the slope of the line. Measures of precision can then be determined in terms of s, the standard error per determination in the detector response, Y , at mean values of X and Y , and T * , the measure of goodness of fit around the line. The results are shown in Table I. In the weight us. peak height correlation, no isobutyraldehyde was present as in the other two methods. All of the r2 values show that the fit about the calculated line is satisfactory and in all three methods the standard error is approximately 10%. Without further calculations, it might be presumed that the three methods are equally precise, and, because the measurement of peak height is more convenient, that this technique would be satisfactory. However, the calibration curve given in Equation 1 is used for predicting the unknown weight of carbonyl, X’, as a function of the detector response, Y , resulting in the inverted equation
I n this form the standard error is now dependent on the carbonyl weight as well as random errors associated with the detector response, Y . Under these conditions, the standard error in Y does not apply and the standard error per determination in weight, s’, is determined as s’ = s / b , which in itself is a measure of the relative precision. The s’ values show that the peak height us. carbonyl weight is the least precise of
the three methods, the other two being apparently equal in precision. Another method of determining the relative precision of several methods is to compare the number of determinations, fii, necessary to obtain the weight of carbonyl within certain limits (14). In this case the limits were set such that the true weight of carbonyl +lo% would be determined 95% of the time. A comparison of methods then consists of comparing f i and s‘ and, as shown in Table I, the weight ratio-area ratio method is decidedly more precise than either the peak height or peak area relationship, the peak area being the next most precise. Thus one determination using the weight ratio-area ratio method will yield results which will include the true weight of carbonyl 95% of the time within +IO%. fi was calculated from data having the largest error. Presumably, a higher degree of precision could be calculated using carbonyl weight values close to the mean value. The use of the internal standard, butyraldehyde D N P , and the exchange mixture of oxalic acid-DMAB permits the determination of aliphatic carbonyls with satisfactory precision and it appears likely that the procedure could be applied to other analyses involving pyrolytic conditions for compound liberation. The statistical analysis described indicates that methods presented with nearly identical correlation coefficients do not necessarily reflect equal precision in the analytical procedures.
ACKNOWLEDGMENT
The authors are indebted to Sara Griffin and Katherine Harper for technical assistance in the accumulation of analytical data required for this work. LITERATURE CITED
(1) Anchel, AI., Schoenheimer, R., J . Biol. Chem. 114, 539 (1936).
(2) Chen, C., Gacke, D., ANAL. CHEM. 36, 72 (1964). (3) Demaecker, J., Martin, R. H., Sature 173. 266 11954). (4) Jdhnson,~G. D., J . ,4m. Chem. SOC. 73, 5888 (1951). ( 5 ) Jones, L. A , , Hancock, C. K., J . Org. Chem. 25, 226 (1960). (6) Keeney, > f . , ANAL. CHEM.29, 1489 (1957). (7) Mattox, V. R., Kendall, E. C., J . Am. Chem. SOC.70, 882 (1948); 72, 2290 (1950). (8) Ralls, J. W., A N ~ L CHEM. . 32, 332 (1960). (9) Robinson, R., lVature 173, 541 (1954). (10) Stephens, R. L., Teszler, A. P., ANAL.CHEM.32, 1047 (1960). (11) Strain, H. H., J . A m . Chem. SOC. 57, 758 (1935). (12) Weybrew, J. A., Jones, L. A , , Tobacco Sci. 6. 164 11962). 3) ’#&brew, J. ‘A,, Stephens, R. L., Ibid., 6, 53 (1962). 4 ) Williams, E. J., “Regression Analysis,” Chap. 6, Wiley, Sew York, 1959. L. A. JONES R. J. MONROE martment of Chemistrv Ndrth Carolina State Uriiversity Raleigh, N . C. PUBLISHED with the approval of the Director of Research of the Xorth Carolina Agricultural Experiment Station, Raleigh, X. C., as Paper No. 1902 of the Journal series.
