremely versatile.

The optical system allows the wavelength of the excitation light and that of the fluorescent light to be chosen to provide the maximum selectivity for a given solute or its fluorescent derivative. The use of this optimization procedure is demonstrated by the high sensitivity detection of the fluoropa derivative of neomycin shown in Figure 4. It is an excellent example of the selection of a specific excitation light wavelength and the complementary emission light wavelength to provide maximum sensitivity.

Figure 4 Detection of neomycin OPA derivative at an excitation wavelength of 365 nm and an emission wavelength of 418 nm. Column: Supelcosil LC-8,15 cm x 4.6 mm, 5 ^m particles. Mobile phase: tetrahydrofuran: 0.0056 mol L~1 sodium sulfate-0.007 mol L~1 acetic acid-0.01 mol L~1 pentanesulfonate, 3:97. Flow rate: 1.75 mL min~1. Post-column reagent: 1 L 0.4 mol L~1 boric acid-0.38 mol L~1 potassium hydroxide containing 6 mL 40% Brij-35, 4mL mercaptoethanol, 0.8 g o-phthalaldehyde. Flow rate 0.4mLmin~1. Mixer 5 cm x 4.6 mm column packed with glass beads. Reactor 10 ft x 0.5 mm knitted Teflon capillary tubing. Reaction temperature 40°C. Sample: 20 mL of a mobilephase extract of a commercial sample. Excitation wavelength 365 nm; emission wavelength 418 nm. (Courtesy of Supelco Inc.)

Figure 4 Detection of neomycin OPA derivative at an excitation wavelength of 365 nm and an emission wavelength of 418 nm. Column: Supelcosil LC-8,15 cm x 4.6 mm, 5 ^m particles. Mobile phase: tetrahydrofuran: 0.0056 mol L~1 sodium sulfate-0.007 mol L~1 acetic acid-0.01 mol L~1 pentanesulfonate, 3:97. Flow rate: 1.75 mL min~1. Post-column reagent: 1 L 0.4 mol L~1 boric acid-0.38 mol L~1 potassium hydroxide containing 6 mL 40% Brij-35, 4mL mercaptoethanol, 0.8 g o-phthalaldehyde. Flow rate 0.4mLmin~1. Mixer 5 cm x 4.6 mm column packed with glass beads. Reactor 10 ft x 0.5 mm knitted Teflon capillary tubing. Reaction temperature 40°C. Sample: 20 mL of a mobilephase extract of a commercial sample. Excitation wavelength 365 nm; emission wavelength 418 nm. (Courtesy of Supelco Inc.)

The principle of optimizing excitation and emission light wavelengths to obtain maximum sensitivity for a multi-component mixture can be quite complex, as shown by the separation of some priority pollutants depicted in Figure 5. The separation was carried out on a column which was 25 cm long, 4.6 mm in diameter and packed with a C18 reversed phase. The mobile phase was programmed from 93: 7 acetonitrile-water to 99 : 1 acetonitrile-water over a period of 30 min. The gradient was linear and the flow rate was 1.3mLmin_1. All the solutes were separated and the compounds, numbered from the left, are given in Table 1. The separation illustrates the clever use of wavelength programming to obtain the maximum sensitivity. The programme used is shown in Table 1.

The wavelength of the excitation light and that of the emission light was changed during chromato-graphic development to provide optimum fluorescent conditions, and thus maximum sensitivity, for each solute. This ensured that each solute, as it was eluted, was excited at the most energetic wavelength and then monitored at the strongest fluorescent wavelength.

It is seen that the analysis involves a somewhat elaborate wavelength programme; nevertheless, if the analysis is sufficiently important, it is readily justified. The system also provides fluorescence and excitation spectra, by arresting the flow of mobile phase when the solute resides in the detecting cell, and scanning the excitation and/or fluorescent light. (This is the

Figure 5 Separation of a series of priority pollutants with programmed fluorescence detection. 1, Naphthalene; 2, acenaphthene; 3, fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene; 7, pyrene; 8, benz(a)anthracene; 9, chrysene; 10, benzo(b)fluoranthene; 11, benzo(k)fluoranthene; 12, benzo(a)pyrene; 13, dibenz(a,h)anthracene; 14, benzo(g/7/)perylene; 15, indeno(123-cd)pyrene. (Courtesy of the Perkin Elmer Corporation.)

