with short chain compounds; there is a progressive decrease of the fluorescence intensity with 1,5-diaminopentylphosphonic, 1,4-diaminobutylphosphonic and especially 1,3diaminopropylphosphonic acids. But this low reactivity ends with the shortest chain compound, 1,2-diaminoethylphosphonic acid, a molecule possessing two vicinal amino groups; this last compound, as in DAB-induced fluorescence, forms a better fluorophore than aminomethylphosphonic acid. The carboxylic analog, 2,3-diaminopropionic acid, curiously, does not form such a good fluorophore. Fluorescamine-Induced Fluorescence. Fluorescamine-induced fluorescence, in the group of simple amino acids, is not very sensitive to molecular structure, as already noted with OPT. Notable exceptions are represented by 1-amino-1-methylethylphosphonicacid and its carboxylic analog. Those two poorly reactive compounds have no hydrogen atom on the carbon atom to which the functional groups are linked; this molecular characteristic is also an unfavorable factor for the production of Ruheman purple, the reaction product of ninhydrin with an amino acid (28). It may be recalled that fluorescamine and ninhydrin are both triketoindane derivatives. In the experiments conducted with fluorescamine, the differences of reactivity between phosphonic and carboxylic compounds are magnified. As in the experiments with DAB, the few sulfur amino acids studied are closer to the carboxylic analogs than to the phosphonic analogs.
CONCLUSIONS The significant fluorescences given by DAB, OPT, and fluorescamine when reacting with the carboxylic amino acids can also be observed when the carboxylic acid group is replaced by the phosphonic group. DAB is especially suitable for the quantitative determination of aminomethylphosphonic acid, diaminophosphonic acids, and also for the natural compound ciliatine, which is poorly reactive with ninhydrin, the classical amino acid reagent. OPT could be considered as a fluorescence reagent covering a larger range of aminophosphonic acids, but its relative insensibility toward molecular structure is restricted by the great instability of the fluorophore. The interest in fluorescamine is linked to the great sta-
bility of the fluorophore; the use of fluorescamine for the determination of ciliatine is limited by the low fluorescence emitted by the chief natural amino phosphonic acid.
ACKNOWLEDGMENT The technical assistance of Mrs. Monique Malgat is gratefully acknowledged.
LITERATURE CITED (1) M. Horiguchi and M. Kandatsu. Nature (London), 184, 901 (1959). (2) M. Kandatsu and M. Horiguchi, Agric. Biol. Chem., 28, 721 (1962). (3) J. S.Kittredge and R. R. Hughes, Biochemistry, 3, 991 (1964). (4) H. Shimizu, Y. Kakimoto, T. Nakajima, A. Kazanawa, and I. Sano, Nature(London), 207, 1197 (1965). (5) V. Chavane, C. R. Acad. Sci. Paris, 224, 406 (1947). (6) M. Labadie and E. Neuzil, in “Composes organiques du phosphore”, Centre National de la Recherche Scientifique, Paris, 1966, p 349. (7) M. Bourhis, H. Jensen, and E. Neuzil, Ann. Pharm. Fr., 28, 561 (1970): 30, 55 (1972). (8) J. S. Kittredge, E. Roberts, and D. G. Simonsen. Biochemistry, 1, 624 (1962). 9. L. Roop and W. E. Roop, Anal. Biochem., 25, 260 (1968). J. Le Pogam, H. Jensen, E. Neuzii, and C. Garrigou-Lagrange, Bull. SOC. Chim. Fr., 12, 3389 (1973). J. Le Pogam, H. Jensen, and E. Neuzii, J. Chromatogr., 87, 179 (1973). N. Seiier, T.Schmidt-Gienewinkel,and H. H. Schneider. J. Chromatogr., 84, 95 (1973). V. H. Ghosh and M. W. Whitehouse, Biochem. J., 108,155 (1968). J. C. Breton, Med. Thesis, Univ. of Bordeaux, 1957, p 194. I. P. Lowe. E. Robins, and G. S.Eyerman, J. Neurochem., 3, 8 (1958). J. Close, quoted by Samejima et ai. (Ref. 18, p 222). M. W. Mc Caman and E. Robins, J. Lab. Clin. Med., 59, 885 (1962). K. Samejima, W. Dairmann, J. Stone, and S. Udenfriend, Anal. Biochem.. 42, 222 (1971): 42, 237 (1971). M. Weigeie, J. F. Blount. J. P. Tengi, R. C. Czajkowski. and W. Leimgruber, J. Am. Chem. SOC.,94,4052 (1972). M. Weigele, S. Debernardo. and W. Leimgruber. Biochem. Biophys. Res. Commun., 50, 352 (1973). G. Hillman, 2.Physiol. Chem., 277, 222 (1943). P. A. Shore, A. Burkhaiter, and V. H. Cohn, J. Pharmacol. Exptl. Therap., 127, 182 (1959). M. Roth, Anal. Chem., 43, 880 (1971). M. Roth and L. Jeanneret, 2. Physiol. Chem., 353, 1607 (1972) M. Roth and A. Hampai, J. Chromatogr., 83, 353 (1973). A. M. Lacoste, A. Cassaigne. and E. Neuzil, C. R. Acad. Sci. Paris, 274, 1418 (1972): 275, 3009 (1972). S.Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele, Science, 178, 871 (1972). E. Neuzii, J. C. Breton, and H. Plagnol, Bull, Mem. Ec. Naf. Med. Pharm. Dakar, 8, 169 (1958).
