strate-buffer-indicator solution [0.02M p-toluenesulfonylarginine methyl ester, 0.0 15M tris(hydroxymethy1)aminomethane a t p H 8.2, and 0.00201, phenol red]. Comparative results using the spot plate method are presented in Table I. Acid Titrations. As a standard method of comparison, potentiometric titrations were performed essentially by the procedure of previous investigators (2, 8 ) . Solutions were the same except for the use of 0.005M tris(hydroxymethy1)aminomethane in place of phosphate buffer. Comparative Results. Table I gives a direct comparison of t h e spectrophotometric, spot plate, a n d conventional titrimetric determinations of trypsin and ovomucoid, as well as a comparative value of the ratio of ovomucoid to trypsin. The agreement is good for the three independent methods. DISCUSSION A N D CONCLUSIONS
The described spectrophotometric method for the determination of trypsin and trypsin inhibitors is rapid and very simple, supplies permanent records and allows for direct determination of activi-
ties from the slopes of the recorded curves. It is superior to the titration method for these reasons, and also because of greater accuracy and reproducibility. The method should be adaptable for determinations of other enzymes which produce changes in pH. Indicators and buffers, however, would need to be appropriately selected for each particular case. The spectrophotometric method for determination of acid production can be adapted for use with instruments not connected to recording devices. This would require manual recording of the changes in transmittance with time, which also seems superior to the titrimetric procedure. The quantitative relationships of trypsin and inhibitors found in this study are comparable to those previously reported (4, 5 ) . On a molar basis the ratios are approximately unity. The optimum working range for trypsin mas 1 to 15 y and for the inhibitors, as ovomucoid or the soybean trypsin inhibitor, 3 to 12 y . The apparent reproducibility was 1 5 % . Amounts of enzymes and inhibitors that could be determined were, of coursp, directly dependent upon the concentration of the buffer. The spot plate method should be useful in supplying approximate values
during studies involving fractionation, inactivation of inhibitors, and the like. LITERATURE CITED
(1) Bergmann, M., Fruton, J. S., Pollok, H., J . Biol. Chem. 127, 643 (1939); (2) Cohen, W., Balls, A. K., Poultry Scz.
34,296 (1955). (3) Feeney, R. E., Ducay, E. D., Silva, R. B.. hlacDonnel1. L. R., Zbid., 31,639 (1952). Green, N. M., J . Bzol. Chem. 205, 535 (1953). Lineweaver, H., Murray, C. W., Ibid., 171, 565 (1947). Seurath, H., Schwert, G. W.,Chem. Revs. 46,67 (1950). Rovery, M., Fabre, C., Desmuelle, P Biochim. et Biophys. Acta 12, 527 (1953). Schwert, G. IT., Keurath, H., Kaufman, S., Snoke, J. E., J . Biol. Chem. 172, 221 (1948). (9) Schwert, G. IT., Takenaka, Y . , Biochzm. et Biophys. Acta 16, 570 (19.55) \----,.
(10) Sugihara, T. F., ~IacDonnell,L. R., Knight, C. -i.,Feeney, R. E., Zbid., 16, 404 (1955). RECEIVED for review September 7 , 1956. -4ccepted December 3, 1956. Published with the approval of the Director as Paper No. 784, Journal Series, Nebraska Agricultural Experiment Station, Lincoln, Neb. Presented at 17th Midwest Regional hfeeting, ilCS, ;\mes, Iowa, November 1956.
Ultraviolet Absorptiometric Determination of Boron in Aqueous Medium Using Chromotropic Acid DONALD
F.
KUEMMEL with M. G. MELLON
Purdue University, lafayette, Ind.
Boric acid causes changes in the ultraviolet absorption spectrum of chromotropic acid in aqueous solution of sufficient magnitude to be of analytical importance. An absorptiometric method was developed for determining boron in the range 0.1 to 2.4 p.p.m. by measuring the decrease in the absorbancy of chromotropic acid caused by the addition of boric acid. Measurements were made at 3 16.5 mp, the point of maximum change, with solutions adjusted to a pH of approximately 7.
