Rorrnd-Tulrle Discussion
Fluorornet ric Analysis Report of round-table discussion held by Division of Analytical Chemistry at 121st Meeting, ACS, Buffalo, N. Y., March 1952 RIoderator: CHkRLES E. WHITE, University of Maryland, College Park, M d . Panel: RZARY H. FLETCHER, U. S. Geological Suraey, Washington, D . C . T . E. FRIEDEMANN, U. S . Army Medical Nutrition Laboratory, Chicago, 111. FREDERICK KAVANAGH, Commercial Solvents Corp., Terre Haute, Znd. E. E. LEININGER, Michigan State College, East Lansing, Mich. J. IC. MURkTA, c'. S. Geological Suri,ey, Washington, D . C .
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LLOROhI17TRl C procedures have been applied in marly phases of chemical analysis. The object of this panel is to present some of the recent advances in fluorometric analysis in the fields of inorganic, mineralogical, biological, and organic chemistry in order t o stimulate a discussion of these topics. The number of applications of fluorometric methods to biological analysis far exceeds those in any other branch of chemistry. One of the most interesting developments in inorganic fluorometric analysis has been in the application of 8-quinolinol as a fluorometric reagent. I t would seem that almost any element that can be precipitated with 8-quinolinol xi11 lend itself to n fluorometric method. The general technique is to extract the metal-8-quinolinol n-ith chloroform and to measure the fluorescence of the resultant solution. This method has proved useful in the determination of traces of aluminum, gallium, and indium. I t permits the use of small original samples and avoids tedious separations. The decrease in the fluorescence of the aluminum 8-quinolinol can be used in the detection of fluorides. Methods for the determination of zinc and lithium using 8-quinolinol have also been developed. DETERMINATIOV OF UR4NIUM
The melts obtained by the fusion of uranium salts with sodium fluoride fluoresce a brilliant yellow green when exposed to ultraviolet light. The intensity of the fluorescence is directly proportional to the weight of uranium in the melt and is used for the quantitative determination of uranium. The reaction is specific for uranium if ultraviolet light of long wave length is used, but other ions may interfere by quenching the uranium fluorescence. The most serious quenchers are cerium, chromium, cobalt, gold, lanthanum, lead, manganese, nickel, neodymium, platinum, praseodymium, and silver. One to 10 micrograms of these elements quenches the uranium fluorescence of a 2-gram melt by 10% (the amount of quenching depends only upon the ratio of weight of quencher to weight of flux). Interference from other ions may be overcome by several techniques. Price's dilution technique uses a sample so small that it contains less than the critical amount of quenchers. I n other methods the uranium and quenchers are separated from each other either by precipitation of the interfering ions from the uranium solution, using a mixture of sodium and potassium carbonates, or by extraction of uranium nitrate from the other ions with an organic solvent using aluminum nitrate as the salting agenb. dluminum salts, which do not quench, are especially useful salting agents because aluminum complexes fluoride, sulfate, and phosphate ions, which if not complexed would prevent complete extraction of uranium. The principal steps in the analysis of a sample for uranium are: decomposition of a weighed sample by acid attack or fusion; solution of the uranium in a known volume of acid; separation of
urdnium and quenchers if necessarj- transference of sample aliquot to platinum container; evaporation and ignition of aliquot; addition of 2 grams of fusion mixture (9% sodium fluoride in equal parts by rreight of sodium and potassium carbonates); fusion of the mixture; and measurement of the fluorescence of the cool melt. The fusion must be made carefully because the fluorescence depends upon the temperature and duration of fusion. If temperatures greater than 700" C. are used, platinum is dissolved from the container and quenches the uranium fluorescence. Various types of fluorometers have been used for the measurement of the fluorescence, The transmission fluorometer developed a t the U.S. Geological Survey is a simple rugged instrument that requiies no elaborate optical system. I n this fluorometer the exciting light and phototube are on opposite sides of the melt, and very close to it. .4s a result of this arrangement, both the instrument blank and the loss of light intensity due to the inverse-square lam- are greatly reduced. Consequently the instrumental sensitivity is high. As little as 0.0005 to 0,001 microgram of uranium in a 2-gram melt can be determined quantitatively. T'ariations in melt thickness that might occur are not critical and cause no significant errors. A completely batterypowered transmission fluorometer has been built for use nrhere no electrical current is available. 1I.H.F. ~