RECEIVEDfor review July 14, 1975: Accepted September 26, 1975. The support granted by the Conseil Scientifique de 1’Universite de Bordeaux I1 is kindly acknowledged.
Fluorescent Detection of Hydrazines via Fluorescamine and Isomeric Phthalaldehydes Robert W. Weeks, Jr.,* Stanley K. Yasuda, and Brenda J. Dean Industrial Hygiene Group, L o s Alamos Scientific Laboratory, University o f California, Los Alamos, N.M. 87545
The use of fluorescarnine, 0-, m-, and p-phthalaldehyde to form fluorescent derivatives Is Illustrated as a class reaction for hydrazine and substituted hydrazines. Results reported herein show that the intensity of product fluorescence and, thus, the likely degree of reaction between a given hydrazine and fluorogen within a few minutes time, is very much a function of the pH of the analyte system. Acid solutions of pH 3-6 showed qualitatively the highest intensity of fluorescence. Using 366 nm wavelength ultraviolet light for irradlatlon of the fluorogenic product, hydrazine was detected at the ng/cm2 level with fluorescamine and with 0- and p
phthalaldehyde. In field survey tests wherein a swipe Is taken, the limit of detection may be even lower because of the effective concentrating of the analyte from a large area onto a relatively small filter paper.
The use of fluorescamine (4-phenylspiro[furan2(3H),l’-phthalan]-3,3’-dione)( I , 2) and of o-phthalaldehyde ( 3 ) for the determination of organic molecules containing amine functionality is well documented. It is the purpose of the present paper to illustrate the utilization of ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
159
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Figure 1. Unaided eye estimate of relative fluorescence intensity as a function of pH for the product of reaction between hydrazine and given phthalaldehyde
Flgure 3. Absorbance (410 nm) and unaided eye estimated relative intensity of color formation as a function of pH for the reaction between Ehrlich's reagent (pdimethylaminobenzaldehyde)and hydrazine
( 0 )c-Phthalaldehyde; (A)mphthalaldehyde; (m) pphthalaldehyde
( 0 )instrument; (H) eye
PH
EXPERIMENTAL
concentration. Visualization and fluorogenic reagents were prepared in the following concentrations: Fluorescamine, 0.35 mg/ml in 1,4-dioxane; o-Phthalaldehyde, 1 mg/ml in acetic acid; m Phthaialdehyde, 1 mg/ml in acetic acid; p-Phthalaldehyde, 1 mg/ml in acetic acid; and p-Dimethylaminobenzaldehyde (Ehrlich's reagent), 0.25 wt % in 0.25 N ethanolic HC1. Solutions of the desired p H were prepared from commercially available buffers and either hydrochloric acid or sodium hydroxide of the appropriate normality. Product absorbance vs. p H was studied by adding 0.3 ml of Ehrlich's reagent solution to 3 ml of buffer containing 1.6 rmol of hydrazine (N2H4) and allowing to react for 2 min before measuring absorbance. The limit of detection tests were performed by placing solutions containing known concentrations of the desired hydrazines in the center of a 7.0-cm Whatman 42 filter paper and then immediately adding 10 drops of ethanol to disperse the hydrazine over approximately a 15 cm2 area. After evaporation of the ethanol, a Pasteur pipet containing the visualization or fluorogenic reagent was drawn from one edge of the filter paper, across the center, and then to the opposite edge in one continuous stroke. (This provided a blank adjacent to the reaction site.) The reagents were allowed to dry. After approximately 5 min, they were viewed in a light-tight black box while being irradiated with either 254 or 366 nm light. (Intensity of 366 nm light = 0.05 pW/cm2 a t surface of paper.) Those st,udies in which fluorescence intensity was monitored as a function of pH were performed as follows: 50 p1 of 500 ppm N2H4 in methanol were placed a t the center of the filter paper and 3 drops of the appropriate buffer immediately placed upon the hydrazine. The paper was allowed to dry and the fluorogenic reagent of interest was applied as above, again the paper was allowed to dry and then visualized with either short or long wavelength UV light.