B
is one of the few common elements whose analytical chemistry leaves much to be desired, particularly in the field of spectrophotometry. The majority of spectrophotometric or colorimetric methods reported for boron involve the use of concentrated sulfuric acid, in which solvent numerous ORON
378
ANALYTICAL CHEMISTRY
organic reagents give color changes in the presence of boron (3, 10, l a ) . 1,2,5,8 - Tetrahydroxyant hraquinone, known commonly as quinalizarin, is a widely used reagent in this class, and is the basis of t h e tentative (“first action”) AOAC method for the determination of boron in soils (6). The most widely used method not requiring the use of concentrated sulfuric acid is that involving the use of 1,7- bis - (4- hydroxy - 3 - methoxyphenyl) - 1,6 - heptadiene - 3,5 - dione, otherwise known as curcumin. The curcumin method depends upon the formation of a colored product upon evaporation t o dryness of a mixture of boric acid, oxalic acid, and curcumin. This colored product is usually extracted into 95y0 ethyl alcohol. Numerous variables, particularly the evaporation temperature, must be carefully controlled for reproducible results. Hom-ever. the curcumin method is
used frequently in plant and soil analysis (4,8), and has recently been applied to the determination of boron in semiconductor materials ( 7 ) . Boric acid forms colorless complexes with a number of polyhydroxy compounds in aqueous medium ( 2 ) . The use of glycerol or mannitol in the alkalimetric titration of boric acid is a practical utilization of these colorless complexes. Many of the compounds reported to form these complexes n ith boric acid absorb ultraviolet radiant energy. It appeared that the addition of boric acid to solutions containing these polyhydroxy compounds would result in changes in their ultraviolet absorption spectra which might have some analytical value. Sndress and Topf (1) have described the changes which occur in the ultraviolet spectrum of a n aqueous solution of disodium 4,5-dihydroxy-2,7-naphthalenedisulfonate upon the addition of
no signs of deterioration 6 days after preparation, and they were used throughout this period in the analytical nork. The 0.0028M solution, on the other hand, has a slight yellow tint upon preparation, which darkens to a definite yellow after a fen- days’ storage in a dark amber bottle. Consequently, the 0.0028M solution should be prepared fresh whenever the 0.00028M solution is to be made from it by dilution. The 2 X solution of sodium acetate trihydrate used to adjust the pH was filtered to remove insoluble matter. Sodium acetate proved more convenient for adjusting the pH than sodium hydroxide, and in addition gave the system some buffer capacity. Stock solutions prepared from different bottles of sodium acetate trihydrate, of the same grade and from the same manufacturer, buffered the system to different pH’s. Consequently, each lot of sodium acetate solution prepared should be checked to see that 10 ml. of the solution in a final volume of 50 ml. will adjust the pH of the system to between 6.8 and 7.0. If necessary, a small amount of 0 . 5 S sodium hydroxide should be added to the sodium acetate stock solution to bring its pH adjusting capacity into the desired range.
15
5
z a a
IO
rb!
s1 B 05
0 290
310
330
350
310
WAVE LENGTH ( m p )
Figure 1. Effect of boric acid on absorption spectrum of chromotropic acid in aqueous solution in pH range 4 to 5
”
0.00014M solution of chromotropic acid, pH 5.1 Solution 0.00014M in chromotropic acid and 0.0431 in boric acid, pH 4.1
____
boric acid. Tlie parent acid of this salt is coniinonly known as chromotropic acid. The contribution of the present investigation was the development of a simple, rapid absorptiometric method for the determination of boron in aqueous solution, utilizing this effect of boric acid on the ultraviolet spectrum of disodium chromotropate. EXPERIMENTAL WORK
Apparatus. All absorption spectra were obtained on a Cary Model 1011RI recording spectrophotometer, using matched 1-em. quartz absorption cells. The slit control dial was kept a t position 1 for the spectra to give a spectral band width of approximately 1 A.