Figure 5 Separation of a series of priority pollutants with programmed fluorescence detection. 1, Naphthalene; 2, acenaphthene; 3, fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene; 7, pyrene; 8, benz(a)anthracene; 9, chrysene; 10, benzo(b)fluoranthene; 11, benzo(k)fluoranthene; 12, benzo(a)pyrene; 13, dibenz(a,h)anthracene; 14, benzo(g/7/)perylene; 15, indeno(123-cd)pyrene. (Courtesy of the Perkin Elmer Corporation.)

Table 1

Fluorescence detector programme

Time (s)

Wavelength of

Wavelength of

excitation light (nm)

emitted light (nm)

0

280

340

220

290

320

340

250

385

510

260

420

720

265

380

1050

290

430

1620

300

500

same technique as that used to provide UV spectra with the variable wavelength UV detector.) In this way, it is possible to obtain excitation spectra at any chosen fluorescent wavelength, or fluorescent spectra at any chosen excitation wavelength. Consequently, even with relatively poor spectroscopic resolution, many hundreds of spectra can be produced, any or all of which (despite many spectra being very similar) can be used to help confirm the identity of a compound.

The above spectrometric arrangement can be considerably simplified and much of the mechanical systems eliminated by employing a diode array sensing device for the fluorescent light. This allows the fluorescence spectrum to be recorded continuously throughout the development of the chromatogram. A specific excitation wavelength must be selected and this is achieved by employing the usual mechanical monochromator. Excitation spectra still need to be obtained by stopping the mobile-phase flow and scanning the excitation light.

Due to the high sensitivities achieved by fluorescence detection, the technique has proved very useful as a detection system in capillary electrochromatog-

raphy and capillary electrophoresis. High sensitivity is achieved by employing a high energy excitation source such as a laser, emitting light at an appropriate wavelength. A typical optical system for fluorescent detection in capillary electrophoresis and capillary electrochromatography is shown in Figure 6. A window is opened in the quartz capillary tube, by removing the polyimide coating from about a millimetre length of capillary tube. The laser beam is arranged to pass through the window and the fluorescent light, emitted normal to the laser beam and the capillary tube, is focused on to a photoelectric cell or photodiode array. A filter can be interposed between the capillary window and the sensor measuring the fluorescent light, to eliminate scattered incident light. The signal from the photo cell is electronically modified in the same way as the normal LC fluorescence detector.

Unfortunately, lasers which have suitable wavelengths for this purpose are somewhat limited. However, lasers of various types are continuously being developed and this offers great promise for the future development of this type of detector.

An example of the use of fluorescence to monitor an electrophoretic separation of the AQC fluorescent derivatives of phenylalanine, methionine and serine are show in Figure 7. In this separation vancomycin was used as the chiral additive. The separation was carried out on a 30.5 cm fused silica capillary, 50 |im i.d., containing 0.1molL~1 phosphate buffer and 5 mmol L_1 vancomycin. The pH of the buffer was 7.0 and the electrophoretic voltage 5 kV.

Fluorescence detection is the most popular high sensitivity detection method presently in use in LC, and will continue to be so for the foreseeable future. The system is basically simple, easy to use and provides at least an order more sensitivity than the generally popular UV detector.

Figure 6 The laser system for fluorescence detection in capillary electrochromatography.
Figure 7 The separation of the enantiomers of the AQC fluorescent derivatives phenylalanine, methionine and serine. Courtesy of LC/GC. (T. L. Bereufer, LC-GC, Vol. 12 No. 10 (1994) 748).
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