Spectrophotofluorometric Analysis of Nonfluorescent Compounds on Paper and Thin Layer Chromatograms SIR: Recently two articles appeared in the literature describing methods for the direct spectrophotofluorometric analysis of aromatic and aza heterocyclic hydrocarbons on thin layer chromatograms (6 8J. These techniques save many man-hours usually spent with the more conventional procedures involving spot extraction followed by spectral examination of the extracts. Direct spectrophotofluorometric analysis is not only applicable to the analysis of compounds that are naturally fluorescent in neutral, acid, or basic environment but should be usable also for those nonfluorescing compounds that can be made to fluoresce on paper or thin layer chromatograms by reaction with an appropriate reagent(s). Many methods for the fluorometric assay and/or detection of nonfluorescing molecules are available in the literature. 938
ANALYTICAL CHEMISTRY
Methods for formaldehyde and its precursors (5, 9 ) , estrogen steroids (Z), vanillin ( I ) , malonaldehyde ( 7 ) , and tryptophan (4, have been modified and shown in this communication to be amenable to the characterization of nanogram amounts on paper and thin layer chromatograms by direct spectrophotofluorometric techniques. EXPERIMENTAL
Apparatus. -411 spectral d a t a were obtained with a n Aminco-Bowman spectrophotofluorometer from the American Instrument Co., Silver Spring, M d . T h e instrument was equipped with a solid sample accessory and with a special holder for strips of glass, plastic, or paper. Slit arrangement No. 2 as described in the manual gave good resolution with minimum noise levels. Thin layer
chromatography plates were obtained from Custom Service Chemicals, Inc., Wilmington, Del. A11 other materials were obtained from the nearest commercial source. Reagents. Chemicals were commercially available and of known purity, or they were purified through distillation or recrystallization to a constant boiling or melting point. General Procedure. Satisfactory procedures are available for obtaining excitation and emission spectra directly from thin layers of adsorbents and on glass and plastic films ( 8 ) . These procedures were followed without modification in this investigation. Formaldehyde Procedure. T o a spot containing 2 pl. of 0.5% J-Acid (6-amino-1-naphthol-3-sulfonic acid) 1 pl. of an aqueous solution of formaldehyde was added; after the moist area stood for 2 minutes, the fluorescence spectrum was recorded.
1
ANAL.-CH20 PRECURSORS
Table I. Fluorescence Analysis on Paper and Thin Layer Chrom atograms
I. GFP CHROMAT. SPOT 2. Ipl 0.5 YO J-ACID IN H2S04 3. llO°C FOR 2 MIN.
Exci- Er,nistation sion Iletecwave- wave- tion lengtha length,a limit,* Compound mr mu ng. 1 Estrone 460 550 Vanillin 420 498 5 ,1Ialonaldehyde 450 510 10 Tryptophan 362 530 50 p-Xitrophenylacetic acid 420 550 1000 a Excitation and emission wavelengths for this determination. Complete excitation and emission spectra are not shown. b Lowest concentration at which characteristic excitation and emission spectra were obtained.
462
n
I:
40k 20
I
/
I1
310
/'
1
i
I
\i
Amp
Figure 1 . Emission (--) and excitation ( - - -) spectra obtained for formaldehyde or its precursors on glass fiber after reaction with J-acid
Procedure for FormaldehydeReleasing Compounds. T h e formal-
dehyde procedure was used as described above, except t h a t 2 minutes a t 100" C. in an oven was required before reaction occurred. Malonaldehyde Procedure. T o a spot containing malonaldehyde in the form of the bisdimethoxyacetal 1 drop of 1% 4,4'-sulfonyldianiline in S,Sdimethyliormarnide containing 17, concentrated hydrochloric acid was added, a n d the moist area was heated 3 minutes in a n oven a t 100" C. The fluorescence from the dry spot was then determined. The fluorescence was again measured after 1 pl. of 29% fetraethyl ammonium hydroxide in methanol had been added. Vanillin Procedure. One drop of a 57, aqueous solution of hydrazine dihgdrochloride was added to a spot of vanillin a n d the area was heated for 3 minutes a t 100" C. p - Nitrophenylacetic Acid Procedure. T o a spot of p-nitrophenylacetic acid 1 drop of 1% solution of p-dimethylaminobenzaldehyde in piperidine \vas added. T h e moist area was then heated 3 minutes a t 110" C. and wet carefully with 2 pl. of toluene just before t,he excitation and emission spectra were obtained. Tryptophan Procedure. T o a spot containing tryptophan 1 drop of a solution of formaldehyde, concentrated hydrochloric acid, and water (1 : 1 : l-v./v.) was added. The moist area \vas then heated for 3 minutes a t 90" C. in an oven. Estrogen Procedure. One microliter of estrone was placed on glassfiber filter paper impregnated with p-toluenesulfonic acid from a saturated aqueous solution. T h e paper was then heated for 3 minutes a t
l l o o c.