Apparatus. Irradiation of samples was effected using a Model CC20 Portable UV Dark Room (Brinkmann Instruments) capable of both short (254 nm) and long (366 nm) wavelength ultraviolet light. The intensity of the long wavelength light on the surface was measured using a Blak-Ray Ultraviolet Meter Model 5221 (UltraViolet Products, Inc.). Whatman 42 filter paper (This is not meant as a product endorsement, but rather as a statement of fact.) was used as a reaction medium for these tests. Absorbance values were measured with a Spectronic 20 (Bausch & Lomb). Reagents. Products used were: 2,4-Dinitrophenylhydrazine, Eastman, reagent grade; Hydrazine, Eastman, 95%; 1,l-Dimethylhydrazine, Matheson, Coleman and Bell, 99%; Phenylhydrazine, J. T. Baker, reagent grade; Methanol, Analytical reagent, (acetone free), Mallinckrodt; Fluorescamine, Hoffmann-La Roche; 1,4-Dioxane, B & A Allied Chemical; Ethanol, U. S. Industrial Chemicals, Co., reagent grade; o-Phthalaldehyde, J. T. Baker, Baker grade; rn-Phthalaldehyde, Aldrich, 97%; p-Phthalaldehyde, Matheson, Coleman and Bell (m.p. 115-116 "C); Glacial acetic acid, Mallinckrodt, reagent grade; Buffers, Beckman, Curtin, J . T. Baker, or VWR; and Filter Paper, Whatman 42,7-cm. Procedure. Solutions of the hydrazines were prepared by dissolving appropriate quantities in methanol to form a stock solution. Aliquots were diluted with methanol to obtain the desired
RESULTS AND DISCUSSION The work of Roth ( 3 ) reported the determination of certain amino acids by reaction with o-phthalaldehyde in basic or weakly acid (pH 6.0) solutions. Likewise, the work of Udenfriend et al. ( I ) on amino acids recommended the use of fluorescamine in the weakly basic range of pH 8-9. The present work reports optimum reaction conditions to exist for mildly acidic reaction systems. Particularly, Figure 1 shows the relative intensity of fluorescence, as estimated by the unaided eye, to give maxima in the pH 4-6 range for the isomeric phthalaldehyde/hydrazine systems. Likewise, the fluorescamine/hydrazine system in Figure 2 shows the estimated relative fluorescence intensity to exhibit a maximum in the pH 3-4 range. Similarly, Figure 3 illustrates the absorption maximum in the pH 4-5 range for the p-dimethylaminobenzaldehyde/hydrazinesystem. Our reason for these studies in the acidic range is based upon our work (6) in which the absorption maximum for the condensation product of an amine with an aldehyde is a
PH
Figure 2. Unaided eye estimate of relative fluorescence intensity as a function of pH for the product of reaction between hydrazine and fluorescamine these reagents and to include m - and p-phthalaldehyde for the detection of hydrazine and its derivatives via analyses not previously reported in the literature. Although this work is limited to spot test detection, the reaction sequences involved should be of value for quantitative analyses using an appropriate spectrophotofluorometer. For comparative purposes, the use of p-dimethylaminobenzaldehyde ( 4 , 5 ) is given for limit of detection values for the hydrazines.
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Table I. Detection Limit of Representative Hydrazines with Various Visualization Reagents Fluorogenic reagent, Compound
Hydrazine
Limit of detectibnO/color of fluorescent product, 366-nm excitation Fluorescamine
o-Phthalaldehyde
0.006/yellow
0.006/blue
m-Phthalaldehyde
0.015/brown quenched spot
p-Phthalaldehyde
0.006/blue
Limit of detection0 with unaided eye/Color of spot with p-dimethylaminobenzaldehyde
0.006/yellow orange
1,l-Dimethyl-
0.1 5/yellow b, c b b 0.1 5/yellow hydrazine Phenyl0.006/yellow 0.006/blue 0.15/blue 0.03/yellow 0.1 5/yellow hydrazine 2,4-Dinitrob b b b Reactant undiscernible from phenylhydrazine product with unaided eyec apg/cm2 of paper surface. b Not detected at the level of 0.15 pg/cm2. C Both the 1,l-dimethylhydrazinelo-phthalaldehyde and the 2,4-dinitrophenylhydrazine/p-dimethylaminobenzaldehyde showed evidence for reaction after a time of the order of 1 day.