GEOCHEMICAL APPLICATIONS
The manner in which geochemical information has been obtained from the study of fluorescent minerals was reviewed. Approximately 10% of the 1500 or so known species of minerals fluoresce. These are divisible into two classes: (1) those that are inherently fluorescent as a pure compound, such as Ca(UO& (PO&. 12H20(autunite), and (2) those that fluoresce only when they contain an impurity element as an activator of fluorescence, such as ZnzSi04:RIn (manganese-activated willemite). advantage is taken of the invariable fluorescence of minerals of the first class by prospecting for them with portable ultraviolet lamps. Important deposits of calcium tungstate (CaWO4, scheelite) have been discovered in this manner in the western states. The more numerous minerals of the second class have been studied by a small group of geochemists in several countries, and a number of activator elements such as ?vln++,E u + + ,Cr+++, and Sm+++have been identified. Whenever feasible, the final proof of the correct identification of the activator has been obtained by synthesizing the fluorescent mineral. The fluorescence is often the only indication that unusual elements are present in the minerals. The intensity of fluorescence generally increases with activator concentration up to an optimum concentration and then decreases. The activators in most minerals of the second class are, as yet, unidentified. The fluorescence properties of minerals may be altered by
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ANALYTICAL CHEMISTRY
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various physical and chemical changes that the minerals undergo subsequent to their formation. Some effects due to mechanical deformation, heat, and long exposure to feeble radioactivity have been recognized. Zoned fluorescence calls attention to fluctuations in the growth history of crystals and also to crystal-chemical forces that cause selective deposition of activator elements on certain faces of crystals. The fluorescence of minerals, therefore, helps in identifying and localizing minerals, provides basic information on the distribution of a number of elements (activators) among minerals, and throws light on the natural history of these minerals in the earth’s crust. J.K.M. RELATION O F pH T O FLUORESCENCE
Many polycyclic and heterocyclic unsaturated organic compounds fluoresce when irradiated under proper conditions. Fluorescence as a function of p H may be characteristic of a compound. The pH-fluorescence curve is an indication of the influence of minor changes in structure upon the probability of internal conversion of absorbed energy into emitted energy. The fluorescent species can be a neutral molecule, a dipolar ion, a negative ion, or a positive ion. The ions may be univalent or polyvalent. A molecule may be fluorescent in more than one state of ionization. If i t is, then the several fluorescent forms may be expected to fluoresce v i t h different efficiencies and different colors. The compounds considered here are those that give visible fluorescence in dilute aqueous solution 1%-henirradiated a t room temperature by the 366 mp lines of the mercury arc in the presence of dissolved oxygen and buffer salts. These conditions usually occur in most fluorometric methods of qualitative and quantitative analysis. Factors which influence the shape of the pH-fluorescence curve are: pH, buffer composition, quenching by dissolved oxygen, efficiency of the fluorescent process, variation with p H of the absorption spectrum of the organic compound, color of the fluorescent light, and characteristics of the measuring instrument. The three common sources of quenching are impurities accompanying the fluorescent substance, salts of the buffer, and dissolved oxygen. Quenching by oxygen is common and is well known to those who measure the fluorescence of carcinogenic hydrocarbons. Unless quenching by oxygen is very large, it is more convenient to obtain maximum quenching by saturating the solution with air than to remove all dissolved oxygen. Quenching by salts of the buffer may be appreciable and also cause a change in shape of the pH-fluorescence curve. An example is quinine sulfate measured in McIlwain’s buffer and in nearly unbuffered sulfate solutions. The two curves coincide in the sulfuric acid range, but the fluorescence in McIIwain’s buffer is less than that of the sulfate solution, showing that ritric acid and phosphate quench fluorescence