RESULTS A N D DISCUSSION
The literature methods required modification for application to direct spectrophotofluorometric analysis. X1though variables such as reaction time, per cent reagent, etc., were investigated, these parameters are not discussed since the primary aim of this communication is to show the wide applicability of this technique. Characteristic spectra obtained for formaldehyde and its precursors are shown in Figure 1. Xormally the most intense band in the excitation spectrum would have been chosen for subsequent measurements of emission; however, since background noise levels from scattered light increase directly as the distance between excitation and emission wavelengths decreases, the moderately intense wavelength a t 375 mp was used for the measurement of all emission spectra. The identification limits for formaldehyde, sym-trithiane, hexamethylenetetramine, glyoxylic acid hydrate, piperonylic acid, 2,4,5-trichlorophenoxyacetic acid, and monochloroacetic acid, were 0.5, 0.5, 1.0, 1.0, 10, 10, and 100 nanograms, respectively. The method \vas so extremely sensitive for formaldehyde that it was necessary to perform the work in an area of the laboratory that was free from traces of cigarett,e tobacco smoke. This method will be used to detect traces of formaldehyde precursors in contaminated atmospheres. The wide applicability of the procedure is shown by the identification limits given for such chemically and physiologically important compounds as estrone, vanillin, malonaldehyde, tryp-
tophan, and p-nitrophenylacetic acid, Table I. The technique as applied to estrone is primarily a characterization procedure, since the test was done on glass-fiber paper impregnated with p toluenesulfonic wid. Estrone could be detected in the microgram range also on a thin layer chromatogram by use of a saturated solution of the reagent. Malonaldehyde was characterized successfully on an aluminum oxide thin layer plate and on glass-fiber paper. The neutral dry spot of the reaction product on alumina fluoresced a t 547 mp with the most intense excitation wavelength a t 468 mp, and a red-orange fluorescence was visually observed. A dry neutral spot on glass-fiber paper fluoresced a t 560 mp with an excitation wavelength a t 470 mp. The alkaline procedure gave the results shown in Table I and was chosen because of its greater sensitivity and good reproducibility. By this technique 5 ng. of vanillin were easily detected on an aluminum oxide thin layer chromatogram and on paper. On glass-fiber paper the detection limit, was 100 ng. with characteristic emission and excitation a t 502 and 420 mp, respectively. On all substrates used, the reaction was visually observed down to 0.1 pg. by the yellow color of the spot. Biologically important tryptophan was easily characterized on glass-fiber paper and on cellulose and alumina thin layer plates a t concentrations just below 50 ng. The yellow-green fluorescence of the reaction products exhibited prominent emission a t 530 and 560 mp with excitation wavelengths a t 310, 362, and 462 mp. h published procedure for the fluorometric assay of tryptophan involves the condensation with formaldehyde followed by oxidation to give p-carboline (9H-pyrido-(3,4-b)indole) (3). In the present procedure, treatment of the spot with hydrogen peroxide after the initial reaction gave no eviVOL. 37,
NO. 7, JUNE 1965
939
dence of similarity to the 258, 300, and 370 excitation and 445 mp emission wavelengths recorded for pure p-carboline on glass-fiber paper wet with 5% aqueous HC1. It was noted, however, that the spectra contained bands analogous to the 365 excitation and 535 emission bands reported by Uphaus, Grossweiner, and Katz (10) for l-methyl-3carboxy-3,4-dihydro-~-carboline isolated from the N-acetyl derivative of tryptophan in trifluoroacetic acid. It seems probable that in this reaction decarboxylation does not occur and the end products are rich in 3-carboxy-3,4dihydro-@-carboline. Also, since tryptamine, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, and 2,3,7trimethylindole gave only weak and erratic positives a t concentrations above 20 pg., the test indicates that the method is specific for tryptophan in the presence of less than 200-fold amounts of 3-substituted indoles. The sensitivity for p-nitrophenylacetic acid was not as good as for the other compounds shown in Table I. The dry spot did not fluoresce, but when wet with 2 pl. of toluene a yellow-green fluorescence with a wavelength maximum a t 550 mp was observed. It appears, therefore, that wetting with an
appropriate solvent can be used to enhance fluoresence, reduce noise from scattered light, and obtain fluorescence spectra more compatible with solution spectra. Many organic compounds present in extracts of air particulate matter do not fluoresce. However, using methods in which fluorogens are formed and techniques described in this communication, air samples can now be quickly screened for the presence of precursors of formaldehyde and malonaldehyde. Unstable phenolic aldehydes believed to be present in automotive exhaust fumes should be capable of being identified through the formation of the more stable and fluorescent azine as described in the procedure for vanillin. Since many aza, and some oxy, heterocyclics have been found in contaminated atmospheres, it seems very probable that these techniques can be used for the detection and characterization of many compounds of biological interest, some of which are probably present in polluted atmospheres. These preliminary results show the potentially wide applicability of the method to the fluorometric detection and characterization of aldehydes, amines, amino acids, steroids, hallucino-
gens, and countless other nonfluorescent compounds. These techniques are being applied to air pollution analysis and should find wide use in other areas of analytical chemistry. LITERATURE CITED
(1) Berguer, K. G., Sperlich, H., Deut. Lebensm.-Rundschau 47, 134 (1959). (2) Epstein, E., Maddock, W. O., Boyle, -4.J., ANAL.CHEM.29, 1548 (1957). (3) Hess, S., Udenfriend, S., J . Phavnacol. Ezptl. Therap. 127, 175 (1959). (4) Prochazka, Z., Chem. Listy 47, 1643 (1953). (5) Sawicki, E., Hauser, T. R., McPherson, S., ANAL.CHEM.34, 1460 (1962). (6) Sawicki, E., Stanley, T. W.; Elbert, W. C., Occupational Health Rev. 16, 8 (1964).
