(14) Cocker, J. D., Nalsall, T.G., Bowers, A., J . Chem. SOC.1956, p. 4262. (15) Coggeshall, N. D., Glassner, A, S., Jr., ANAL.C a w . 21, 550 (1949). (16) Dannenberg, H., Dannenberg-von Dresler, D., Chem. Ber. 89, 1326 (1956). (17) Evans, R. F., Smith, J. C., Strauw, F. B J . Inst. Petrol. 40, 7 (1954). (18) Frjedel, R. A,, Orchin, X, “Ultraviolet Spectra of $somatic Compounds,” Wiley, Sew York, 1951. (19) Heilbronner, E., Froehlicber, U., Plattner, P1. A., hlelv. Chim. Acta 32, 2479 (1949). (20) JaffB, 33. H., Orchin, M., “Theory and Applications of Ultraviolet, Spectroscopy,” p. 303, Wiley, h’ew York, 1962. (21) Karr, C., Jr., Childers, E. E., Warner, W. C., ANAL.CHEM.35, 1290 (1963).
(22) Karr, C., Jr., Childers, E. E., Warner, W. C., Estep, P. A,, Ibid., 36, 2105 (1964). 1231 Karr. C.. Jr.. Comberiati. J. R.. A., J :
J., J . Miller, :1959).
126) Mieveris. H.’ B.. ‘Plitt, J. R., J . Chem. Phyi. 17, 47d (1949): (27) Kruber, O., Raeithel, A., Chem. Ber. 8 5 , 327 (1952). ( 2 8 ) Morton, R. 9., deGouveia, A. J. A,, J . Chem. SOC.1934,p. 916. (29) hlosby, W. L., J . Am. Chem. SOC. ’9’7, 755 (1955). (30) Mosby, 1%’. L., Ibid., ’95, 3348 (1953). (31) Neimark, M. E., Kogan, I. E., Bragilevskaya, 31. M., Sb. h’auchn. ~
CHARLES J. ROGERS, CECIL W. CHAMBERS, and ~~~~A~
Tr. Ukr. NauchnAssled. Uglekhim. Inst. 14, 105 (1963). (32) Ruaicka, L., Schinz, H., Mueller, P. H.. Helv. Chhim. Acta 2’7. 195 11944). --,_ (33) Schoepf, C., Klein, D.; Hofmann, E., Chem. Ber. 8’7, 1638 (1954). (34) qnyder, L. R., h N A L . CHEM. 33, 1530 (1961). (35) Snyder, L. R., J. Chromalog. 6, 22 (1961). (36) Snvder. L. R.. Warren, II. D.. ~ b i d . , ” i‘34q1964). ~, (37) Stoll, M., Seidel, C. F., Wilhalm, B., Hinder, bl., Helv. Chim. Acta 39, 183 (1936). ~~
RECEIVEDfor review July 14, 1966. Accepted September 23, 1966. Presented before the Division of Fuel Chemistry, 152nd Meeting, ACS, New York, N. Y., September 1966.