of quinine. Quenching by oxygen and buffer salts mav be unimportant t o the final results of qualitative and quantitative analysis because it will be the same for standard and sample. Quenching is important, however, when an absolute measure of fluorescenre is required as in quantum yield experiments and in determining ionization constants from the shape of pH-fluorescence curves. Four examples of compounds n-hich fluoresce in these different states of ionization are quinine, anthranilic acid, riboflavin, and thiochrome. Because there is no fluorescence without prior absorption of light, data for correlating absorption of energy with emission of energy were obtained by measuring the absorption spectra of the four compounds a t several values of p H in the buffers used in obtaining the pH-fluorescence curves. The relative absorptions for the 366 mM lines were computed by calling the maximum absorption 100. The shape of the anthranilic arid pH-
fluorescence curve is that expected if a dipolar ion is the main fluorescing species, but the long tail of the curve suggests that an anion also fluoresces. The relative absorption of 366 mp light also parallels the fluorescence curve. The quinine curve is about what would be expected if a constant fraction of the 366 mp energy absorbed is emitted as fluorescent light. Both absorption and fluorescence follow fairly closely the relative concentration of the quinine doubly charged cation; the anion is very weakly fluorescent. The principal influence of p H in both quinine and anthranilic acid is on absorption of exciting energy. The pHfluorescence curve for riboflavin is interesting because absorption of the 366 mp lines is nearly independent of pH but fluorescence is not. There is a color change from greenish yellow a t p H 10 to bluish a t p H 11.3. Unlike the two previous examples, probably it is the conversion of absorbed light into fluorescent light, not the absorption itself, which is the function of pH. The shape of the curve suggests that the fluorescing structure is a neutral molecule or dipolar ion. The pH-fluorescence curve and the relative light absorption for thiochrome indicate that the main influence of change of p H on fluorescence is not upon the absorption of light. The p H of the solution may affect markedly the color of the fluorescent light, which may vary from dark purple to deep red with blue the most common color. A change in color with change in pH indicates change in structure of the fluorescing substance. This color change is an important factor in determining the shape of the pH-fluorescent curve. If the photocell of the fluorometer is sensitive priniarilj- to the blue, the pH-fluorescence curve will have a different shape from that determined mlth a photocell that is also sensitive to red light. Photocell spectral sensitivity and the characteristics of light filter over the photocell modify the shape of the pH-fluorescence curve and must be considered in comparing the curves obtained by different experimental arrangements. Many fluorescing systems change under the influence of the light exciting fluorescence. The change can take the form of increase in fluorescence, decrease in fluorescence, or change in color of fluorescent light in various combinations. An example of the utility of fluorometric methods may be cited in the identification of the blue-fluorescing substance in the 15-mm. portion of the oat root tip lying between 3 and 18 mm. from the tip. This part of the root had a wet weight of about 0.5 mg., yet the sensitivity of the method was such that a complete pH-fluorescence curve could be made on the extract from 30 root tips. The crude, unpurified chloroform extract of the root sections had a pH-fluorescence curve nearly identical with that of scopoletin, which was shown to be the blue fluorescing F.K. substance in that portion of the oat root. ANALYSIS O F BIOLOGICAL MATERIALS
Fluorometry is an important analytical technique which has assumed prominence within the past 15 years. Its most dramatic and pvidest application occurred in the early years of the past decade, when it was used in vitamin assays, particularly of thiamine and riboflavin in foods and body fluids. Since then the interest in vitamin assays has $vaned, and instead, nevi interests have arisen in the application of fluorometry t o inorganic analysis and the determination of hormones, metabolic intermediates, drugs, and decomposition products in prepared foods. T h e widest application of fluorometry still is t o samples of biological origin, and it is in the analysis of such samples that the limitations of the techniques become most evident and often lead t o serious error. Some of the difficultics are: The ranges and the variations of concentration in biological materials are often very n-ide. Thus, it may be necessary to repeat the analysis until the photometric response is within the instrumental range. Obviously, an instrument with a wide range
