periments showed that, if the quinalizarin content were increased, the slopes of thecurves would become steeper and the curves would converge at a higher point. The absorbance values with titanium Iwsent are aa repeatable as those with titmium absent if the quinalizarin content is held constant. This fact is utilized in making correction for titanium interference.
ACKNOWLEDGMENT
The author wishes to acknowledge the cooperation of his coworkers, R. E. Kohn and R. L. Chance, in the preparation of this paper. LITERATURE CITED
(1) Berger, K. C., Truog, E., IND. ENG. CHEM., ‘4NAL.ED. 11,540 (1939).
MacDougall, Daniel, Biggs, D. A., ANAL.CHEX 24, 566 (1952). Rudolph, G. A4.,Flickinger, L. c., Steel 112, 114 (April 5, 1943). Smith, G. S., Analyst 60, 735 (1935 ) . Weinberg, Sidney, Procter, K. L.. Plliller, O., ANAL. CHEM. 17, 419 (1945). RECEIVEDfor review May 9, 1956. Accepted Xarch 27, 1957. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1957.
Characteristics of Boron-Benzoin Complex Improved Fluorometric Determination of Boron CHARLES E. WHITE and DONALD E. HOFFMAN’ Department o f Chemistry, University o f Maryland, College Park, Md.
b The boron-benzoin complex used in the determination of boron is stabilized a t its maximum intensity for a 20minute period with glycine buffer and for a somewhat longer time with isopropylamine. The complex is decomposed b y extended exposure to ultraviolet light. The maximum fluorescence emission is from 450 to 520 mk and the maximum excitation is from 310 to 400 mp. The complex contains 1 gram atom of boron to 1 mole of benzoin.
B
is an excellent reagent for boron. Details of the qualitative and quantitative procedure for this determination have already been published (6). From a quantitative standpoint a disadvantage in the use of this reagent was the tendency for the fluorescence to reach a maximum intensity in a period of about 3 minutes and then decrease rapidly after 5 minutes. The fluorometer used in the original measurements was one which did not have a shutter imposed between the light and the sample, and permitted the continuous exposure of the sample to the exciting radiation, The fluorescence is stable for a much longer period if exposure of the sample is confined to the brief interval during which a reading is being made. A means of increasing the stability of the fluorescence and other characteristics of the boron-benzoin complex have been determined. ENZOIN
APPARATUS AND REAGENTS
The fluorometer was constructed in Present address, Chemistry Department, University of Delaware, Newark, Del.
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Figure 1. Variation of fluorescence intensity with time under continuous irradiation A. 8.
0.1 ml. of 0.6N sodium hydroxide 0.4 ml. of glycine buffer (pH 12.8)
this laboratory and consisted of a sample holder, a 1P28 photomultiplier tube attached to a sensitive microammeter (R.C.A. W.V. S a ) , and a power source for the phototube (4). The usual source of ultraviolet light was a General Electric Co. H100B4 lamp; however, for the excitation spectra the radiation from a xenon arc mas passed through a Bausch & Lomb grating monochromator (KO. 33-8640-01). A Beckman Model DU spectrophotometer equipped with a photomultiplier tube was used to measure the absorption spectra. All inorganic compounds were reagent grade. Commercial 957& ethyl alcohol was redistilled to remove a nonvolatile fluorescent impurity. Boron solution was prepared by dissolving boric acid crystals in water in proper amounts to afford concentrations of 1 to 10 y of boron per ml. and 50 y per ml. Benzoin (Eastman Kodak Co.) n a s recrystallized from benzene and dissolved in redistilled 95% ethyl alcohol to produce qolutions of 0 5 0 2, and 0.137,. Glycine buffer was prepared for a pH of 12.8 by thc method of Clark ( I ) .
Two stock solutions were necessary. Solution A contained 7.505 grams of glycine, 5.85 grams of sodium chloride, and 1 liter of water; solution B contained 2 grams of sodium hydroxide pellets dissolved in 500 ml. of water. For the buffer 50 ml. of A was mixed with 430 ml. of B. These solutions were stored in polyethylene bottles. Isopropylamine (Matheson, Coleman, & Bell Co.) was distilled to remove a nonvolatile fluorescent impurity. A stock solution of quinine sulfate ivas prepared by dissolving 0.01 gram (USP, Jlerck &- Co., Inc., New York) in 1 liter of nater a n d diluting further Rith 0.l.Y wlfuric acid as desired. -4 solution of a fluorescence in the rai’ge of the determinations in progress was used to set the fluorometer for comparabk rtadings over a period of time. Boron-free glassware and quartz test tubes were used in routine work. d l 1 alkaline solutions were stored in polyethylene containers. Glass-stoppered borosilicate glass test tubes, graduated a t 25 mi., n-we used for fluorometric nwasurements. I n thesc cases the solutions wtre never in the test tubes for more than 1 hour because alkaline soluVOL. 2 9 ,
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Figure 2. Variation of fluorescence intensity with varying amounts of potassium hydroxide A.
