Analyst, August 1994, Vol. 119 1907

Fluorescent Detection of Hydrazine, Monomethylhydrazine, and 1 ,I -Dimethylhydrazine by Derivatization With Aromatic Dicarbaldehydes

Greg E. Collins and Susan L. Rose-Pehrsson* Naval Research Laboratory, Chemistry Division, Code 61 10 Washington, DC, 20375-5342, USA

A chemical derivatization scheme has been developed for the sensitive and selective determination of hydrazine, monomethylhydrazine (MMH) and 1,l-dimethylhydrazine (UDMH) by fluorescence spectrometry. Incorporation of hydrazine into an aromatic framework by derivatization with o-phthalaldehyde (OPA), naphthalene-2,3- dicarbaldehyde (NDA), or anthracene-2,3- dicarbaldehyde (ADA) creates an efficient fluorophore the emission wavelength of which is red-shifted from the original reagent. The fluorescence emission for each of the different derivatizing reagents (OPA, NDA, and ADA), is minimal and nearly within the noise of the background. The hydrazine derivatives, on the other hand, are intensely fluorescent and characterized by a broad fluorescence emission centred at 376 nm for OPA, 500 nm for NDA, and 549 nm for ADA. For the NDA hydrazine derivative, a linear concentration dependence is observed from 50 ng 1-1 to 500 yg 1-1 of hydrazine (correlation coefficient, r > 0.999). The response time necessary to give 90% of a full- scale response is <2 min. The response of the ADA reagent is similar; however, its response time is faster ( < O S min), and its detection limit is higher (150 ng 1-1). By careful control of the pH and the aromatic dicarbaldehyde chosen, it is possible to differentiate quantitatively between the hydrazine, MMH, and UDMH levels present in mixed samples. The detection limits for MMH and UDMH using the NDA reagent are 120 ng 1-1 and 40 yg 1-1, respectively.

Keywords: Fluorescence; derivatization; hydrazine; naphthalene-2,3-dicarbaldehyde

Introduction Hydrazine (Hz), monomethylhydrazine (MMH), and 1,l- dimethylhydrazine (UDMH) are high-energy propellants used in large volumes for the space shuttle programme and for other aerospace operations (see Fig. 1). They also have a number of commercial applications, including their role as essential building blocks to the synthesis of various polymers, pesticides, pharmaceuticals, and chemotherapeutic agents. 172

The large volumes of Hz being used in the public and private sectors have generated concern for the health and safety of persons in close contact with these chemicals. The toxicolog- ical problems associated with the inhalation or ingestion of the hydrazines have been monitored in laboratory animals, and they include damage to internal organs, creation of blood abnormalities, irreversible deterioration of the nervous system, and documented teratogenic and mutagenic effects.3.4

* To whom correspondence should be addressed.

Therefore, the American Conference of Governmental Indus- trial Hygienists (ACGIH) has recommended that the threshold limit values (TLVs) for Hz, MMH, and UDMH be lowered from 100,200, and 500 ppb, respectively, to 10 ppb in air.5 Compliance with these standards will necessitate the development of new sensors for detecting these low levels of hydrazine in the workplace.

Several different analytical techniques have been used for the detection of hydrazines. Examples include coulometry ,6 potentiometry,7 titration,8 colorimetry,g dosimetry,10,11 flu- orescence,12~13 mass spectrometry,14 ion-mobility spec- trometry,15 gas chromatography,l6 and liquid chromato- graphy.17 Each of these techniques has merits in specific applications; however, none has combined the sensitivity, selectivity, and real-time monitoring capability necessary for determining the new TLVs for hydrazines in the workplace. We have been investigating analytical methodologies to meet these new requirements. The similarities in properties between Hz (N21-&) and ammonia (NH3) suggest that many of the reagents designed for the fluorescent derivatization of amines, amino acids, and peptides may also be applicable to the trace analysis of Hz. Among the different derivatizing agents developed for use in fluorescent amino acid analysis, emphasis has focused recently on the use of the aromatic dicarbaldehydes o-phthalaldehyde (OPA) and naphthalene- 2,3-dicarbaldehyde (NDA) (see Fig. 1). o-Phthalaldehyde and NDA react with peptides and primary amino acids in the presence of nucleophiles, such as 2-mercaptoethanol and the cyanide ion, respectively, to form highly fluorescent deriva- tives containing isoindole rings. 18-20 We recently demon- strated that NDA is an effective derivatizing reagent for the fluorescent detection of extremely low levels of Hz in solution.21 In this paper, the selectivity of the NDA reagent for the fluorescent detection of Hz, MMH, and UDMH is examined, as well as the reactivity and analytical characteris- tics of two other dicarbaldehydes in the homologous series, OPA, and anthracene-2,3-dicarbaldehyde (ADA) (see Fig. 1). Through proper choice of the pH, excitation and emission monitoring wavelengths, and specific dicarbaldehyde reagent, we will show that it is possible to quantify selectively the Hz, MMH, and UDMH levels in mixed samples.

