Harvey Diehl

Bathophenanthrolinedisulphonic acid and bathocuproinedisulphonic acid, water soluble reagents for iron and copper

Abstract Bathophenanthrolinedisulphonic acid and bathocuproinedisulphonic acid have been isolated in solid form as sodium salts and characterised. Both materials retain the high sensitivity of the parent materials as spectrophotometric reagents, for iron and copper respectively. They are water soluble and unlike the parent reagents may be used in aqueous perchlorate solutions. Interferences are about the same as with the parent reagents, but the pH range for maximum colour formation extends two pH units further into the alkaline region. Iron II tribathophenanthrolinedi-sulphonic acid is an excellent oxidation-reduction indicator, changing sharply from red to green in the cerate titration of iron II ; it may be used in solutions containing perchloric acid.

The infrared spectra and structures of the three solid forms of fluorescein and related compounds.

The three solid forms of fluorescein, yellow, red and (newly isolated) colourless, have been shown to differ distinctly in structure. By spectroscopic methods, principally infrared absorption, the colourless form has been assigned the lactone structure, the red solid the p-quinone structure, and the yellow solid a zwitterion structure with a positive charge distributed over the oxygen-bearing ring.

Studies on fluorescein-V The absorbance of fluorescein in the ultraviolet, as a function of pH.

The ultraviolet absorption spectum of an aqueous solution of highly-purified yellow fluorescein at ionic strength 0.10 has been measured at various pH values in the range from 0.15 to 8.70. The maxima at 227, 249 and 295 nm change little with pH, but the maximum found at 437 nm in acid medium changes greatly in absorbance and position on addition of alkali, resolving first into two maxima, at 455 and 475 nm, and finally becoming a single large maximum (at pH 8) at 490 nm. A unique feature of the absorption at 437 nm is that all four prototropic forms of fluorescein, H(3)Fl(+), H(2)Fl, HFl(-) and Fl(2-), absorb at this wavelength. The total absorbance at this wavelength first falls rapidly as the pH rises from 0.15, reaching a minimum at pH 3.63, then increases to a maximum at pH 5.3, and finally falls to steady value at pH > 8.0. The absorbance as a function of pH is defined by seven constants: three dissociation constants (K(H(3)Fl) = 6.61 x 10(-3), K(H(2)Fl) = 3.98 x 10(-5), K(HFl) = 4.36 x 10(-10)) and four molar absorptivities ((H(3)Fl) = 4.94 x 10(-4), (H(2)Fl) = 1.20(5) x 10(4), (HFl) = 2.16 x 10(4) and (Fl) = 7.61 x 10(3) 1.mole(-1).cm(-1)). Solutions of yellow fluorescein in water undergo rapid deterioration on exposure to daylight or fluorescent lighting but are stable in the dark.

Studies on fluorescein—VII: The fluorescence of fluorescein as a function of pH

The relative fluorescence of fluorescein over the pH range 3-12 has been measured at 516 nm, with excitation at 489 nm. The relative fluorescence is essentially zero at pH 3, increases slowly between pH 4 and 5, rises rapidly between pH 6 and 7, reaches a maximum at pH 8, and remains constant at above pH 8. The curve of relative fluorescence as a function of pH lies somewhat above the corresponding curve describing the fraction of fluorescein present as the doubly charged anion, Fl(2-), indicating much weaker fluorescence of the singly charged anion, HFl(-), and very much weaker fluorescence by the neutral species, H(2)Fl. The fluorescence data have been used to calculate a value for the third dissociation constant. Because of the complexity of the system, one unknown dissociation constant and three (relative) fluorescence constants, a series of three variable regressions on the data was made. The final values were K(HFl) = 4.36 x 10(-7) (mu = 0.10) for the third dissociation constant and K(H(2)Fl) = 0.8; kappa(HFl) = 5.7; kappa(Fl) = 100.0 for the relative fluorescence constants.

