Rhodamine Reagent

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

Note Rhodamine B is a universal reagent that can be used on silica gel, talc, starch [5] and cellulose layers, just as on urea [1] or silver nitrate-impregnated [7] phases. Liquid paraffin-impregnated silica gel and RP layers are less suitable, since the background to the chromatographic zones is also intensely colored. It is often possible to increase the detection sensitivity by placing the plate in an atmosphere of ammonia after it has been sprayed or dipped, alternatively it can be oversprayed with sodium or potassium hydroxide solution. 

The reaction was not particularly sensitive on paraffin-impregnated kieselguhr layers because of background coloration. For quantitation it was better to use the five-fold more sensitive rhodamine 6G reagent (q.v.). 

Note Rhodamine 6G is a universal reagent which can also be incorporated in the TLC layers [4, 9] or added to the mobile phase [4], The spray reagent can also be made up in water [8], acetone [4, 6] or ammonia solution (c = 2.5 mol/1) [5]. The visual detection limit is most favorable when the water from the mobile phase or the detection reagent has not completely evaporated from the layer. This can be recognized by the fact that the background fluorescence has not turned from red to pink [4]. 

The reagent used for the deteetion may be speeifie to a special functional group or specific lipids or may be a nonspecific reagent that makes all hpids visible. The most commonly used reagent that is nonspecific for any lipid group is 0.1% (w/v) 2, 7 -dichlorofuorescein in 95% methanol. This is mainly useful when the plates have been developed in acidic solvents. The lipid spots or bands can be visualized as yellow spots or bands under UV light. After the plates are developed in alkaline solvents, an aqueous solution of Rhodamine 6G (0.01%) can be used, and lipid spots can be seen as pink spots under UV. Because both these methods are nondestructive, they can be effectively used in PTLC so that the separated sample bands can be scraped off and used for further analysis. 

Rhodamine B 67 is frequently used in the quantitative determination of DNA or RNA and fluorescent labeling for DNA [190-192]. This dye was assembled onto the surface of a quartz substrate by electrostatic interaction between the fluorescence reagent RB and y-aminopropyltriethoxysilane (APES), and the Quartz/ APES/RB film was constructed (Fig. 1) [193]. 

The fluorescent properties of NHS-rhodamine are similar to TRITC. The wavelength of maximal absorbance or excitation for the reagent is 544 nm and its emission maximum is 576 nm, exhibiting a visual color of orange-red. Its molar extinction coefficient at 546 nm in a methanol environment is 63,000M 1cm 1. Other components in solution as well as the pH (in aqueous buffers) can change this value. 

Lissamine rhodamine B sulfonyl hydrazine is soluble in DMF. The reagent may be dissolved in this solvent as a concentrated stock solution before adding a small aliquot to an aqueous reaction medium. The compound itself and all solutions made with it should be protected from light to avoid decomposition of its fluorescent properties. 

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