Experimental

Apparatus

Fluorescence measurements were made using an SLM Model 8000 double-beam scanning spectrofluorimeter. A Rhodam- ine B reference was used to correct for instrument response and fluctuations in the lamp intensity (450 W xenon arc lamp).

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online / Journal Homepage / Table of Contents for this issue

1908 Analyst, August 1994, Vol. 11 9

Chemicals and Stock Solutions

All chemicals were used as received. Naphthalene-2,3-dicar- baldehyde (99%) and anthracene-2,3-dicarbaldehyde (== 100%) were obtained from Molecular Probes, and o-phtha- laldehyde (97%) from Aldrich. Stock solutions of OPA (10-2 mol 1-I), NDA (10-2 mol 1-I), and ADA (8 x 10-4 moll-1) were prepared in anhydrous ethanol. The Hz, monomethylhy- drazine, and 1,l-dimethylhydrazine solutions were prepared daily by diluting the anhydrous hydrazines in purified water (Millipore) to provide the following stock concentrations: 1 pg 1-, 10 pg 1-1,100 pg 1-1,1 mg 1-1 , lO mg 1-1,100 mg 1-1, and lo00 mg 1-1. All pH studies used 0.1 moll-' buffer solutions of boric acid (pH 6-11) or sodium dihydrogenphosphate (pH 3-5), each buffer solution being adjusted to the desired pH with NaOH or HCl. Solutions with a pH > 11 were prepared using NaOH, while those with a pH < 3 were prepared with HN03 or H2S04.

Procedure

In general, 2 ml of buffer solution were pipetted into a silica cuvette, along with 10-100 pl (Eppendorf microburette) of either the OPA, NDA or ADA stock solutions, giving reagent concentrations of 0.02 mmol 1-1 for ADA, and 0.5 mmol 1-1 for NDA and OPA. All measurements were taken at ambient temperature. Subsequent spiking of these solutions with the different hydrazines was accomplished by adding 1&100 p1 of the dilution standard, mixing the solution, and then returning the solution to the sample cell holder of the spectrofluo- rimeter. The final concentration of hydrazines in solution ranged from 50 ng 1-1 to 50 mg 1-1. Unless mentioned otherwise, fluorescence measurements were made at the pH best suited for the particular reagent and hydrazine being examined, i.e., pH 2.5 for the detection of Hz with NDA or OPA, pH 8 for the detection of MMH with NDA; and pH 11 for the detection of Hz with ADA. The following excitation and emission wavelengths (hex and hem) were used for the detection of the fluorescent derivatives formed: OPA (hex = 318 nm, he, = 376 nm); NDA (hex = 403 nm, hem = 500 nm); and ADA (hex = 476 nm, he, = 549 nm).

Kinetic Investigations

Kinetic studies examining the reactions of the hydrazines (Hz, MMH or UDMH) with the dicarbaldehyde reagents were conducted by monitoring the rate of fluorescent product formation using the spectrofluorimeter. The kinetic, pH profiles for the derivatization reactions between the aromatic dicarbaldehydes, OPA, NDA, and ADA, and the hydrazines, were carried out with the reagent concentrations in large

excess ([Hz] < < < [OPA], [NDA], or [ADA]), as the case would be in an actual analytical application. More specifically, the following concentrations were used in the various pH studies discussed in this paper: (i) [OPA] = 2.3 x 10-3 mol 1-1, [Hz] = 1.4 X 10-5 mol 1-1; (ii) [NDA] = 2.4 X 10-4 moll-1, [Hz] = 7.4 X 10-7 moll-1; (iii) [NDA] = 2.4 x 10-6 mol 1-1, [MMH] = 5.2 x 10-7 mol 1-1; (iv) [NDA] = 2.4 x 10-6 mol 1-1, [UDMH] = 3.1 x 10-4 mol 1-1; and (v) [ADA] = 1.9 X 10-6 mol 1-1, [Hz] = 7.4 x 10-8 mol 1-1.

