Rhodamine 110 chloride

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). 

As shown in Figure 6.21, excellent linearity was obtained, as represented by the high coefficient of correlation obtained for the least square linear regression. Similar results were obtained for the evaluation of autosampler accuracy when other analytes (propyl paraben and rhodamine 110 chloride) were employed in the determinations. Liu et al.9 conducted similar evaluations for the samples employed in the evaluation of the drug release rate profile of OROS with similar results to those discussed above. 

To evaluate linearity, limits of detection (LOD), limits of quantitation (LOQ), and sensitivity, an experiment assessed the responses for different concentrations of two analytes of interest. The analytes employed were methyl paraben and rhodamine 110 chloride. Consecutive 5.0 /jL injections of a series of serial dilutions (four replicates) of this standard mixture containing the analytes described were carried out via a cartridge packed with C18 stationary phase and per-column dimensions of 0.5 mm circular cross section and 80 mm length. 

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. 

FIGURE 6.23 Standard calibration curve obtained for rhodamine 110 chloride. Peak area values represent average value for four replicates. Error bars represent + one standard deviation (%RSD is very small error bars may not be visible at all concentration values). 

Acid rhodamines are made by the iatroduction of the sulfonic acid group to the aminoxanthene base. The preferred route is the reaction fluorescein (2) with phosphorous pentachloride to give 3,6-dichlorofluoran (fluorescein dichloride) (23), which is then condensed with a primary aromatic amine in the presence of 2inc chloride and quicklime. This product is then sulfonated. For example, if compound (23) (fluorescein dichloride) is condensed with aniline and the product is sulfonated. Acid Violet 30 Cl45186) (24) is produced. 

Analysis. The abiUty of silver ion to form sparingly soluble precipitates with many anions has been appHed to their quantitative deterrnination. Bromide, chloride, iodide, thiocyanate, and borate are determined by the titration of solutions containing these anions using standardized silver nitrate solutions in the presence of a suitable indicator. These titrations use fluorescein, tartrazine, rhodamine 6-G, and phenosafranine as indicators (50). 

Xanthene Dyes. This class is best represented by Rhodamine B. It has high fluorescent brilliance but poor light and heat stabihty it may be used in phenohcs. Sulfo Rhodamine is stable and is useflil in nylon-6,6. Other xanthenes used in acryhcs, polystyrene, and rigid poly(vinyl chloride) are Solvent Green 4, Acid Red 52, Basic Red 1, and Solvent Orange 63 (see Xanthene dyes). 

Rhodamine B chloride [3,5-his-(diethylamino)-9-(2-carboxyphenyl)xanthylium chloride] [81-88-9] M 479.0, m 210-211"(dec), Cl 45170, A,max 543nm, Free base [509-34-2] Cl 749, pK 5.53. Major impurities are partially dealkylated compounds not removed by crystn. Purified by chromatography, using ethyl acetate/isopropanol/ammonia (conc)(9 7 4, Rp 0.75 on Kieselgel G). Also crystd from cone soln in MeOH by slow addition of dry diethyl ether or from EtOH containing a drop of cone HCl by slow addition of ten volumes of dry diethyl ether. The solid was washed with ether and air dried. The dried material has also been extracted with benzene to remove oil-soluble material prior to recrystn. Store in the dark. 

Smith, S. N. and Steer, R. P. (2001). The photophysics of Lissamine rhodamine-B sulphonyl chloride in aqueous solution implications for fluorescent protein-dye conjugates. J. Photochem. Photobiol. A Chem. 139, 151-156. 

Remove unreacted NHS-rhodamine and reaction by-products by gel filtration or dialysis. Lissamine Rhodamine B Sulfonyl Chloride... 

Figure 9.16 Lissamine rhodamine B sulfonyl chloride reacts with amine-containing molecules to produce stable sulfonamide bonds.

Lissamine rhodamine B sulfonyl chloride is relatively insoluble in water, but may be dissolved in DMF prior to the addition of a small aliquot to an aqueous reaction. Do not dissolve in DMSO, as sulfonyl chlorides will readily react with this solvent (Boyle, 1966). The compound has a maximal absorptivity at 556 nm with an extremely high extinction coefficient of up to 93,000M em-1 (in methanol) in highly purified form. Its emission maximum occurs at 576 nm, emitting red luminescence. 

Lissamine rhodamine B sulfonyl chloride has been used in numerous applications, including multiple-labeling techniques in microscopy (Wessendorf, 1990), for confocal microscopy... 

The following protocol is a general guide for labeling biological macromolecules with Lissamine rhodamine B sulfonyl chloride. Optimization of the fluorophore incorporation level (F/P ratio) may have to be done for specific labeling experiments. 

Dissolve Lissamine rhodamine B sulfonyl chloride (Invitrogen) in DMF at a concentration of l-2mg/ml. Protect from light and use immediately. 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower-ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B derivatives of the basic rhodamine molecule. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes derivatives of this fluorescent probe among the best choices of labels for use in double-staining techniques. 

Modification with Lissamine Rhodamine B Sulfonyl Chloride... 

Figure 23.7 Avidin (or (strept)avidin) can be labeled with Lissamine rhodamine sulfonyl chloride to form a fluorescent probe.

To the filtered seawater (500 ml about 1.5 xg U) is added 0.05 M ferric chloride (3 ml), the pH is adjusted to 6.7 0.1 and the uranium present as (U02(C03)3)4- is adsorbed on the colloidal ferric hydroxide which is floated to the surface as a stable froth by the addition of 0.05% ethanolic sodium dodecyl sulfate (2 ml) with an air-flow (about 10 ml min-1) through the mixture for 5 min. The froth is removed and dissolved in 12 M hydrochloric acid-16 M nitric acid (4 1) and the uranium is salted out with a solution of calcium nitrate containing EDTA, and determined spectrophotometrically at 555 nm by a modification of a Rhodamine B method. The average recovery of uranium is 82% co-adsorbed WO4- and M0O4- do not interfere. 

Estradiol 5-Dimethylaminonaphthalene-l -sulfonyl chloride Lissamine rhodamine B sulfonyl chloride 87... 

Lissamin rhodamine B sulfonyl chloride chemiluminescence reagent, 5 852-853 Lister, Joseph, 11 7 Literature research, in fine chemical... 

The most significant parent structure for pigments of this group is obtained by a slightly modified route by simply using phthalic anhydride instead of aldehyde. Reaction with m-diethylaminophenol at 180°C in the presence of sulfuric acid or zinc chloride and subsequent oxidation with iron(III)chloride thus affords a dye known as Rhodamine B (132), the basis of Pigment Violet 1 ... 

Figure Bll.2.1 shows the normalized autocorrelation functions of various micelles loaded with octadecyl rhodamine B chloride (ODRB) at pH 7 (PBS buffer)3 . The differences in size of the micelles are clearly reflected by the differences in diffusion times td- The translational diffusion coefficients are reported in Table Bll.2.1, together with the hydrodynamic radii and the aggregation numbers.

---