A. CLARKE
Cincinnati Wafer Research Laboratory, Robert A mTaft Sanifary Engineering Cenfer, Cincinnati, Ohio
b
Furfurals resulting from the dehydration of carbohydrates in strong hydrochloric acid condense with resorcinol to yield stable derivatives. These derivatives produce intense fluorescence in basic solution. The fluorescent compounds are presumably xanthenone derivatives of t h e corresponding sugar that fluoresce a t 508 mp when activated a t 488 mp. An analysis can ~ e a~ ~ b e completed in 20-30 ~ i n u and linear relationship is produced for carbohydrates in the nanogram ran
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for a rapid and sensitive method for microdeterniination of carbohydrates, the Seliwanoff reaction ( 1 ) was investigated. Although this reaction is used for detecting ketoses, theoretically, under proper conditions, all. carbohydrates should react. I n strongly acidic solution carbohydrates are hydrolyzed to monosaccharides and dehydrated to furfurals, which condense with resorcinol and presumably yield xanthenoce derivatives. The rate of formation of these derivatives varies with different sugars, concentration of acid, and temperature. An adjustment of the pH produces intense fluorescence, which may be employed for the quantitative determination of carbohydrates. The o-phenylenediamine fluorometric method (9) commonly used for determining carbohydrates is not as sensitive as the method described here, and requires 3 hours of heating the reaction to measure microgram quantities. N A SEARCH
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FLUORESCENCE WAVELENGTH,rnp
Figure 1 . Curves showing fluorescence intensity of 1 .O prnole/ml. of arabinose, rhamnose, and galactose
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Heated for 15 minutes ut 1 10R-150C.in 3 ml. of concentrated hydrochloric acid
VOL. 38, NO. 13, DECEMBER 1966
e
1351
0.50
Figure 2. Curves showing the effects of concentrated hydrochloric acid on a 1.0 X I O v 3 pmole/ml. arabinose solution after final dilution Reaction in 2.5 ml. of hydrochloric acid,
0;5 ml. of hydrochloric acid,
hydrate solution. T o each tube were added 5.0 ml. of concentrated HC1, 0.5 ml. of resorcinol reagent, and distilled water to achieve a total volume of 6.5 ml. A. reagent blank was prepared containing 5.0 ml. of concentrated EIC1, 0.5 ml. of resorcinol reagent, and distilled water to adjust the volume to 6.5 ml. Caps made of Teflon (DuPont) were placed tightly on the tubes, and the tubes were placed in a 108-10" C. oil bath for 30 minutes. After cooling, the contents of each tube were transferred t o 100-ml. beakers and the pH was adjusted to 8.5-9.5 with NaQH. These volumes were then adjusted to 100 ml. with distilled water. The fluorescence was measured and the reagent blank subtracted from all readings. There was a linear relationship observed for concentration us. fluorescence intensity in the range of 3.75-15.0 ng. RESULTS AND DISCUSSION 0.00 0.0
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I n the reaction of a sugar with resorcinol, a t least three fluorescent deriva-
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TIME, minutes
EXPERIMENTAL
Apparatus. Fluorescence mas measured with a Aminco-Bowman spectrophotofluorometer equipped with a n RCA 1P21 phototube. The slit widths for the analysis were those described in arrangement No. l except that the photomultiplier slit was 3//18 inch. The recorded fluorescence intensity was that observed at 508 mp when activated at 488 mp and is expressed as meter multiplier times transmission. Reagent. Resorcinol reagent was prepared by dissolving 50 mg. of resorcinol in 20 ml. of 66% HC1 solution. Carbohydrates. These solutions were prepared by dissolving 0.1 pmole of each sugar in a liter of water. The following sugars were tested: d(+) glucose, d(+) galactose, d(+) mannose, I(+) rhamnose, D(-) ribose, L( -) arabinose, and I ( -) fucose. Preparation 01 Standard Curve. h solution was prepared containing 1.5 X 10-6 pmole/ml. of n-ribose. Into a series of four 25-ml. tubes were placed 0.25- to 1.0-ml. aliquots of the carbo-
Figure 3. Curves showing the temperature dependence of a reaction of resorcinol and a ribose solution in 5 ml. of concentrated hydrochloric odd Final carbohydrate concentration was 1 .O X lo-* !.&mole/ml. peratores were 85' C., 0 ; 95' C., A; and 1 1 5 ' C., 0
1852
e
ANALYTlCAL CHEMI§TRY
Tem-
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tives are formed. These were detected by extracting the derivatives from an acid solution a t pH 2.0-3.0 with nbutanol, and separating them by thin layer chromatography using silica gel plates. Solvent systems were butanol-acetic acid-water (5:4:1), or 95% ethanol. Plates wcre removed from the tank after the solvent had moved 10 cm., dried under a hood, and exposed to ammonia. Fluorescent derivatives were observed by placing the plates under ultraviolet light. One of the fluorescent derivatives that traveled with the solvent front was the result of an intermolecular reaction of resorcinol as evidenced by extraction and separation of the reagent blank. Definitive proof of structure of the other derivatives has not materialized. In the analysis of equal molar concentration of sugars, the fluorescence intensity varied with different sugars. Figure 1 shows that the intensity of fluorescence proceeds in the order of arabinose, a pentose > rhanmose, a methylpentose > galactose, a hexose; this order held with varying degrees when other pentoses and hexoses were tested.