Studies on fluorescein—II: The solubility and acid dissociation constants of fluorescein in water solution

The solubility of yellow fluorescein and of red fluorescein as a function of pH has been measured in water at ionic strength 0.10. The pH of minimum solubility is the same for both, 3.28. The intrinsic solubility, defined as the solubility of the undissociated species, H(2)Fl, and assumed to be constant and independent of pH, was calculated from the observed solubilities on the low-pH side of the minimum: S(i, yellow) = 3.80 x 10(-4)M: S(i, red) = 1.45 x 10(-4)M. The first dissociation constants were evaluated from the intrinsic solubilities and the observed solubilities on the low-pH side: both fluoresceins yielded the same value, pK(H3Fl) = 2.13. In using the observed solubilities on the high-pH side of the minimum to evaluate the intrinsic solubility and the second dissociation constant it was necessary to modify the existing theoretical approach by taking into account the presence of the fully dissociated anion. Appropriate mathematical treatments were devised to handle the more complex equations. Both fluoresceins yielded the same value for the second dissociation constant, pK(H2Fl) = 4.44. Both fluoresceins give the same yellow colour in saturated solution and the results just reported for the pH of minimum solubility and for the dissociation constants also indicate that for each of the three prototropic forms of fluorescein present in solution, H(3)Fl(+), H(2)Fl, and HFl(-), only one structure exists.

Studies on fluorescein—VI: Absorbance of the various prototropic forms of yellow fluorescein in aqueous solution☆

The absorbance of yellow fluorescein in water, at ionic strength 0.10, as a function of pH at 437, 455, 464, 475 and 490 nm has been resolved into four components, the absorbances of the individual prototropic forms of fluorescein in water. The molar absorptivity of each species at each of the five wavelengths is reported. A novel type of isosbestic point is described.

Determination of iron in wine using 2,4,6-tripyridyl-s-triazine

Abstract Procedures are described for the determination of iron in wine using 2,4,6-tripyridyl- s -triazine, a new ferroine reagent. One procedure involving wet ashing with nitric and perchloric acids gives results comparable to those obtained using the usual 1,10-phenanthroline method while a direct extraction procedure often gives low but reproducible results indicating the presence of “complexcd iron” in the sample.

Coulometric assay of the primary standards potassium dichromate and ammonium hexanitratocerate

The high-precision coulometric titration apparatus of Eckfeldt and Shaffer (Leeds & Northrup Company) has been used for the precision assay of two primary standard materials for oxidation-reduction work. The purity found for NBS 136b Potassium Dichromate. 99.975 %, standard deviation 0.002 %, checks the earlier value of Marinenko and Taylor. Ammonium hexanitratocerate, if low in thorium, is also an excellent primary standard, two recent commercial preparations having purities of 99.972 and 99.984%, a higher standard deviation, 0.005 %, reflecting a minor difficulty in end-point detection caused by platinum oxide formation on the indicating electrodes.

On the properties of Calcein Blue.

A satisfactory method for the preparation of Calcein Blue has been devised. Elemental analysis, equivalent weight by neutralization, and the NMR spectrum show the compound to be 4-methylumbelliferone-8-methyleneiminodiacetic acid.0.25H(2)O. The ultraviolet absorbance and fluorescence have been studied as a function of pH and, combined with potentiometric titration and solubility date, have yielded for the acid dissociation constants the values pK(1) = 3.0, pK(2) = 6.9, and pK(3) = 11.3. These acid functions are identified respectively as carboxyl, phenol, and ammonium ion, the free Calcein Blue being a zwitter-ion. Calcein Blue fluoresces in both acidic and basic solution when excited at a suitable wavelength. The fluorescence of the doubly-charged anion formed on the neutralization of the phenol group, when excited at 360 nm, reaches a maximum at pH 9, and decreases to zero with the neutralization of the ammonium ion; the wavelength of maximum emission is 455 nm. In the presence of calcium, the fluorescence increases with alkalinity up to pH 9 and then remains constant. The calcium derivative is a 1:1 compound, formation constant 10(7.1). The fluorescence of Calcein Blue at all pH values is quenched by copper(II). The calcium derivative is changed on standing in highly alkaline solution, presumably by ring opening, to another fluorescent material; thus Calcein Blue, although satisfactory as an indicator, is not useful for the direct fluorometric determination of calcium.

Determination of calcium in lithium salts

Abstract Two procedures are described for the determination of traces of calcium in lithium salts. In both procedures the calcium is titrated with very dilute EDTA and the end-point is determined fluorometrically with Calcein as indicator. This titration is carried out in the presence of the lithium salt in one procedure; in the other, a preliminary separation of the calcium is made on the chelating exchange resin, Dowex A-1.