Rhodamine 110 chloride fluorescence detection

FIGURE 6.17 Chromatogram overlay for 24 consecutive runs performed on a single column. (A) results of overlay for the chromatograms obtained with UV absorbance detection. Peaks are identified as (with increasing retention time) uracil (dead volume marker), methyl paraben, and propyl paraben. (B) results of overlay for chromatograms obtained from fluorescence detection (peak identified as rhodamine 110 chloride). 

Signals for methyl paraben were monitored with UV detection at 254 nm. The signal for rhodamine 110 chloride was monitored via fluorescence detection with an excitation filter of 482 nm (35 nm bandwidth) and emission filter of 535 nm (40 nm bandwidth). A gradient method (same as the one in Figure 6.16) was used. The compositions of mobile phases A and B were 5 95 H20 CH3CN with 0.1 HCOOH and CH3CN with 0.085% HCOOH, respectively, with a total flow rate of 300 fiL/ min (corresponding to 12.5 /rL/min for each column). 

LOD is defined as the lowest concentration of an analyte that produces a signal above the background signal. LOQ is defined as the minimum amount of analyte that can be reported through quantitation. For these evaluations, a 3 x signal-to-noise ratio (S/N) value was employed for the LOD and a 10 x S/N was used to evaluate LOQ. The %RSD for the LOD had to be less than 20% and for LOQ had to be less than 10%. Table 6.2 lists the parameters for the LOD and LOQ for methyl paraben and rhodamine 110 chloride under the conditions employed. It is important to note that the LOD and LOQ values were dependent upon the physicochemical properties of the analytes (molar absorptivity, quantum yield, etc.), methods employed (wavelengths employed for detection, mobile phases, etc.), and instrumental parameters. For example, the molar absorptivity of methyl paraben at 254 nm was determined to be approximately 9000 mol/L/cm and a similar result could be expected for analytes with similar molar absorptivity values when the exact methods and instrumental parameters were used. In the case of fluorescence detection, for most applications in which the analytes of interest have been tagged with tetramethylrhodamine (TAMRA), the LOD is usually about 1 nM. 

Another study employed CE for the determination of the stoichiometry of the conjugation reaction between immonuglobulin and Lissamine rhodamine-B sulphonyl chloride (LRSC). The chemical structure of the dye is shown in Fig. 3.162. Separation of the unconjugated dye from the conjugated end product was performed by CE using an uncoated fused-silica capillary column (60 cm X 75 //m i.d.). The running buffer consisted of 10 rnM borate and 0.5 mM sodium dodecyl sulphate. The separation voltage was 20 kV and analytes were detected by a fluorescence detector. It was concluded from the results that the CE method combined with... 

Stannic chloride (Rgt. No. 236) is now only rarely used for detection. If no success is obtained with fluorescence layers, iodine vapour (Rgt. No. 141) or rhodamine B reagent (No. 220) can be used for non-destructive detection. 

Carbofinan. atrazine, metolachlor, and their byproducts were separated on HPTLC plates containing fluorescent indicator. Several single and dual solvent systems were investigated for resolution by one-dimensional development. The quantification of the compounds was carried out by densitometric scanning. 1-pyrene carboxaldehyde detected chlorpyrifos and its byproducts with a sensitivity of 0.5-0.05 pg, but dansyl chloride, NBD-Cl, DPH, and Rhodamine 6G were also investigated and compared to fluorescence quench detection (131b) (Table 10). 

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