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V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2 of sensitivity, which can be set readily to several narrow ranges, T o d d be desirable. The fluorescent compound of a fluorescent derivative may have strong light-quenching properties. Examples of such substances are the porphyrins, the colored adrenaline derivatives, and the colored pyridinium derivatives of nicotinic acid and other pyridine compounds. The fluorescence response is linear only :tt high dilution. Many biological materials contain impurities and colored substances which may have strong light-quenching properties. The interference due t o these substances may be minimized either by isolation procedures or, often more simply, by dilution. Obviously, the use of instruments of high sensitivity is indicated. Such an instrument has been used by the author since 1942 and is now available commercially from the Central Scientific Co., Chicago. This instrument is now known as the Cenco-Friedemann-Liebeck fluorometer and is a multirange type mith two photocells, one of which is especially sensitive in the blue part of the spectrum and the other in the red. The effect of dilution on the fluorescent properties of caramelized sugar and, generally, the interfering effect of colored materials in filtrates taken for analysis were discussed. At progressively smaller concentrations, but a t constant hydrogen ion concentration, some substances like quinine exhibit decreased fluorescence; others. like riboflavin, do not. The hydrogen ion concentration is very important, especially a t low concentrations. Multirange instruments are helpful in the study of the efficiency of adsorbing agents for use in chemical assay. Thus the recover) of thiamine after absorption on Decalso, a zeolite x-hich is nidely used in the analysis of thiamine, diminishes rapidly a t progressively higher dilutions. The effect, although present, is not apparent in thci single-range instruments in current use. On the other hand, the recovery of riboflavin from Supersorb, a magnesium silicate which is used in riboflavin assag, is quantitative over the range of 1.6 to 0.004 microgram per ml. T.E.F. FLUOROMETRIC DETERXIINATION OF CITRIC AND MALIC 4CIDS
The fluorometric method for the determination of citric acid by the formation of ammonium citrazinate ( 4 ) has proved successful. When the method was devised, it 11-as believed that the citric acid wxs changed to aconityl chloride with thionjl chloride. K h e n the aconityl chloride is treated with ammonia gas, the expected product is aconitamide, Khich is transformed to citrazinic acid by ring closure upon heating with 767, sulfuric acid a t 165" C. Evidence has been found that the reactions do not follox- this path completely. The aconityl chloride formed in the reaction with thionyl chlorid~mas hydrolyzed to aconitic acid, which was then determined by polarographic reduction in hydrochloric acid solution (6). On the basis of the amount of aconitic acid found, it was impossible to account for the yield of citrazinate as determined by the fluorescence intensity. Therefore it is suggested that part of the citric acid is transformed to citrazinate by a different path, possibly involving citryl chloride and citramide. The control of conditions a t each step in the procedure is important. For example, it is essential that the moisture be removed from the sample completely, since it reacts with thionyl chloride and reduces the fluorescence intensity. -4fter the reaction n i t h thionyl chloride, the excess reagent must be removed completely without allowing the entrance of moisture. This is accomplished by alternate evacuation and flooding with dry air. The only drying agent n-hich was completely satisfactory under the drastic conditions employed n-as Dehydrite. The completeness of ring closure TT as shon-n to be particularly dependent upon conditions. The ammonium citrazinate solution is not suitable for setting the fluorometer to a reading of 100, but a standard solution of sodium salicylate served fairly satisfactorily. The method for the determination of malic acid ( 5 ) depends