The Cary absorption cells fitted with tapered necks for ground-glass stoppers, intended for use with volatile solvents, proved useful in working with the solutions of high salt content (0.4M in sodium acetate). The solutions were transferred to the absorption cells by means of 50-ml. beakers. Any liquid adhering to the outside of the cells was conveniently removed by dipping the cell in distilled water while holding the cell by the neck during this rinsing procedure. These details are
mentioned here because of the difficulties encountered using the conventional cylindrical cells without necks, which are difficult to hold and to clean when a thin film of concentrated salt solution evaporates on the outside of the cell. Reagents. The boric acid used was a C.P. Baker’s analyzed product. The sodium acetate trihydrate was a Baker and Adamson K.F. grade. The disodium 4,5-dihydroxy-2,7-naphthalenedisulfonate was obtained as a practical grade dihydrated salt from Eastman Organic Chemicals. Any further references to chromotropic acid or its solutions imply this dihydrated disodium salt as the starting material. Preparation of Stock Solutions. ilqueous 0.00028M stock solutions of the disodium chromotropate were prepared both by dilution of 50-ml. aliquots of 0.0028M solutions, and by direct weighing of 0.056-gram portions of the reagent, with subsequent dissolution in distilled water and dilution to 500 ml. The 0.00028M solutions have a p H between 4.5 and 5.0, depending upon the p H of the distilled mater used in their preparation. After a few hours’ standing in the daylight, these solutions develop a slight yellow color. This coloration can be avoided by storing the solution in a dark amber bottle, and keeping this bottle in the dark when not in use. The 0.00028M solutions stored in this manner showed
Tlie standard 0.002V boric acid solution, containing 0.022 mg. of boron per ml., 11-as prepared by suitable dilution of a 0.02X stock solution. Because of the report ( 5 ) that the species present in boric acid solutions changes upon standing, thiee different 0.02M solutions were prepared during this work. S o differences in behavior toward the chromotropic acid system were detected, however, betn-een the freshly prepared solutions and the solutions n hich had stood for a f w weeks.
PRELIMINARY STUDIES
I n absolute ethyl alcohol solution 0.25M boric acid causes only slight changes in the absorption spectrum of a 0.0001M solution of chromotropic acid. I n aqueous medium a t a p H of 4 t o 5, boric acid causes large changes in the absorption spectrum of chromotropic acid, shifting the peaks to longer wave lengths and causing a large increase in absorption in the 350- to 360-mp region. Figure 1 s h o w the effect of 0.04M boric acid on the ultraviolet spectrum of chromotropic acid in this p H range. The acidic boron complex formed, as well as the excess boric acid not involved in complex formation, lowers the p H of the solution. However, hydrochloric acid, added to solutions of chromotropic acid in sufficient quantity to lower the p H to 3.5 and 0.8, causes negligible changes a t the absorption maxima of the reagent, and only small changes at VOL. 29, NO. 3, MARCH 1957
379
the absorption minima. This indicates that the large changes caused by boric acid are not due merely t'o a p H effect. The aqueous chromotropic acid system is much more sensitive to boric acid in the p H range 7 to 10 than in the range 4 to 5 . This increased sensitivity is due both to t'he formation of larger amounts of the complex for a given amount of boric acid, and to a larger absorption change for a given amount of comples. Figure 2 shows the effect of 0.002 aiid 0.00028N boric acid on the spectrum of chromotropic acid at a p H of approximately 7 . Boric acid causes a decrease in the absorption of the reagent in t h r 355- to 380-mp region, with the point of masimum change occurring a t 361.5 mp. The spectra of the i,engent a t p H 7.6, 9.0, and 10.0 are not much different from that given in Figure 2 for the reagent a t p H 6.9, with only small differences a t the 360- and 346-mp peaks. However, to ensure reproducible conditions for the formation of the complex, which is dependent upon pH, the mixtures of chromotropic and boric acids were adjusted to a pH of 6.8 to i . 0 by the addition of 2 M sodium acetate. Calibration Procedure a t pH 7. Solutions for the calibration curve for this system were prepared by adding 25 nil. of t h e 0.00028111 solution of the reagent and 10 nil. of t h e 2M sodium acetate solution to various aliquots of the standard 0.00211 boric acid solution, followed by dilution to 50 ml. with distilled water. The reagent and buffer solutions ivere added by pipet, and in the order indicated. Figure 3 s h o w the plot of the absorbancy decrease a t 361.5 mp us. the boron content of the standard solutions prepared as described above. Each point on the curve is the average of two values, obtained on tn-o separate days using different stock solutions of boric acid, sodium acetate. and reagent. The reagent stock solutions used for this calibration work Tyere prepared by dilution of 0.002831 solutions. The absorbancy decreases used for thc calibration curve were obtained on the Car. instrument. K i t h the standard solutions in the reference cell and a reagent blank (reagent plus buffer) in the sample cell. differential-type spectra are obtained for the solutions containing boron. \\-herein the absorbancy decreases appear as sharp peaks rising above the base line. After the solutions have beeii placed in the proper absorption cell, the instrument is balanced or zeroed a t 400 nipu:where neither the blank nor solutions containing boron show any absorption, before scanning down scale to 350 inp. Figure 4 shows the differential spectra in the 350- to 390-mp region given by the same solu-