B.
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tions dissolved appreciable quantities of boron from such glass in several hours.
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EXPERIMENTAL
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The general procedures outlined previously (6) were followed. The effect of 7 5 compounds on the boron-benzoin fluorescence was studied. The list included several commercial antioxidants, various solvents, inorganic bases and salts, buffers, hydroxy compounds, organic amines, aldehydes, acids, and esters. Many of the organic compounds fluoresced in their natural states and produced blanks of high intensity. Some of the compounds showed encouraging results and were selected for further study. Inorganic Bases and Buffers. The effect of acetate buffers (pH 6 . 2 and 7.4), carbonate-bicarbonate buffer ( p H 9.7), and glycine buffers ( p H 10.1, 11.5, 12.0, and 12.8) was examined. The results of tests with the glycine buffer of pH 12.8 were satisfactory. The following procedure was used to evaluate the effect of an added agent. A 50-7 quantity of boron and 2 ml. of 0.5% benzoin solution were diluted to about 20 ml. with ethyl alcohol. Quantities of glycine buffer varying from 0.1 to 0.5 ml. were added, and the solution was diluted to 25 ml. with alcohol. The fluorescence intensity was measured on the microammeter, which was set for each determination with a quinine sulfate standard to permit a comparison of results. No additional alkali was necessary because the glycine buffer contained sodium hydroxide. I n every instance the intensity readings were more stable than those obtained with the use of the original procedure in which sodium hydroxide was . used in place of the buffer. The stability is shown in Figure 1. The solution containing sodium hydroxide produced steady readings for only 1 minute, while the solution containing buffer produced steady readings for 10 minutes. The buffer helped to stabilize the fluorescence of solutions containing from 1 to 5 y of boron under continuous irradiation. Exact measurements of quantities were necessary to obtain differences of intensity between the blank and successive concentrations of boron. The addition of 1 ml. of water in excess of the amount already present and the following order of addition of reagents were helpful in producing a significant difference of intensity between successive concentrations: 1 ml. of water, 1 ml. of boron solution, about 15 ml. of 95y0 ethyl alcohol, 3 ml. of 0.5% benzoin solution, 1106 *
ANALYTICAL CHEMISTRY
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Figure 3. Variation of fluorescence intensity with different amounts of glycine buffer A. 5 minutes a h e r mixing.
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Figure 4. Variation of fluorescence intensity with time with various bases and continuous irradiation A. 0.2 ml. of 0.6N sodium hydroxide.
B. 2 ml. of isopropylamine C. 2 ml. of isobutylamine
and 0.2 ml. of glycine bufier, diluted to a total volume of 25 ml. with alcohol. The blank reached a maximum value of 42, and the solution with 1 y of boron gave a reading of 60 and remained steady for 30 minutes under momentary irradiation. I n a similar run samples containing 1 to 5 y of boron with the
glycine buffer were subjected to ultraviolet irradiation only long enough to obtain readings. The results were coiistant for 5 to 35 minutes. Experiments where 0.2 ml. of 0.1N sodium hydroxide was substituted for the buffer and the samples were given only momentary irradiation also showed constant read-
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Figure 5. A. B.
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Absorption spectra of boron-benzoin complex
0.1 3% benzoin with 0.05 ml. of 0.9N potassium hydroxide in 25 mi. 0.1 3% benzoin with 0.05 ml. of 0.9N potassium hydroxide and 10 y of boron in 25 mi.
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Figure 6. Variation of fluorescence intensity with various molar ratios of boron to benzoin A.
