The Anthrone Reagent Application to Determination of the Heptuloses L. P. ZlLL Biology
Division, Oak Ridge National Laboratory, Oak Ridge, Tenn.
Evidence is presented that implicates nitrate ion in the widely recognized instability of the anthrone reagent. Increased stability of the reagent is obtained by the use of low anthrone and acid concentrations and by the addition of thiourea, as proposed by Roe. The anthrone reaction may be applied to the quantitative determination of the heptuloses.
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investigations in n-hich a single, rapid reaction could be applied to the detection and determination of many sugars separated by the ion exchange chrometography of their borate complexes (6, 19). During the previous investigations, observations were made on the instability of the reagent, the stability of the color formed in the reaction, and the extension of the method to the determination of the seven-carbon sugars. These observations are reported in this paper.
HE anthrone reagent, which was originally used for the de-
termination of glycerol ( 1 4 ) ,found a rapidly expanding application to diverse problems of carbohydrate determination n ith the demonstration by Dreyn-ood (3) that anthrone is almost a group-specific qualitative reagent for carbohydrates. Subsequently, illorris (9) showed that anthrone can be used for the quantitative determination of hexoses. Since that time, eutensive literature has dealt with the applications of the method and with the control of intrinsic variables encountered in the reaction. Outstanding factors of the method are simplicity of procedure and great sensitivity> 1% hereas instability of the reagent has been repeatedly mentioned as a disadvantage. Although the anthrone reaction is nonspecific for single sugars, this nonqm4firity hac presented certain advantages in previous
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Figure 1. Absorption spectra of aged anthrone reagents 0.5% anthrone and 1% thiourea in 66% &SO4 (XaXOs added in solid form t o gire ca. 0.0002% nitrate) freshly prepared and standing 10 minutes after addition of NaNOs 0.2% anthrone in 95% H&04 (containing 0.00005% nitrate) aged for 16 days a t 4 O C. - 0.5% anthrone and 1% thiourea in, 66% &So4 (undiluted acid containing 0.0000570 nltrate) aged for 18 days a t 4 O C.
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PREPARATIOIY AND STABILITY OF ANTHRONE REAGEXT
Crystalline anthrone x a s obtained from the Matheson Co., Inc., East Rutherford, N. J. It has been reported that anthrone obtained from various commercial sources varied widely n ith regard to its stability in sulfuric acid (16). However, hlatheson anthrone is uniform in acid stability, when prepared in acid samples having a single batch number. Anthrone from other sources was not investigated. Great variability of reagent darkening vith time n-as encountered xhen portions of a single anthrone sample \\'ere dissolved in samples of acids having differing amounts of impurities, as indicated by the assay values given on the bottle. This variation in stability v a s traced to the nitrate ion concentration of the sulfuric acid. The concentration of this interfering anion varied widely among the various batches from a *ingle qource and those from different commercial sources. On the basis that discoloration of the reagent was the result of an oxidative change, Roe ( 1 1 ) proposed an anthrone reagent containing 1% thiourea and 0.05% anthrone in 66% sulfuric acid. The thiourea, functioning as an antioxidant, was assumed to retard or prevent the formation of extraneous color in the reagent. This reagent and the commonly employed 0.2% anthrone in 95% sulfuric acid were investigated. Samples of these t\vo reagents were prepared and allowed to stand in the refrigerator a t approximately 4" C. for 16 dajs. The absorption spectra of these two samples n-ere then determined, freshly prepared reagents being used as blanks. These curves represent the spectral properties of the material or materials causing discoloration in older anthrone reagents (Figure 1). A possible interpretation of the bathochromic absorption shift in the more concentrated acid is given in the discussion. Evidence of the implication of nitrate ion in the darkening of the reagent is shovn in Figure 1. Nitrate ion added to 10 ml. of anthrone reagent prepared according to Roe (11) produces in 10 minutes a t room temperature an absorption spectrum identical to that obtained by aging of the reagent for several days. To avoid introduction of water, the nitrate ion was in the form of a 50-7 crystal of sodium nitrate, producing a concentration of ea. 0.0002%. The absorption spectrum strongly indicates that the darkening of the reagent is dependent on the presence of nitrate ion. To determine the role of thiourea in the retardation of this darkening, two samples of the Roe reagent (one with and one nithout thiourea) mere prepared and allowed to stand in a refrigerator a t 4" C. for 15 days; the absorption spectra then were determined n-ith fresh reagent for blanks. These spectra demonstrate that the absorption a t 428 mp, in the presence of thiourea, is somewhat less than in its absence (Figure 2 ) . .I more pertinent observation is that lower anthrone and acid concentrations provide a much greater decrease in the darkening of the reagent. An identical change is obtained when freshly