380
ANALYTICAL CHEMISTRY
290
310
350
330
370
WAVE LENGTH ( m p )
Figure 2. Effect of boric acid on absorption spectrum of chromotropic acid in aqueous solution a t pH 7
rlll solutions 0.4M in sodium acetate and 0.00014.11 in chro-
niotropic acid 50 boric acid added, pH 6.9 . . 0.00028M in boric acid, pH T.0 - - _ _ 0.002M in boric acid, pH T.0
tions used to obtain the conventional spectra of Figuic 2. Although the concentrations of boron which give the spectra of Figure 4 are above the upper limit of calibration, the differential spectra for these concentrations ale shown for comparison. The reagent blank is placed in the sample cell, rather than in its usual position in the reference cell, because of the decrease in the absorption of the reagent being measured in this system. The intense radiant energy source and photomultiplier detector system of the Cary instrument enable slit widths of 0.25 nim. or Ion-er to be used in obtaining these differential spectra. Because of the critical setting of the wave length (eliminated on the Cary because of the scanning procedure) and the necessitjof zeroing the instrument on highly absorbing solutions, other manual spectrophotometers a t hand were not considered for this work. However, instruments such as Beckman Models B and DU, for which photomultiplier attachments are available, could probablj lic used if sufficient care is exercised in setting the m v e length dial.
Stability of System. The mixtures of chromotropic acid and sodium acetate, with or without boric acid, develop pink t o red hues after a f e n minutes' standing in strong sunlight. Because of this light sensitivity, exposure to daylight was kept a t a niininium during the preparation of the saniples. The reagent and sodium acetate solutions weie added to two or three samples a t a time; the samples were diluted to volume, mixed, and placed in a convenient laboratory cabinet while other samples n ere being prepared and until the group was ready to be run on the instrument. KOsign of color was observed 11-hen this piocedure was followed, and the system has been shown to be stable for a t least 18 hours if unnecessary exposure to light is avoided. Stability of the solutions for longer periods was not investigated. As no color was observed in solutions xhich had stood in the fluorescent light of the laboratory at night, precautions such as those mentioned above were not so necessary for samples run at this time, although prolonged exposure to the fluorescent lights was also avoided.
rn3. 8 / 5 0
The intense ultraviolet source of the instrument also appeared to have a slight effect on the system. Because of this, a fresh port'ion of the sample solution should be placed in the reference cell if a repeat run on the same sample is desired. The same portion of the reagent blank can be used in the sample cell for obtaining a number of differential spectra, as the reagent hlank appears to be relatively insensitive to the ultraviolet sourre. For a rathrr large series of samples, however, it is reconiniended that a fresh portion of t h r reagent blank be placed in the sample cell for every four or five samples 1~1111 on the instrument. Accuracy and Precision. A number of samples of varying boron contents n-ithin t h e calibration limit \ \ m e r u n over a 22-clay period t o test t h e accuracy and precision of the proposed method. These t'est samples n-ere prepared in the same manner as the solutions used for calihration-that is, by addition of the reagent and sodium a(.(.tate solutions to various aliquots of tlie standard boric acid solut'ion. Thc niajority of boron concentrations invol\-ed were different from tliose used for tlici mlibration ( u r w . Differential-t'ype spectra were obtaintd in the 400- t o 350-nip rrgion for tlicee test solutions, using a freshly prcpare(l reagent hlank for each group of samples. The allsorbancy decreases ohtained in this manner were referred to the calibration curve in Figure 3 to arrive a t the boron content of the solutions. The samples n-ere run in nuniwous groups of four to seven during thc 22-clay period, using 14 different' 0.00028.11 stock solutions of the reagent. T\wlve of these werc p ~ e pared by wigliiiig out individual 0.056-gram portions of the rragent, the othcr two hy dilution of 0.0028M stock solutions. Results of this study are summarized in Table I.
ml.