( 7 ) Sawicki, E., Stanley, T. W., Johnson, H., Chemist-Analyst 52, 4 (1963). (8) Sawicki, E., Stanley, T. W., Johnson, H., Microchem. J . 8 , 257 (1964). (9) Sawicki, E., Stanley, T. W., Pfaff, J. D., Anal. Chim. dcta 28, 156 (1964). (10) Vphaus, R. A,, Grossweiner, L. I., Katz, J. J., Science 129, 641 (1959).
THOMAS W. STANLEY EUGESESAWICKI
Division of Air Pollution Robert A . Taft Sanitary Engineering Center I:. S. Dept. of Health, Education, and Welfare Cincinnati, Ohio
Polarography of Dithiodimalic Acid SIR: The study of organic sulfhydryl and disulfide compounds is of interest because of their relation to proteins. Kolthoff and coworkers thoroughly studied the polarography of cystine and cysteine (3-5). The polarography of thiomalic acid has also been investigated (2). The present paper summarizes findings on the polarography of dithiodimalic acid (TSST) , a disulfide dimer of thiomalic acid.
EXPERIMENTAL
Reagents. TSST was prepared by the oxidation of thiomalic acid with ferric alum, similar to the method for preparation of dithiodiglycollic acid (7). Apparatus. A Leeds & Northrup Electro Chemograph T y p e E was used throughout. h manual setup with circuit similar to t h a t used by Kolthoff and Lingane was also used a t times. iill potentials were measured against the saturated calomel electrode. The characteristics of the DME were m = 2.403 mg. second-’; t = 3.57 seconds (open circuit); and h = 35 em. The pH was measured with a Leeds & Northrup pH meter using a generalpurpose glass electrode. 940
ANALYTICAL CHEMISTRY
RESULTS
Polarograms of l O - 3 X TSST were taken in buffers of pH 1-9. Well defined cathodic waves were obtained a t pH 1 and 3. The wave height was much reduced a t pH 5 and no wave was observed a t higher p H values. 4 t pH 1 and 3, diffusion currents proportional to the concentration were observed. ht p H 5 alone, a prewave was obtained preceding the main wave. Triton X100 (7.9 x or thymol (4.9 X 10 -5M)completely suppressed the maximum. The diffusion coefficients a t pH 1.3 and 3.1 were 4.81 X lo-* and 4.807 x low8,respectively. The corresponding zero current potentials were 0.07 and 0.09 volt. I n both cases TSST concentration was 5 X 10-4M. DISCUSSION
By following the usual calculations, the value of CY was found to be 0.185. The reduction of TSST ( 1 , 6) can be represented by El/2
=
~
where the different symbols have their usual significance. Applying the equation for perchloric acid buffer a t p H 1.3 cm. the value of KO, is 4.235 X second -I. ACKNOWLEDGMENT
The author expresses his thanks to
R. C. Kapoor, Head, Chemistry Dept., Jodhpur University, for helpful suggestions during the progress of this work. H e is also grateful for the gift of thiomalic acid which was supplied by Evans Chemetics, Inc., New York, N. Y. LITERATURE CITED
( 1 ) Delahay,
P., “New Instrumental Methods In Electro-Chemistry,” Interscience, New York, 1954. ( 2 ) Kapoor, R. C., Tiwari, S. K., Proc. Natl. Acad. Sci. ( I n d i a ) 28, 52 (1959). ( 3 ) Kolthoff, I. &I., Barnum, C., J. Am. Chem. SOC.62, 3061 (1940). (4) Kolthoff, I. M., Barnum, C., Ibid., 63, 520 (1941). ( 5 ) Kolthoff, I. M., Stricks, W., Tanaka, N.,Ibid., 77, 4739 (1955). (6) Koutecky, J., Collection Czech. Chem. Commun. 18, 597 (1953). ( 7 ) Leussing, D. L., Kolthoff, I. &I., J . Electrochem. Soc. 100, 334 (1953).
S. K . TIWARI Prince of Wales Chemical Laboratories Aligarh Muslim University Aligarh, India