upon heating with 2-naphthol in 92% sulfuric acid. The blue fluorescence n-as believed to be due to the formation of 5,6-benzocoumarin. This has been corroborated by Goodwin and Kavanagh ( 2 ) ,who obtained identical pH us. fluorescence curves for 5,6-benzocoumarin and a solution of malic acid treated n-ith 2-naphthol and sulfuric acid as described. The fluorometric determination of malic acid by the methods of Barr ( 1 ) and Hummel (3)depends upon the formation of coumarin derivatives. Fluorometric methods for a number of organic groups and compounds are possible and many applications of the methods already in existence might be made. Methods depending upon the syntheses of fluorescent organic compounds might be expected to require rather close attention to detail; however, in spite of this, many useful methods are possible. E.E.L. DISCUSSIOY
The determination of adrenaline provoked much discussion. Aluminum oxide does not seem to be the best agent for adsorbing protein to permit the determination of adrenaline. The use of a small sample gives the most satisfactory results and isobutyl alcohol has proved to be a good extracting agent. K i t h the use of the dilution technique and an instrument of proper sensitivity, very satisfactory results can be obtained. The browning of sugars and the discoloration resulting when foods are treated with sulfuric acid may cause a decrease or an increase in the intensity of the fluorescence of the products under determination. However, experiments in R hich dilute solutions xere used n-ith a multirange fluorometer seem to indicate that the fluorescence is always increased by this browning of food materials. The decrease in fluorescence in more concentrated solutions is probably due to the absorption of the ultraviolet radiation by the foreign material in the first few millimeters of the cell. This absorption can easily be observed by visual observation of the solution. I n reply to a question on the uranium method, it was stated that ordinary reagent grade aluminum nitrate is satisfactory for use nhen the uranium quantity is greater than 10-8 gram. With samples containing quantities smaller than this, it is necessary to purify most of the chemicals used. A question concerning the effect of phosphate on the fluorometric determination of fluoride with aluminum-8-quinolinol provoked considerable discussion. Charles Horton made the following observations: I n the fluorometric method for determining traces of fluoride with the 8-quinolinol extraction method, equilibrium is reached more quickly if an acetic acid solution of the reagent is added before extraction than if the reagent is placed in the extracting solvent. This method for the determination of fluorides depends on the relative stability of the metal-organic complex as compared to the metal-fluoride complex. TTTO or more of the fluoride complexes are generally present. Phosphate, sulfate, or other ions which interfere may form comple~eswith the fluoride, the metal, or the organic compound and affect the amount of t h e fluorescent compound prePent. In the titration of large amounts of fluoride, the photofluorometric method using quercetin or morin is superior to the use of Alizarin Red S in the visual method. Commercial morin is generally unsatisfactory as a reagent. It may be purified by ion exchange or sublimation. ?*lorinof good quality can be obtained from Schuchardt in Germany. Frank Grimaldi observed that in the determination of traces of aluminum in minerals with the 8-quinolino1, with the extraction fluorescence technique, it is very important to have a great excess of the reagent in order to prevent phosphate interference. Therefore, it is better to add the solution containing aluminum to the acetic acid solution of the 8-quinolinol than to employ the reverse procedure. I n the analysis of phosphate rock a sample of about 0.1 mg. is used and the complex is extracted n i t h chloroform a t a p H of 4.6; O . O l ~ oaluminum oxide can be determined in the 0.1-
ANALYTICAL CHEMISTRY
1968 mg. sample. This method is specific for aluminum and as much as 10% iron does not interfere. A discussion concerning the source of ultraviolet radiation for the study of the fluorescence of minerals seemed to indicate that the high pressure and low pressure mercury vapor lamps with their essential radiation a t 3650 and 2537A., respectively, are satisfactory for most purposes. The use of a monochromator to produce narroiv bands for the excitation of the fluorescence of minerals does not seem warranted.
LITERATURE CITED
(1) Barr, C. G., Plant Physiol., 23,443 (1948) (2) Goodwin, R. H., and Kavanagh, F., d r c h . Biochem, 27, 152 (1950).
(3) Hummel, J. P., J . Bid. Chem., 180, 1225 (1949). (4) Leininger, E., and Katz, S., ASAL. CHEY..21, 810 (1949). (5) I h i d . , p. 1375. (0) Schwaer, L., Collection Czechoslac. Chem. Commun., 7 , 320 (1935). THEreport on the determination of uranium concerns work done on behalf of the U.S. Atomic Energy Commission and is published with the permission of the commission.