Figure 3. Calibration curve for chromotropic acid system in aqueous solution in pH range 6.8 to 7.0
DISCUSSION
360
375
360
315
WAVE LENGTH b p . )
Figure 4. Differential type spectra of same solutions used to obtain spectra in Figure 2
-4. Solution 0.00028M in boric acid R . Solution 0.002M in boric acid
Table 1.
Results of Accuracy and Precision Tests on Chromotropic Acid Method for Boron (All concentrations expressed as mg. of boron/50 ml.)
Range of Boron Concn. 0.009-0.043 0.048-0.087
0.091-0.115 a
x o . of
Individual Concn.0 11 11 6
s o . of
Separate Detn. 16 24 23
Deviations from Ihown 1-alms Range Average -0.002-+0.002 0.0011 -0 006-+ 0.007 -0.007-+0.010
0.0028
0,0044
Sumber of different boron levels investigated within each concentration range.
The react'ion of boric acid with chromot'ropic acid is apparently an equilihrium reaction resulting in the forniatioii of a complex, and is sliift,ed tomud the formation of giwter quantities of the complex by fuitiicr nihlitions of boric acid. The plot of the absorbancy change (increase or ilec.rease) us. tlie boron concentration a t hoth a pH of 7 and in the rangc 4 t o 5 is not a straight line, and approaclirs a logarithmic wlationship. The specti,uni in Figure 2 given by the mixtui,c containing a 0.002M concmentrntion of boric acid is very similar to the spwtrum in Figure 1 of the mixture containing a 0.01M ('onccnt'ration of boric. acid. Conversion of the reagent into the complex is nearly conipletc in t,hcsc, tn-o niistures (as intlicated by a "lerc>liiigoff"' of the ahsorbancy changes a t approsimately these VOL. 2 9 , NO. 3, MARCH 1957
381
concentrations), and the fact that they give similar spectra suggests that the spectrum of the complex is not affected by p H changes in the range 4 to 7 . However, the amount of complex formed is dependent upon pH, 20 times as much boric acid being required in the p H range 4 to 5 as a t p H 7 to form approximately the same amount of complex. Nothing could be concluded from the experimental work as to the nature of the complex present in the mixtures of chromotropic and boric acids. The generally accepted structures for complexes of boric acid n-ith poly-hydroxy compounds such as chromotropic acid are given below. Both complexes could be present in the mixtures, and contribute to the observed absorbancy changes. The proton released in the formation of the Type I1 complex would account for the lowering of the p H noted in Figure 1.
Type I Reagent stock solutions prepared by dissolving 0.056-gram portions of the reagent in 500 ml. of distilled water did not give a very reproducible system for the higher boron concentrations investigated. Varying absorbancy decreases were observed r i t h different stock solutions for the same amount of boron present. This lack of reproducibility among reagent stock solutions is believed due in part to errors involved in weighing such a small amount of reagent, coupled with the fact that the reagent itself was an impure. practical grade material. The preparation of a concentrated stock solution, using a large Fveight of the reagent, followed by dilution to the concentration desired, is in effect taking a n average of many small portions of the solid reagent. Stock solutions prepared in this manner would be expected to lead t o a more reproducible system as far as the quantity of reagent is concerned. Alternative paths to the same end of greater reproducibility would be to obtain a better grade reagent, or to purify the practical grade material by suitable recrystallization techniques. Although the light sensitivity of the system is an inherent disadvantage of the method, it causes no great inconvenience in processing the samples, if the proper precautions are taken. The proposed chromotropic acid method is definitely less tedious and time-consuming than methods used a t present.