function of pH and reaches maxima in acidic solution. These figures substantiate the pH dependence of the condensation product and the reasonable pH correlation achieved by an unaided eye vs. instrumental readings. This has also been verified by pertinent experience of other workers (7). Analogous mechanistic considerations (8, 9) for the condensation of aldehydes with hydrazines, show merit for the extension of studies into the acidic range for the hydrazine species. The present work showed (Figure 1) the reaction of the phthalaldehydes with hydrazine to give qualitatively better fluorescence in mildly acidic solutions. The work of Roth (3) has shown, however, that not all species of a given class of compounds will yield maximum fluorescent intensities a t the same pH. It is, thus, important to establish a pH/fluorescence/visualization profile for each compound of interest and to include pH values a t both strongly acidic and strongly basic conditions. Table I shows the limit of detection values obtained using various reagents to detect representative hydrazines. For 2,4-dinitrophenylhydrazine(2,4-DNPH) at levels of 0.15 wg/cm2 and above, there was no evidence for reaction with either p-dimethylaminobenzaldehyde or with the isomeric phthalaldehydes. Indeed, a t 2,4-DNPH levels of 0.6 pg/cm2 and above, a yellow-brown color may be observed and the compound may be considered self-indicating, In the cases of 2,4-DNPH plus either isomeric phthalaldehydes or fluorescamine, possibly reaction has occurred, but the presence of two nitro groups may have effected a quenching of fluorescence. Ample documentation (IO) is available to substantiate the effect whereby electron withdrawing groups decrease fluorescence. For comparison, the limit of detection values for the hydrazines are also given using Ehrlich's reagent, p-dimethylaminobenzaldehyde (5). It is important to note the existence of an upper, as well as a lower, limit of detection in fluorescence determinations. This limitation is imposed by self-quenching or concentration quenching and is typically of the order of
M (11) for solution work. In the present case, an upper limit for the determination of hydrazine with o-phthalaldehyde was shown to be 3.1 pg/cm2. At this level, there was a dark spot a t that area where fluorescence would have been observed at lower concentrations. The value of this least upper bound defines the range of a given determination and is important for survey detection work. The present work affords alternative analytical pathways for the determination of hydrazine and substituted hydrazines. Certain examples are cited wherein lower limit of detection values are obtained by the procedures given herein than are obtained by establishing analytical techniques.
LITERATURE CITED (1)
S. Udenfriend, S. Stein, P. Bohlen, and W. Dairman. "Chemistry and
Biology of Peptides", Ann Arbor Science Publishers, Ann Arbor, Mich., 1972, p 655. (2) J. Sherma and G. Marzoni, Am. Lab., 6 (IO),21 (1974). (3) M. Roth, Anal. Chem., 43, 880 (1971). (4) S.Vickers and E. K. Stuart, Anal. Chem., 46, 138 (1974). (5) F. Feigl (translated by R. E. Oesper). "Spot Tests in Organic Analysis", Seventh English Ed.. Elsevier Publishing Co., New York, N.Y., 1966, pp 277-278. (6)R. W. Weeks, Jr., and C. K. Trusty, Los Alamos Scientific Laboratory, unpublished data, 1975. (7) E. Rinde and W. Troll, Institute of Environmental Medicine, New York University Medical Center, New York, N.Y., personal communication, 1975. (8) P. Y. Sollenberger and R. E. Martin "The Chemistry of the Amino Group", S. Patai. Ed., John Wley & Sons, New York, N.Y., 1968, p 368. (9) J. D. Roberts and M. C. Caserio, "Basic Principles of Organic Chemistry", W. A. Benjamin, Inc.. New York, N.Y., 1965, p 450. (10) 6. L. Van Duren and T. L. Chan, Adv. Anal. Chem. Instrum., g, 419 1197 11. .I (1 1) G. G. Guilbault. "Practical Fluorescence", Marcel Dekker. Inc., New York, N.Y. 1973, p 27. I . - .
RECEIVEDfor review July 7, 1975. Accepted September 18, 1975. Work supported by the National Institute for Occupational Safety and Health and performed at the Los Alamos Scientific Laboratory operated under the auspices of the Energy Research and Development Administration. Contract No. W-7405-ENG-36 and Interagency Agreement IA-74-35.
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