The reaction order dependence on Hz was obtained from a plot of the observed rate constant versus the concentration of Hz, under conditions such that [Hz] > > > [NDA], or [ADA], or [OPA]. The actual concentrations employed in these studies were the following: (i) [OPA] = 4.5 x 10-6 moll-1, [Hz] = 1.40 X 10-5-2.9 X 10-4 moll-1, pH = 2.5; (ii) [NDA] = 2.4 X 10-8 mol 1-1, [Hz] = 1.5 x 10-4-2.8 X 10-3 mol 1-1, pH = 2.5; and (iii) [ADA] = 1.9 x 10-7 mol 1-1, [Hz] = 1.2 x 10-6-1.4 x 10-5 mol 1-1, pH = 6.

The kinetic order for the different dicarbaldehydes was obtained by plotting the observed rate constant versus the concentration of reagent in solution, wherein [NDA], or [OPA], or [ADA] > > > [Hz]. The specific concentration levels used were: (i) [OPA] = 4.8 X 10-5-1.25 X 10-3 moll-1, [Hz] = 4.3 X 10-6 mol 1-1, pH = 2.5; (ii) [NDA] = 4.8 x 10-5-5.9 x 10-3 mol l-l,[Hz] = 7.4 x 10-7 moll-1, pH = 2.5; (iii) [ADA] = 3.9 x 10-7-3.7 X 10-6 mol 1-1, [Hz] = 7.5 x 10-8 mol 1-1, pH = 13.5.

Mass Spectrometry

Mass spectrometry was used to identify the fluorescent products formed in the reactions of OPA, NDA, and ADA with Hz, in addition to the reactions of NDA with MMH and UDMH. These studies were carried out using either a Varian Model ITS40 gas chromatograpNquadrupole ion trap, or a Finnigan Model TSQ70 quadrupole mass spectrometer employing NH3 chemical ionization (Cl) or fast atom bom- bardment (FAB). Concentrated samples (10-3 moll-1) of the hydrazine derivatives were prepared by adding equimolar amounts of the hydrazine and the derivatizing reagent in 33% ethanol-water solutions (optimal pH conditions were used in each instance). The gas chromatographic separations of the NDA-hydrazine product from the intermediates and starting reagent were achieved using a DB-5 (5% phenylmethyl-95% dimethylpolysiloxane) column and a ramping temperature profile of 60-260 "C at 15 "C min-1. Electron impact (70 eV) ionization was used in the gas chromatography- mass spec- trometry (GC-MS) experiments. Thermal desorption chem- ical ionization with NH3 ( 4 6 6 . 6 Pa) or FAB (using 7 keV Xe atoms) were utilized as soft ionization sources in the verifica- tion of the parent peaks for the different hydrazine deriva- tives. A direct-exposure probe was used in the thermal

0 0 0 OPA NDA ADA

H\ /CH3 N-N

H\ /" N-N

H\ /H N-N

H / 'H H I 'CH, H' \CH3

Hz MMH UDMH

Fig. 1 Molecular structures of the derivatizing agents (OPA, NDA and ADA) and the hydrazines studied.

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online

Analyst, August 1994, Vol. 1 19 1909

desorption experiments. Samples were coated directly onto an emitter wire and introduced into the ion source. A ramping current was applied to the emitter wire, increasing the temperature at the probe tip at a rate of approximately lo00 "C min-1. Glycerol was used as the matrix in the FAB experiments.