Maximum amount of boron, 1 8 pmoles
6. Maximum amount of boron, 27 p o l e s
ings for :i like period but were slightly loner than those with the buffer. Potassium and lithium hydroxides gave results comparable to those with sodium hydroxide. I n all of these cases where the alkali was used without a buffer, the readings were constant with momentary irradiation but not with continuous irradiation. Potassium hydroxide was used in later experiments rather than sodium hydroxide because of its ease of solubility in water and alcohol. An interesting observation was made in connection with the above experiments. The addition of small amounts of base partially restored the fluorescence after it had decreased on standing. Ammonium hydroxide 17-as unsatisfactory; with this base the boron samples produced a green fluorescence which turned blue in a short time similar to a blank. The above results indicated that with
momentary irradiation glycine buffer. sodium hydroxide, or potassium liydroxide might be used with equal facility. However, Figures 2 and 3 show that the amount of potassium hydroxide must be much more carefully controlled than that of the glycine buffer. In each case the alkali or buffer was added to a practically neutral solution of boron. I n the analysis of an unknown it mould be necessary t o titrate an aliquot of the solution to find the amount of alkali necessary to attain the practically neutral solution before the addition of the glycine buffer. The curves show that volumes of glycine buffer ranging from 0.4 to 0.9 ml. were suitable for a final 25-m1. volume with the quantity of boron used. Organic Bases. Several organic amines were substituted for inorganic bases. Isopropylamine, n-butylamine, isobutylamine, n-amylamine,
sec-amylamine, ethylenediamine, and propylenediamine were investigated. Isopropylamine and isobutylamine produced the most satisfactory results. The other amines either fluoresced in their natural states or produced low intensities in solution. Results showed that isopropylamine produced the most stable intensity values. Figure 4 shows a comparison between solutions containing sodium hydroxide, isopropylamine, and isobutylamine. Each of the three solutions contained 1 nil. (50 y) of boron solution and 2 ml. of 0.5% benzoin. The intensities were measured from solutions subjected to continuous irradiation. Solvents. Several solvents were investigated. The authors (6) of the original fluorometric method reported that methyl alcohol was satisfactory for high concentrations of boron. Isopropyl alcohol produced a linear relationship between intensities and concentrations of boron up to 50 y . The highest intensity values were ohtained with 95% ethyl alcohol. These results were confirmed, and other alcohols were tested as the reaction medium. The fluorescence was produced in n-propyl, n-butyl, and isoamyl alcohol, but the intensities were weaker than those in ethyl alcohol. Dioxane, Cellosolve, chloroform, and ethyl ether were also unsatisfactory. Yo appreciable fluorescence was obtained in acetone when sodium hydroxide was used as the base; liowvrver, a fluorescence of moderate intensity wab produced when isopropylamine was used. The maximum solubility of boric acid in acetone a t 25" C. is 0.47 mole yo; this low solubility limited the use of acetone. Formamide and N,N-dimethylformamide are both good solvent media for the boron-benzoin reaction. Sodium hydroxide, potassium hydroxide. and isopropylamine all serve as suitable bases for the boron-benzoin fluorescence in formamide. Isopropylamine produced an intensity in formamide that !\-as comparable with that in ethyl alcohol for identical concentrations of boron. Isopropylamine did not produce an appreciable fluorescence with boron and benzoin in S,S-dimethylformamide. Sodium hydroxide caused a greenish-yellow fluorescence with boric acid and benzoin in X,S-dimethylformamide. The intensity was comparable with that in forninmide. ,4 greenish-yellow fluorescence was produced in dimethyl sulfoxide. None of these solvents seemed to have any immediate advantage over ethyl alcohol. However, in some cases it may prove profitable to use Ar,X-dimethylformamide because of its ability to dissolve a wide variety of materials. Fluorescence Emission and Fluorescence Excitation Spectra. The VOL. 29, NO. 7,JULY 1 9 5 7
1107
fluorescence emission and excitation spectra for the boron-benzoin complex were determined with the fluorometer in conjunction with a Bausch & Lomb monochromator. The details of these experiments and the spectral curves obtained have been published elsewhere (5). The emission spectrum has a strong band between 450 and 520 m r with a maximum a t 280 mp. The excitation is most pronounced from 310 to 400 mp and shows a peak a t 370 mp. Absorption Spectra. The absorption spectrum of benzoin with potassium hydroxide compared t o t h a t of the boron-benzoin complex is shown in Figure 5. Rather concentrated solutions (0.13%) of benzoin and the complex were necessary to show absorption in the region of the spectrum above 300 m l . The curves show that the complex absorbs much more strongly below about 370 mp than does benzoin alone. I n more dilute solutions the complex shows a n absorption peak a t 270 mp and the benzoin curve has a peak a t 245 mp. The absorption curve‘ correlates well with the emission and excitation values given above. The excitation maximum coincides with a region of high absorption and the emission curve overlaps the absorption curve of longest wave length. These are general characteristics for fluorescent substances. Boron-Benzoin Ratio. Freeman and White ( 2 ) have shown that the method of Vosburgh and Cooper ( 3 ) for the determination of the ratio of metal to ligand may be applied to
fluorescent solutions. Curves for the results with various mole ratios of boron and benzoin are shown in Figure 6. These curves indicate that 1 molecule of benzoin is attached to 1 atom of boron in the complex. This result leads to the obvious conclusion that, in analysis, a t least 1 mole of benzoin for 1 of boron is required t o achieve a straight line relationship between fluorescence intensity and boron concentration. CONCLUSIONS
I n the quantitative fluorometric determination of boron with benzoin, a shutter should be placed between the source of ultraviolet light and the solution. The sample should be irradiated only while a reading is taken. A high pressure mercury vapor lamp such as H100A4 or H100B4 with a filter such as Corning No. 5860 is a satisfactory excitation source. The secondary filter should transmit between 460 and 510 mp, such as a Wratten B2 or 2A. Solutions should not be exposed unnecessarily to ultraviolet radiation during the period of preparation and during the intervals between readings. The boron solution should be adjusted to approximate neutrality and the reagents should be added in the following order and ratio: 1 ml. of boron solution (1 to 25 y), 0.5 ml. of water, 0.5 ml. of glyrine buffer (pH 12.8), 1.5 ml. of ethyl alcohol, 3 ml. of 0.5y0benzoin, and alcohol to make a total of 25 ml. All solutions should contain the same ratio
Determination of Urinary D. F.