1577
A N A L Y T I C A L CHEMISTRY
1578
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Figure 2. Absorption spectra for aged anthrone reagent Samples kept a t 4" C. for 15 days
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0.05% anthrone in 66% Hd30r (0.00005% nitrate) 0.06% anthrone and 1%. thiourea in 66% &SO4 (O.Ooooo% nitrate)
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prepared anthrone reagents are heated a t 100" C. for various lengths of time (Figure 3). The effeqt of increased nitrate ion concentration is clearly shown by the enhanced formation of color in the acid that contains 0.00005% nitrate ion as compared to that containing 0.00002% nitrate ion. The former concentration is the maximum allowable in reagent grade sulfuric acid under the revised specifications for reagent chemicals of the AMERICAN CHEMICAL SOCIETY (1). Reagents prepared in 95% sulfuric acid having a nitrate ion concentration of 0.0001% darkened within a matter of hours, whereas those prepared from acid with 0.00002% nitrate ion were stable for several days a t room temperature and for much longer times when stored in a refrigerator a t 4' C. Addition of nitrate ion (as sodium nitrate) to raise the nitrate concentration to 0.0007% caused immediate and perceptible darkening of the reagent. The absorption spectra obtained by extended heating of various bugars with anthrone have a maximum a t approximately the same wave length as that formed by heating the reagent alone (ca. 427 mp). It was desirable, therefore, to determine the magnitude of color production by the reagent a t this wave length, under the heating conditions necessary to produce this maximum with a sugar. Figure 4 presents the absorbance of a series of blanks that were heated for various times and read against an unheated blank. I t is apparent that, in the reagent in concentrated acid, the absorption a t ca. 420 mM prohibits its use a t this wave length. However, the reagent proposed by Roe possesses a fairly low and uniform background absorption that, though not desirable, does not prevent the use of the reagent a t these wave lengths. The presence or absence of thiourea apparently exerts little effect a t this elevated temperature. Discussion. It has been noted in this laboratory that anthrone reagents permitted to stand undisturbed a t room temperature for extended times develop the typical discoloration in only the topmost parts of the liquid, whereas the bottom part remains almost as yellow as the freshly prepared reagent. This would appear to implicate atmospheric oxygen in the process of discoloration. It has been reported that significant amounts of anthraquinone and dianthrone are formed when anthrone is dissolved in sulfuric
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Figure 3. Absorption spectra obtained by heating anthrone reagents having different nitrate ion concentrations A. B.
0 2 7 @anthrone in 95% H z S O I having 0.00002Ye nitrate 0.Z7c anthrone in 95% H z S O I having 0.000057c nitrate
Spectra obtained *th 15-minute heating _ _ - - - Spectra obtained with 45-minute heating
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20 30 40 50 60 HEATING TIME (rnin) Change in absorption at ea. 420
Figure 4. rnw with various times of heating
All samples read in Evelyn colorimeter using 420-mr filter against unheated blanks prepared in same manner 0 10 ml. of reagent (0.2% anthrone in 95% HzSO4) Plus 5 ml. of water 0 10 ml. of reagent (0.05% anthrone in 66% HzSOd plus I ml. of water A 10 ml. of reagent (0.05% anthrone and 1 % thiourea in 66% HtSOd) plus 1 ml. of water
V O L U M E 28, NO. 10, O C T O B E R 1 9 5 6
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This interference is also attributed to the nitration of the compounds being measured. Further support for discoloration of aromatic compounds in sulfuric acid containing nitrate ion is found in the method reported by Holler and Huch (6). Quantitative colorimetric determination of nitrate by the nitration of 3,4xylenol to form 6-nitro-3,Pxylenol was carried out a t room temperature in 80% sulfuric acid. Nitration products of the various xylenols have absorption maxima between 410 and 446 mp, a range that includes the maximum observed for nitration of the anthrone reagent.
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APPLICATION OF ANTHRONE REAGENT TO DETERMINATION OF HEPTULOSES
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During a study of the action of acid on sedoheptulosan (2,7anhydro-8-D-altroheptulopyranose),it was found that not only the free sugar sedoheptulose (D-altroheptulose), but a furfural derivative, 5-( I,2-dihydroxyethy1)-2-furfura1dehyde1 was formed (20). As it has been demonstrated that the initial formation of furfural or a furfural deiivative is required for the anthrone test ( l a , IS),it appeared that a positive anthrone reaction should be obtained with the heptuloses. The orcinol test, which is commonly used for the determination of sedoheptulose and sed* heptulosan, exhibits a linear absorption with concentration only up to ea. 50 y (10, 80); thus a method having a larger range is desirable.