Results and Discussion Reactions of OPA, NDA and ADA With Hz Scheme 1 shows the derivatization scheme for Hz with NDA. The initial reaction between Hz and NDA involves the nucleophilic attack of an electron lone pair of the Hz and an aldehyde group on the NDA. The postulated, alcoholic intermediate (2) is probably unstable, quickly losing a molecule of water to form the hydrazone, molecule 3. The proximity of the remaining lone pair of electrons on the hydrazone to the second aldehyde group, enables a similar set of reactions to complete the cyclization of the NDA reagent to form 2,3-diazaanthracene (5) . Although cyanide, CN-, has been commonly used in the reaction of NDA with primary amines in the formation of fluorescent, isoindole derivatives, the presence or absence of cyanide had no effect upon the reaction shown in Scheme 1. As demonstrated later in this section, 2,3-diazaanthracene (5) is non-fluorescent , whereas its conjugate acid (6) is intensely fluorescent.

There are probably two different acid-catalysed pathways operational in Scheme 1: (i) protonation of an aldehyde group in either 1 or 3 creates a new functional group, which is more susceptible to attack by the nucleophilic hydrazine (1 -+ 2 and 3 + 4); and (ii) protonation of the hydroxyl group of either 2 or 4 to form the weakly basic and significantly improved leaving group, -OH+2 (thus catalysing 2 -+ 3 and 4 + 5 ) . Gas chromatography-mass spectrometry has been used to corro- borate the formation of the hydrazone, 3 [C12H100N2: mlz = 198 (25%), 186 (12%), 154 (36%), 126 (100%)], as well as the final condensation product, 5 [C12HsN2: mlz = 180 (loo%), 153 (20%), 126 (70%)]. o-Phthalaldehyde and ADA react with hydrazine in an analogous fashion to the derivatization scheme shown in Scheme 1 for NDA. Chemical ionization mass spectrometry has verified the formation of phthalazine (CsH&: mlz = 130) and 2,3-diazanaphthacene (C16H10N2: mlz = 230) in the two respective reactions.

The incorporation of hydrazine into an aromatic framework such as benzene, naphthalene or anthracene, creates an efficient fluorophore the emission of which is significantly

0

0

1

red-shifted with respect to the fluorescent spectrum of the derivatizing reagent. Fig. 2 contains a set of emission spectra collected for OPA (hex = 318 nm), NDA (hex = 403 nm), and ADA (hex = 476 nm) following the sequential addition of 0, 4.3, 25, and 105 pg 1-1 of Hz to the solution (with the exception of OPA, where an additional aliquot of 440 pg 1-1 of Hz were added because of its lower sensitivity). Prior to the addition of Hz, the emission observed with the respective he, was minimal and nearly within the background for each of the reagents. The hydrazine derivatives, on the other hand, are characterized by a broad, intense fluorescence centred at 376 nm for OPA, 500 nm for NDA, and 549 nm for ADA. The fluorescence intensities increase in proportion to the concentration of Hz in solution.

Reaction order studies were conducted for each of the reagents (OPA, NDA, and ADA) in their reaction with Hz. The results are entirely consistent with the derivatization sequence presented in Scheme 1. In each instance, a plot of the observed rate constant, kobs, versus the concentration of Hz in solution showed excellent linear correlation, suggesting that the over-all reactions are first order with respect to Hz ( r = 0.99 for OPA, r = 0.99 for NDA, and r = 0.99 for ADA). Similarly, a linear dependence was observed between kobs and the concentration of NDA and ADA (of constant [Hz]), indicating that both of these reactions are first order in the dicarbaldehyde concentration ( r = 0.98 for NDA, and r = 0.99 for ADA). Increasing concentrations of OPA (with [OPA] > > > > [Hz]) produced a bell-shaped curve. The decrease seen in the apparent rate constant at very high concentrations of OPA is likely to arise from either a competing side reaction that forms a non-fluorescent deriva- tive at the emission wavelength being probed, or strong absorption (screening) by OPA or an OPA dimer.

As expected, pH strongly influences the rates of formation of the fluorescent derivatives arising from the reaction between hydrazine and the dicarbaldehydes. Fig. 3 shows the pH dependence of the three hydrazine reactions. While the reaction rates for NDA and OPA with Hz possess relative maxima near pH 2-3, the ADA reaction shows a vastly different pH dependence, with a reaction rate maxima at pH 11. In addition, the rate of reaction for ADA with Hz is nearly three orders of magnitude higher than that found for Hz with NDA or OPA. Despite the similarity in structure of the reagents in this homologous series, it is evident that the nature of the reaction for the formation of the fluorescent derivative is fundamentally different for the ADA reagent. An explana- tion for this disparity will be given later.