Effect of Acid Concentration and pH. I n the reaction of carbohydrates
with resorcinol, the rate of formation of the xanthenone derivatives increases as the acidity of the solution increases. Figure 2 illustrates the effects of increasing the acid concentration twofold in the presence of arabinose. The derivatives resulting from this reaction give virtually no fluorescence in strong acid solution. The fluorescence increases as pH approaches neutrality, and reaches a maximum a t pH 8.5-9.5. Evidence from carbohydrates tested indicates that all of the fluorescent derivatives have approximately the same activation and fluorescence wavelength maxima of 488 and 508 mp (uncorrected). Influence of Temperature. The influence of temperature on the production of fluorescence from carbohydrates was determined. Ribose was studied a t three different temperatures over a period of 45 minutes. The temperature dependence of this reaction is shown in Figure 3. When the most favorable condition of 5 ml. of concentrated hydrochloric acid and temperatures of 115-20O C. were em-
ployed for 15 minutes, the complete analysis of pentoses in amounts as low as 2.0 ng. could be performed in 20-30 minutes. Hexoses require a 30-minute heating period to determine nanogram amounts and the precision of this method is i:1%. Precaution must be taken to keep the Teflon caps on the tubes sealed during the heating period, and that all samples should be removed together from the heating bath since there is a continuous synthesis of the fluorophors with heating time. This study has demonstrated that the sensitivity of the method described here is 200 times greater than that of the o-phenylenediamine method. Because this method is simple and requires only 20-30 minutes to complete an analysis, it is applicable for rapid analysis of many carbohydrate samples. LITERATURE CITED
(1) Seliwanoff, T., Be?. 20, 181 (1887). (2) Spikner, J. E., Towne, J. C., ANAL.
CHEM.34, 1468 (1962). RECEIVEDfor review August 11, 1966. Accepted September 16, 1966. Mention of commercial products does not constitute endorsement by The Federal Water Pollution Control Administration.
Activation Analysis of Silicon by Convention Carrier Separations and by Computer Reduction of Gamma Spectra K. GAIL HEINEN and GRAYDON LARRABEE Texas Instruments lnc., Dallas, Texas The results of the analysis of silicon for various dopants in the concentration range 0.55 p.p.b. to 415 p.p.m. by neutron activation analysis are reported. A rapid technique of computer reduction of complex gamma spectra has been shown to yield results with comparable precision and in some cases better accuracy than the classical radiochemical separation techniques. The precision or reproducibility was shown to be =!=5.34/,of the amount present even in the low p.p.b.-region. Comparison of analytical and electrical results indicate an overall accuracy that is no worse than &35% (comparing with electrical results). The computer method of analysis yields results which average 6.1 % higher than the radiochemical separation procedures. This agreement between the two radiochemical methods is considerably better than that expected based on other workers ,
statistical analysis of activation analysis procedures. Results are presented to show that arsenic is lost during silicon dissolution and previously published works on radiochemical determination of arsenic are probably in error.
A
TECHNIQUES for the analysis of high purity silicon for trace impurities have been discussed by Cali (1) and Kane (8). It is generally accepted that neutron activation analysis is the only analytical procedure with sufficient sensitivity t o determine trace impurhies at the levels of interest in semiconductors. I n 1955 Morrison and Cosgrove (IS) and in 1960 Makasheva et al. (1%) determined impurities in silicon at the part per million level using gamma ray scintillation spectrometry. To increase the sensitivity of the activation analysis of silicon subsequent
SALYTICAL
workers found it necessary to utilize elaborate radiochemical separation procedures and then beta count the separated impurity. Several comprehensive procedures for the radiochemical separations of impurities in silicon have been presented in the literature. Among these is a scheme for separating 23 elements by Thompson, Strause, and Leboeuf (16) and 12 elements by Kant, Cali, and Thompson (10). Gebauer and Martin (6) described procedures for separating 30 elements with emphasis on half lives. A11 these procedures analyze for many elements which are of little interest in semiconductors (Le., not electrically active). This nimiety makes the radiochemical separation procedures needlessly complex and time consuming. The elements of most interest are the so-called dopants and/or impurities that are electrically active in silicon. These include the group IIIA and VA VOL. 38, NO. 13, DECEMBER 1966
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