Fluorescence chromatograms

Treatment of the chromatogram with a reagent results in the production of colored or fluorescent chromatogram zones, which are used to evaluate the success of the separation and for quantitative analysis For this purpose it is necessary that the color or fluorescence intensities remain stable for about 30 minutes... 

The required exposure times are difficult to estimate. They are best found by trial and error. Documentation of fluorescence quenching at A = 254 nm usually only requires one trial. The exposure time found to be adequate here is normally suitable for all following exposures of fluorescence quenching if the exposure conditions are maintained constant (camera type, film type, distance of objective and lamp, aperture etc.). The exposure time required for fluorescent chromatograms is primarily dependent on the intensity of the fluorescence and, therefore, has to be optimized for each chromatogram. It is best to operate with a range of exposure times, e.g. aperture 8 with exposures of 15,30,60,120 and 240 seconds. Experience has shown that one exposure is always optimal. 

Sinigrin (JiRt 35—40) appeared as a yellow fluorescent chromatogram zone on a dark background under long-wavelength UV light (2 = 365 nm). The detection limit was 25 — 50 ng substance per chromatogram zone. 

For the determination of sterols and their esters the chromatogram is immersed in a 10% aqueous copper(II) sulfate solution and then heated to 105°C for 30 min. In this case green-yellow fluorescent chromatogram zones are visible in long-wavelength UV light (X = 365 nm) [6]. 

Under long-wavelength UV light (A = 365 nm) the 2-(2-hydroxyphenyl)-benztri-azoles yielded yellow-green fluorescent chromatogram zones, which were, in the cases of Tinuvin P (hRi 20 — 25) and Tinuvin 343 (2-[2-hydroxy-3-(l-methylpropyl)-5-ter/-butylphenyl]benztriazole hR( 45 — 50), suitable for quantitation. 

Glycine (hRf 20 — 25), alanine (hRf 35 — 40), valine (hRf 55 — 60) and leucine (fiRf 65 — 70) appeared as blue-green fluorescent chromatogram zones in long-wave-... 

Note Sulfuric acid is a universal reagent, with which almost all classes of substance can be detected by charring at elevated temperatures (150—180°C). The production of colored or fluorescent chromatogram zones at lower temperature (< 120 C) and their intensities are very dependent on the duration of heating, thf... 

Funk et al. have used a low-pressure mercury lamp without filter to liberate inorganic tin ions from thin-layer chromatographically separated organotin compounds these were then reacted with 3-hydroxyflavone to yield blue fluorescent chromatogram zones on a yellow fluorescent background [22]. Quantitative analysis was also possible here (XoK = 405 nm, Xji = 436 nm, monochromatic filter). After treatment of the chromatogram with Triton X-100 (fluorescence amplification by a factor of 5) the detection limits for various organotin compoimds were between 200 and 500 pg (calculated as tin). 

A whole range of separated celandine extract components are visible as intensely fluorescent chromatogram zones however, chehdonine does not emit fluorescent light at this stage, but fluorescence quenching is likely to occur under short-wavelength UV light (X = 254 nm) (Fig. lA and IB). 

Now chelidonine produced an intense green fluorescent chromatogram zone in addition there were other intensely fluorescent zones in the track of the celandine extracts — some of which were not previously visible or had another color shade (Fig. IC). In addition the general fluorescence was increased as a result of the UV irradiation. Figure II illustrates the corresponding fluorescence scans. 

Observation under UV light (A, = 254 or 365 nm) reveals intense fluorescent chromatogram zones on a dark background. 

Note Primary amines yield fluorescent chromatogram zones even before the application of reagent 3. Secondary amines do not yield fluorescent derivatives until they have been treated with reagent 3. Hence, the reagent sequence allows the stepwise detection of primary and secondary amines. Taurine is preferred as the essential component of reagent 3 over the multiplicity of other possibilities because it produces intense fluorescence it is also not very volatile and is readily available. Amides and substances with peptide linkages, eg. hippuric acid, are not detected, neither are secondary amines that are volatile at high temperatures. 

Observation under short- and long-wavelength UV light (X, = 254 nm or 365 nm) reveals yellow to orange fluorescent chromatogram zones on a yellow-green fluorescent background. 

Under long-wavelength UV light (X = 365 nm) yellow-orange fluorescent chromatogram zones are observed on a pale light-blue fluorescent background. 

On excitation with long-wavelength UV light (X = 365 nm) ketoprofen (hcf 35-40) and flurbiprofen (h/ f 50-55) appeared as yellow or blue fluorescent chromatogram zones on a pale blue fluorescent background. The detection limits of, for instance, flurbiprofen were 10 ng subtance per chromatogram zone. 

Cephaeline (h/ f 6-11) appeared as blue and emetine (h/ f 10-15) as yellow fluorescent chromatogram zones on a dark background when examined under long-wavelength UV light (X. = 365 nm) (Fig. 1). Emetine — like cephaeline — also yielded a blue fluorescence on RP-2 and RP-18 layers. 

After ca. 30 min, when the chloroform had evaporated, fluorescent chromatogram zones appeared on a dark background on excitation with long-wavelength UV light (X = 365 run) 2,3-diaminonaphthalene (h/ f 70-75), red and 2,3-diaminopyridine (h/Jf 55-60), blue. The detection limits were 3 ng substance per chromatogram zone. 

---