0

2

H

H

0

3

H

6

H

5

Scheme 1

H OH

4

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online

1910 Analyst, August 1994, Vol. 119

I/ I 1 I

Fig. 4 illustrates the dramatic differences in reaction rates and apparent sensitivities between the three dicarbaldehydes examined. For comparison, each derivatization reaction was performed using the optimal pH for fluorescent detection (OPA and NDA, pH 2.5; ADA, pH 11) and excitation/ emission wavelengths for each reagent. The plot displays the observed fluorescence signals following the addition of 45 yg 1-1 of hydrazine to the OPA, NDA, and ADA solutions. In agreement with Fig. 3, the production rate of the fluorescent, ADA-Hz derivative is substantially faster than those of the NDA or OPA derivatives. The reaction nears completion after only 10 s. The formation of the NDA-Hz

8.00

Q)

6.00 (I)

?? 7 E

> 4.00

.- c - 2.00

a"

OPA ,.: NDA '..., ADA

0 325 375 425 475 525 575 625 675

Wavelength/nm Fig. 2 Emission spectra obtained for OPA, NDA, and ADA following the addition of 0,4.3,25, and 105 pg 1-l of Hz (the addition of 440 pg 1-1 of Hz is also indicated for OPA).

40

30 .- tn - -L 20

a E 10

0

i 2.0~ lo4

1.5 x lo4 - I -

1.0~10~ L

2 5.0 x lo3

E

n -1 0 1 2 3 4 5 6 7 8 9 1011 121314"

PH Fig. 3 Plot of the formation rate constants (k) of the fluorescent OPA, NDA, and ADA derivatives of Hz as a function of pH. A, NDA-Hz; 0,OPA-Hz; and e, ADA-Hz.

c .C 3.00

derivative, on the other hand, takes place over a significantly longer time scale (10 min), while possessing the largest, apparent fluorescence quantum efficiency. This latter result manifests itself in an improved sensitivity of the NDA reagent for the detection of Hz. The sensitivity for Hz detection using our instrumentation increases in the order OPA < ADA < NDA, giving detection limits (assuming a signal-to-noise ratio of 3 : 1) of 1025, 150, and 50 ng 1-1, respectively.

Further examination of the reaction between Hz and NDA indicates that the NDA-Hz derivative is a fluorescent, pH indicator that is fluorescent only under acidic conditions (i. e., pH < 4). It follows logically that the fluorophore detected at a A,, of 500 nm is the conjugate acid of 2,3-diazaanthracene (5, Scheme 1) or species 6, Scheme 1. If the pH is adjusted to a neutral or basic pH, the NDA-Hz derivative (5, Scheme 1) becomes undetectable at the specified excitation and emission wavelengths. One can repeatedly cycle between the fluores- cent and non-fluorescent forms of this derivative (5 6, Scheme 1) by changing the pH. The rate of reaction step 5 e 6 (Scheme 1) is extremely rapid, suggesting that the rate limiting step is 3 4 (Scheme 1). As expected from Fig. 3, the OPA-Hz derivative behaves similarly to the NDA-Hz deriva- tive, exhibiting fluorescence only under acidic conditions. In contrast, the ADA-Hz derivative is fluorescent at basic pH (pH > 7). Apparently, the fluorophore in the ADA-Hz system is the conjugate base, i .e., the anthracene equivalent of 5 (Scheme 1).

Because the observed rate constant for the formation of the fluorescent NDA-Hz derivative (6, Scheme 1) at pH 2.5 is nearly 1000 times lower than that for the fluorescent ADA-Hz derivative at pH 11, we investigated the reactivity of NDA with Hz under neutral and basic conditions. The purpose of this study was to determine if the formation of the neutral, non-fluorescent NDA-Hz derivative (5 , Scheme 1) proceeds at a rate comparable to the formation of the neutral and fluorescent ADA-Hz derivative. An experiment was con- ducted in which an aliquot of Hz was added to a solution of NDA at a pH of 8 and allowed to react for 2 min; this was immediately followed by the addition of sufficient acid to lower the pH to 2.4. The reaction was monitored using the spectrofluorimeter and compared directly with a control in which the same Hz aliquot was added to a solution of NDA at a pH of 2.4. Fig. 5 shows the results obtained in both instances, demonstrating that acidic conditions prior to the formation of 2,3-diazaanthracene (5, Scheme 1) result in a significant suppression of the formation rate for the fluorescent, conju- gate acid, 6 (Scheme 1).