ADAMS, R.
K. KOPPE,
and
D. J.
of water to alcohol so t h a t the total amount of water in 25 ml. of the final solution is in the vicinity of 2 ml. The benzoin present should be in excess of 1 mole of benzoin for 1 of boron. The fluorescence intensity should be read between 5 and 25 minutes after the solutions are mixed thoroughly. ACKNOWLEDGMENT
The authors wish to express their appreciation to the Research Gorp. for the partial support of this research through a Frederick Gardner Cottrell grant. Acknowledgment is due also to John S. Magee, Jr., for determination of the values used in Figure 6. LITERATURE CITED
(1) Clark, W. SI., “Determination of Hvdroeen Ions.” D. 206. Williams z d W&ins, BiltiAore, i928. (2) Freeman, D. E., Jr., White, C. E., J. Am. Chem. SOC.78, 2688 (1956). (3) . , Vosbureh. W. C.. Coouer. G. R., J . Am, Chem. Soc.’63, 437 (1941).
(4)White, C. E., Chapter on Fluorescence Analysis, “Symposium on Trace Analysis,” Yoe, J. H., Wiley, New York, in press. (5) White, C. E., Hoffman, D. E., Magee, J., Jr., Spectrochim. Acta, in press. ( 6 ) White, C. E., Weissler, A., Busker, D., AXAL.CHEM.19,802 (1947).
RECEIVED for review November 12, 1956 Accepted February 9, 1957. Taken from a thesis by Donald E. Hoffman in partial fulfillment of the requirements for the degree of master of science, Universitr of Maryland. August 1955.
FIuoride
MAYHEW‘
Division o f lndustrial Research, Washington State institute of Technology, The State College o f Washington, Pullman, Wash.
b Recovery of fluoride from urine samples in excess of approximately 10 p.p.m. analyzed by currently accepted procedures was found to b e inversely proportional to the fluoride level. Significant improvements in recovery are achieved through elimination of the chloride removal step by employing the high salt-thorium fluoride titration and calculation of results from a standard fluoride recovery curve prepared by distillation of known quantities of fluoride added to normal urine samples.
T
determination of urinary fluoride is an essential step in establishing the current dietary fluoride intake of HE
Present address, Minnesota Mining and Manufacturing Co., St. Paul, Minn.
1108
ANALYTICAL CHEMISTRY
farm animals (3, 5,9,10, IS). The procedure used in the analysis of animal material for fluoride content is the Willard and Winter perchloric acid distillation (It?), with a subsequent titration of an aliquot of the distillate using thorium nitrate in the presence of Alizarin Red S indicator. Bovine urine normally contailis 0.02 to 0.870 of chloride ( I 5 ) , which distills from perchloric acid at 137’ C. and interferes seriously with the thorium nitrate titration (2). This chloride interference is commonly prevented by adding sufficient silver perchlorate to the sample in the distillation flask (2, 4, 6-8, IO, 11) or by conducting a preliminary silver chloride precipitation and filtration prior to transferring the ashed sample to the distillation flask (IC), for final distillation. Addition of
silver perchlorate to the distillation flask frequently produces a voluminous precipitate of silver chloride, inducing serious bumping during the diitillation procedure. Precipitation and filtration of the copious volume of silver chloride prior to distillation present a difficult and time-consuming washing problem and provide opportunity for adsorption of fluoride with resultant low fluoride recoveries. The Smith and Gardner modification (12) of the salt-acid-thorium titration of Williams (17) for microgram quantities of fluoride has the outstanding advantage of introducing a relatively high concentration of sodium chloride into the titration solution, thus controlling the interference of the chloride ion. By application of this high salt titration procedure it becomes possible
