The fluorescence characteristics of hematoporphyrin have been studied at 26°C as a function of concentration and pH. The pk values are 4.7 and 8.2 as determined in water and water-ethanol mixtures. The fluorescence spectrum consists of a peak at 625 nm and a second, less intense band at 675 nm. These emission bands are assigned to the Qy (0 → 0) and Qx (0 → 0) states. The four absorption bands in the visible region are assigned to π → π* transitions as follows: 502 nm, (1 ← 0); 536 nm, Qy (0 ← 0); 557 nm, Qx (1 ← 0); and 605 nm, Qx (0 ← 0). Corresponding excitation wavelengths include 505,540,570, and 620 nm. Polarized fluorescence is used to establish molecular association as a function of concentration. The polarization factor is used to verify the change from D2h to D4h symmetry as a function of pH.
eliminated redily by addition of mcorbic acid. Consequently, 0.2 ml. of a 2.5% methanolic solution of ascorbic acid was invariably used for the preparation of the measuring solutions.All the interfering and noninterfering ions (Table 11) can be quantitatively separated by anion exchange (4, 7 ) , SO that thorium can be determined in samples of greatly varying composition. Calibration Curves. IN 10 ML. of MEASURINQ SOLUTION. The solutions contained 0 to 250 yg. of thorium dissolved in 1 ml. of IN hydrochloric acid, 0.2 ml. of the methanol-ascorbic acid solution, and 1 ml. of 0.25% dyestuff solution. These solutions were made up to volume with methanol and measured against a reagent blank solution at 490 mp. Beer's law holds from 0 to 200 pg. of thorium. IN 6.2 ML. OF MEASTJRING SOLUTION. The solutions contained 0 to 150 pg. of thorium dissolved in 1 ml. of the methanol-hydrochloric acid mixture, 0.2 ml. of the ascorbic acid solution, 1 ml. of 0.125'% dyestuff solution, and 3 ml. of methanol. These solutions were measured against a simultaneously prepared reagent blank solution at 490 my. Beer's law applies from 0 to 120 pg. of thorium. Sensitivity and Accuracy. Two micrograms of thorium per 10 m1.-1.0 pg. of thorium per 5.2 ml. of measur.ing solution-can still be determined. The average deviation of absorbance over the whole concentration range Table 111. Comparison of Two Dye
Analyses for trace elements in biological fluids are uniquely susceptible to extreme errors unless special precautions are taken during collection, storage, and analysis. The integrity of the specimen may be compromised before it is analyzed, by contamination during collection and processing or by attenuation of the analyte concentration during storage. If this happens, determined values are not valid even though the method of analysis is extremely sensitive and highly accurate. Obstacles to obtaining precise and accurate analytical data arising from these factors are discussed. We consider control procedures applicable at all stages for ascertaining the sources of error and eliminating them.
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