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Figure 5. Absorption spectra of products formed by reaction of sedoheptulosan with anthrone 0.2% anthrone in 95% HzSOc Absorption spectra obtained by heat of mixing 10 ml. of anthrone reagent with 5-ml. sample containing ZOO y of sedoheptulosan _ - _ - - Same sample after heating for 10 minutes at looo C. B . 0.05% anthrone and 1% thiourea in 66% HIS~~.. Three curves are for 10 ml. of reagent and 1 ml. of sample containing 200 y sedoheptulosan after various times of heating. Heat of mixing only - - _ - - 20-minute heating a t 100' C. 60-minute heating a t 100" C. A.
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acid having a concentration greater than 85% and is allowed to stand a t 30" C. for 2.5 hours (7). Furthermore, bubbling of air through this solution for 12 hours resulted in almost quantitative conversicn to dianthrone (7). On the basis of these considerations it would appear that the initial step is oxidation of anthrone to dianthrone, which is partially prevented by thiourea, followed by nitration to produce the actual extraneous color. The bathochromic absorption shift observed in the concentrated acid may be a result of the change in acid concentration ("solvent effect")or, more probably, of the introduction of more than one nitro group on the molecule. The latter assumption is supported by the observation that absorption maxima move progressively to longer wave lengths as the number of double bonds in the molecule increase (8). It appears that inhibition of the nitration reaction is not possible. However, several factors can be controlled to retard the darkening of the reagent: Sulfuric acid having the lowest nitrate concentration available should be used; low acid concentration in the reagent will prevent the optimum conditions for nitration; storage in a refrigerator a t 4' C. strongly retards the reaction rate of nitration; and addition of thiourea retards the oxidation of anthrone to products that produce discoloration during nitration. The reagent proposed by Roe is satisfactory, except for its decrease in sensitivity as compared to the high sensitivity of the concentrated reagent. Whetsel (16, 1 7 ) has pointed out the interference caused by nitrate ion in sulfuric acid on the spectrophotometric determination of anthraquinone, benzanthrone, and 2,2'-dibenzanthronyl.
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Figure 6. Variation of absorbance with concentration of mannoheptulose and sedoheptulosan in anthrone reaction 20-minute heating of 1-ml. sample with 10 ml. of Roe reagent for each poiqt. Samples read in an Evelyn colorimeter using 515-mp filter 0 Mannoheptulose 0 Sedoheptulosan
Figure 5 gives the absorption spectra obtained upon application of the anthrone reagent to samples of sedoheptulosan. The absorption maximum a t ea. 515 mp was chosen for use in quantitative determinations. Maximum color development a t this wave length is obtained with 20 minutes' heating a t 100" C. The magnitude of response and the linearity of absorption with concentration are given in Figure 6 .
A N A L Y T I C A L CHEMISTRY
1580 Discussion. The application of the anthrone reaction to the heptuloses is another illustration of the nonspecificity of this reagent within the carbohydrates. It is possible that conditions can be established for the determination of all five-, six-, and seven-carbon sugars. Hydrolysis and dehydration by the sulfuric acid solvent in the reagent are primary factors in the application of this method to a wide range of compounds. Because the over-all accuracy of a colorimetric method depends in part on the susceptibility of the color to fading, the transient blue-green color produced by pentoses has been used in only two recorded cases ( 2 , 4 ) . Rapid loss of color was retarded by rigorous temperature control. Anthrone reagent prepared in 66% sulfuric acid provides a blue-green color with pentoses that is comparable in stability to that of the hexoses. The maximum absorption a t 625 my obtained in the reaction of hexoses with anthrone also represents an unstable intermediate, as longer heating causes a large decrease in this absorption. K i t h glucose (15),it has been shown that this absorption loss is superseded by the appearance of an absorption maximum at 425 my. In the present work the variation of absorption a t this wave length, with sugar concentration, was found to be linear; however, because of the additional heating time required and the background color produced by nitrate ion, this method has not been used.
presented in this paper. Thanks are also extended to A. A. Benson, who supplied the sample of mannoheptulose.
ACKNOWLEDGMENT
RECEIVED for review February 4, 1966. Accepted June 22, 1956. Work performed under Contract W-7406-eng-26 for the U. S. Atomic Energy Commission. Preliminary report glven a t meeting of Federation of American Societies for Experimental Biology, April 16 to 20, 1956 (abstract, 18).