Spectrophotometric Determination of Small Quantities of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichlorom phenoxyacetic Acid Us i ng Partition Chromatog rap hy NATHAN GORDON’, Insecticide Division, Livestock Branch, Production and Marketing Administration, U . S . Department of Agriculture, Beltsville, Md., and MORTON BEROZA, Bureau of Entomology and Plant Quarantine, U . S . Department of Agriculture, Beltsville, M d .
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HE recent widespread use of 2,4-dichlorophenox~-acetic acid (2,4-D) and 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) as weed killers has unfortunately given rise to instances of contamination of liquid insecticide concentrates used on plants BUSceptible t o these compounds. For example, large quantities of cotton were reported to be seriously damaged as a result of utilizing these contaminated liquid insecticide concentrates. A method which could detect small quantities of the contaminants as well as distinguish between them would be of use both in control and in regulatory enforcement work. Previous methods have been published for the determination of small quantities of 2,4-D. A bioassay method involves the use of plants (10). A colorimetric qualitative test for 2,4-D has been described by Freed ( 2 ) and a modification of this test has been used to determine 2,4-D in milk (6). A method for determining 2,4-D in soil leachates by means of countercurrent distribution has also been described (11). These methods lack specificity, are time-consuming, and do not differentiate between 2,4-D and 2,4,5-T. After the present method was completed a method for the determination of 2,4-D by chromatography on kieselguhr containing a strong phosphate buffer was reported (9). The amount of 2,4-D is determined by titration. The method described below involves the separation of the acids, after a suitable extraction procedure, b y partition chromatography of the Martin and Synge type ( 7 ) . After separation, the 2,4-D and 2,4,5-T are determined spectrophotometrically by measuring the ultraviolet absorbancy of the acids a t 284 and 289 mp, respectively. I n the chromatography of acids, Isherwood ( 4 ) recognized that narrower zones could be obtained by repressing the ionization of acids. He accomplished this by using 0.5 N sulfuric acid as the immobile phase. Lugg and Overell (6) used formic and acetic acids to repress the ionization of acids in paper chromatography and thus obtained narrower zones. This principle was utilized in the development of the method below; the method may be generally useful for the separation of acids which absorb in the ultraviolet
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* Present address, Offioe of D.C.
t h e Chief Chemical Officer, Washington 25,
A hydrolysis step is included to convert any ester, amide, or salt of 2,4-D or 2,4,5-T to the free acids. The hydrolysis of the esters, amides, and salts is complete after 1 hour’s refluxing with 25% sodium hydroxide if wetting agent is present. REAGENTS
Ether, USP. Carbon tetrachloride, C.P. Distill before use, %-Hexane, commercial grade. Purify following the method of Graff, O’Connor, and Skau ( 3 ) , passing the n-hexane through silica gel, and retaining only the portion sufficiently transparent to be read in the spectrophotometer a t 230 mp. Distill the effluent before use. Glacial acetic acid, C.P. Absolute methanol, C . P . Both CON P R ESSED c used as received. AIR Formic acid(Eastman 139). ADAPTOR Distill before using to prepare the 90% formic acid. Immobile Solvent. Add S EPARATO RY 2 ml. of 1 to 5 90% formic FUNNEL acid-glacial acetic acid mixture to 18 ml. of 90% methMOBILE anol and mix. S0 LVENT Mobile Solvent (n-hexane saturated with immobile solvent). Add approximately 400 ml. of purified n-hexane to 20 ml. of the immobile solvent contained in a 500-ml. separatory funnel and agiCOLUMN tate vigorously for 5 minutes. Allow the layers to separate. 2.1 CM. Draw off the immobile solvent 21 CM. and reserve for use. Silicic acid. Analytical reagent grade KO.2847 supplied by Mallinckrodt Chemical Works was used in the chromatographic separations. SINTERED Silica gel. Mesh size 28GLASS DISK 200, used for the purification of n-hexane. Supplied by IO-ML. GRADUATE Davison Chemical Co., Baltimore, &Id. A Pure 2,4-dichlorophenoxyacetic acid (2,4-D). PreFigure 1 . C h r o m a t o pared by recrystallizing the graphic Column
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1r--r