382
ANALYTICAL CHEMISTRY
It is applicable to a wider range of boron concentrations, but, conversely, does not have the sensitivity of many of the established methods. However, utilization of 5-cm. absorption cells would probably increase the sensitivity of the chromotropic acid method to the point where it would be comparable to other methods. Welcher (11) states that titanium, magnesium, aluminum, nitrous acid, chromates, dichromates, and numerous other oxidizing agents give colored products with chromotropic acid. Yoe and Sarver (IS) reported that zirconium, uranium, and iron can be determined using the colored systems which chromotropic acid gives with these elements. Vanadium also gives a colored system with this reagent (9). Because of these interferences, and the possibility of separating boron by distillation a s methyl borate, the effect of extrane-
Type I1
ous ions on the chromotropic acid system was not investigated. It is known, however, that silicon causes interference in the method. Low results obtained using a certain lot of 2M sodium acetate solution were traced to the presence of silicon in this solution. The silicon originated from the 0 . 5 N sodium hydroxide solution which had been added to the sodium acetate solution to adjust its buffering capacity. The 0.5N alkali had been stored in a borosilicate glass bottle for some time, and contained appreciable quantities of silicon leached from the bottle. Silicon does not affect the shape of the differential spectra in any way other than reducing their intensity. This element apparently, through some unknown mechanism, affects the equilibrium between chromotropic acid, boric acid, and the complex with the result that less complex is formed. The results given in Table I indicate that the proposed method is sufficiently accurate and precise to be used for the determination of boron present in quantities ranging from 0.005 to 0.12 mg. per 50 ml. (0.1 to 2.4 p.p.m.). Boron concentrations higher than the calibration limit in Figure 3 can be determined by this method, particularly if a Cary recording spectrophotometer is used, which can easily measure absorbancies up to 1.50 units. However, because of the parabolic nature of the calibration curve, the system is inherently less ac-
curate and precise a t these higher boron levels, where relatively large differences in boron concentration give only small differences in absorbancy. The purity of the chromotropic acid used also becomes increasingly critical where large quantities of boron are to be determined. Because of the loss in accuracy and precision, and because of the difficulties encountered in preparing reproducible stock solutions of the reagent, the system was calibrated only u p to 0.12 mg. of boron per 50 ml. RECOMMENDED PROCEDURE
Adjust the aqueous solution containing boron, obtained by suitable previous preparative treatment of the sample. to a p H between 4.5 and 6.5. The volume of this solution should be 15 ml. or less after the p H adjustment. Add 25 ml. of a 0.00028M solution of disodium chromotropate and 10 mi. of 2M sodium acetate, dilute to 50 ml., and mix. Avoid exposure to strong light after mixing. Place the sample in the reference cell, and the blank (reagent plus buffer) in the sample cell. Zero the Cary instrument a t 400 mp, and then scan down scale to 350 mp. Obtain the boron content of the sample by referring the absorbancy of the differential peak a t 361.5 mp to a calibration curve, previously constructed by carrying samples of known boron content through the same procedure. LITERATURE CITED
(1) Andress, K., Topf, W.,2. anorg. u. allgem. Chem. 254, 52-64 (1947). (2) Boeseken, J., in “Advances in Carbohydrate Chemistry,” vol. 4, pp. 189-210, ilcademic Press, New York, 1949. (3) Cogbill, E. C., Yoe, J. H., Anal. Chim. Acta 12, 455-63 (1955). (4) Dible, W. T., Truog, E., Berger, K. C.. XSAL. CHEW 26, 418-21 (1954):
(5) Grebenshchikov, I. V., Favorska, T. A,, J . Russ. Phys. Chem. SOC.61, 561-74 (1929). (6) Horwitz, W., ed., “Official Methods of Analysis of the Association of Official Agricultural Chemists.” p. 38, 1955. ( 7 ) Luke, C. L., ANAL.CHEX.27,1150-3 I
fl95.5) \----/.
(8) Naftel, J. A., IXD.E N G . CHEnI., AXAL.ED. 11, 407-9 (1939). (9) Sandell, E. B., “Colorimetric Determination of Traces of hletals,” pp. 264-5, Interscience, Nen- York, 1950. (10) Snell, F. D., Snell, C. T., “Colori-
metric Me_thodsof Analysis,” vol. pp. 103-4, Van Nostrand, New York, 1949. (11) Welcher, F. J., “Organic Analytical Reagents,” vol. 1, pp. 243-8, Van Nostrand, New York, 1947. (12) Welcher, F. J., Ibicl., vol. 2, p. 336; vol. 4, pp. 432,465,539. (13) Yoe, J. H., Sarver, L. A,, “Organic Analytical Reagents,” p. 219, FTiley, Xew York, 1941. 2,
RECEIVEDfor review April 25, 1956. Accepted Sovember 27, 1956.