The author wishes to acknowledge the valuable assistance of D. A. Mondon during various phases of the experimental work
Cellophane-Dye Dosimeter for ERNEST J. HENLEY
and
LITERATURE CITED
(1)
hfERICAN
1956. (2) Bridges, R. R., ANAL.CHEW24, 2004-5 (1952). (3) Dreywood, R., IND. ENG.CHEY., AXAL.ED. 18, 499 (1946). (4) Gary, S . D., Klausmeier, R. E., ANAL.CHEM.26, 1958-60 (1954). (5) Holler, A. C., Huch, R. V.,I b i d . , 21, 1385-9 (1949). (6) Khym, J. X., Zill, L. P., J . Am. Chem. SOC.74, 2090-4 (1952). (7) Koalov, V. V., Kudelina, K. I., J . Gen. Chem. U.S.S.R. 17, 302-8 (1947). (8) Lewis, G. K., Calvin, RI., Chem. Rem. 25, 273-328 (1939). (9) 1Iorris. D. L.. Science 107. 254-5 (1948). (IO) Xordal, A, Klevstrand, R:, Anal. Chim’. Acta 4, 411-21 (1950). (11) Roe, J. H., J . Biol. Chem. 212, 335-43 (1955). (12) Sattler, L., Zerban, F. W,, J . Am. Chem. SOC.72, 3814 (1950). (13) Battler, L., Zerban, F. W.,Science 108, 207 (1948). (14) Schutz, F.,Papier-Fabr. 36 (Tech. Tl.), 55-6 (1938). (15) Scott, T. A., Jr., Rlelvin, E. H., ASAL. CHEM. 25, 1656-61 (1953). (16) Whetsel, K. B., I b i d . , 25, 1334-7 (1953). (17) I b i d . , 26, 1974-7 (1954). (18) Zill, L. P., Federation Proc. 15, 391 (1956). (19) Zill, L. P., Khym, J. X., Cheniae, G. XI., J. Am. Chem. SOC.75, 1339-42 (1953). (20) Zill, L. P., Tolbert, K.E., Ibid., 76,2929-33 (1954).
IO’ to IO’ Roentgen Range
DAVID RICHMAN’
Department o f Chemical Engineering, Columbia University, N e w York 27,
Experimental work in radiation chemistry and biology necessitates careful measurement of the radiation dose used. This paper describes a recording integrating chemical dosimeter, capable of measuring gamma and beta doses from 200,000 to 10,000,000 roentgens. Calibration curves are presented for 0.8-, 2-, and 3-m.e.v. beta radiation, and for a cobalt-60 gamma source over a dose rate range of 106 to 107 roentgens per hour. The dosimeter is fabricated from commercially available cellophane, and is convenient, inexpensive, and stable.
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OR process control, equipment design, and safety it is necessary to measure the effects of atomic radiation quantitatively. Most well-developed systems now in common use are biological monitors, operating a t levels up to 50 roentgens. In the higher ranges (loj to 107 roentgens) there are several chemical systems in use, most of which require analytical chemical techniques before dose exposure can be ascertained. Many have been suggested and actually calibrated Yith some degree of care; these include: ( a ) oxidation-reduction dyes in agar ( 2 , 13); ( b ) benzene or sodium benzoate in water ( 3 ) ; 1
Preaent address, Brookhaven National Laboratory, Upton, Long Island,
ti.Y.
CHEMICAL SOCIETY, “Reagent Chemicals,” p. 400,
N. Y.
(c) degree of polymerization of various monomers ( I d ) ;
( d ) reduction of methylene blue dyes in water (4,1 7 ) ; (e) phosphate and cobalt glasses (8, 16); (f) depigmentation of rats (11); ( 9 ) poly(viny1 chloride) films, in which a redox dye is incorporated (6); ( h ) ferrous-ferric oxidation in 0.8N sulfuric acid (10, 19); (i) ceric-cerous reduction in 0.8Nsulfuric acid ( 7 , 19). For monitoring applications in radiation sterilization or radiation chemistry, cost, convenience, and reliability are the chief criteria. Of the nine dosimeters described only the poly(viny1 chloride) films and phosphate and cobalt glasses approach these specifications. Unfortunately, both have severe limitations. The phosphate glass is subject to fading, is expensive, and has a poor radiation response above 1,000,000 roentgens. The poly(vinyl chloride) films are expensive and not generally available. In view of the shortcomings of the present dosimeters, an investigation of some commercially available polymeric systems which could be adapted for this use was initiated. Of the many tested, a Du Pont moisture-proof, heat-sealable cellophane, containing a dimethoxydiphenylbisazobis-8-amino-l-naphthol-5,7disulfonic acid dye (14) proved to be most suitable. One of the advantages of this system is the stability of the dye. It is relatively insensitive to pH, light, and heat. Films stored in the dark for periods of 2 years do not change optically. They fade only on exposure to temperatures over 60’ C. for long periods of time-e.g., 10 hours gives a readable change.