At pH < 5, more than 99% of Hz in solution is singly protonated (N2Hs+) .The removal of the lone pair of electrons from the amine nitrogen by protonation has a drastic impact

-.VV I B 3.50

3.00

Q 2.50 3 2.00 E a .2 1.50

Q)

b

c - 2 1.00 0.50

0 0 100 200 300 400 500 600 700 800 900 1000

Time/s Fig. 5 Comparison of the time response for the formation of the fluorescent NDA-Hz derivative following the addition of 23 pg I-' of Hz to A, NDA (pH 2.4) and B, NDA (pH 8 for 2 min., followed by conversion to pH 2.4). See text for details.

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online

Analyst, August 1994, Vol. 11 9 1911

upon the rate of the reaction 3 + 4 (Scheme 1). The pK, for hydrazine (K, is the acid dissociation constant) is 7.9, indicating that the rate of formation of the neutral complex, 2,3-diazaanthracene (5 , Scheme l) , will be significantly faster at a pH 3 8. The optimal pH for the formation of 2,3-diazaanthracene ( 5 , Scheme 1) is a trade-off between the hydrogen ion concentration necessary to catalyse the conden- sation reactions, and that required to prevent protonation of Hz and the formation of the weaker, nucleophilic inter- mediate. This study reveals that the time response for the formation of the fluorescent NDA-Hz derivative can be dramatically improved by carrying out the reaction under basic conditions, followed by a rapid pH change to acidic conditions in forming the fluorescent, conjugate acid. Using this reaction sequence, one can take advantage of the extreme reactivity of the Hz molecule under basic conditions.

The energies of the excited state levels for phthalazine (2,3-diazanaphthalene), the hydrazine derivative of OPA, have been determined elsewhere, and these values will be applied to explain the fluorescence, pH dependence for the three azines.22 The lowest excited singlet state for phthalazine is snn* (electronic state arising from the n + n* transition), followed by the higher energy, snx* (electronic state arising from the n -+ n* transition). The low fluorescence quantum yield obtained from the neutral form of phthalazine is a result of intersystem crossing between snx* and a lower energy, triplet state, t,,*, the longer lifetime of the latter being more conducive to non-radiative energy losses. Grabowska and Waluk23 have demonstrated that the relative arrangement of the sn,* and snn* levels changes upon protonation of phthalaz- ine. The increase in energy of the snn* level is due, presumably, to the fact that the lone pair of electrons associated with the aza group are now incorporated within a CJ

bond with the proton. Because the probability of intersystem crossing between electronic states of the same type ( i e . , sx,* + t,,*) is 2-3 orders of magnitude lower than the probability of intersystem crossing between electronic states of dissimilar type ( i e . , s,,* -+ tx,*), the fluorescence quantum yield is sharply enhanced following protonation of phthalazine.24 A similar argument can be used to explain the fluorescent properties of 2,3-diazaanthracene (5 , Scheme 1). Although the electronic state levels for 2,3-diazaanthracene (5 , Scheme 1) have not yet been determined, 9,lO-diazaphenanthrene has been examined extensively, and the spectroscopic levels for this molecule follow the same relative order seen for phthalaz- ine .25

The dependence of fluorescence on pH is distinctly different for the derivative formed in the reaction of ADA with Hz, 2,3-diazanaphthacene. In this instance, it is the neutral form of the derivative formed at basic pH that is strongly fluorescent. One possible explanation for this observation may be that the lowest energy, triplet state for 2,3-diazanaphthacene is now the t,,* state. The lack of intersystem crossing between sn,* and tn,* would help explain the intense fluorescence seen from this derivative. Subsequent protonation of the azine would theoretically interchange the levels of the singlet states, sn,* and snn*, thereby introducing a pathway for intersystem crossing and non-radiative decay processes. Because the reactivity of Hz is higher under basic conditions, and because a

0

II -H,O 0

fluorescent derivative is formed directly under these pH conditions, the rate of reaction for the formation of the fluorescent ADA-Hz derivative is substantially higher than that of the fluorescent NDA-Hz derivative formed under acidic conditions.

Reactions of NDA and ADA With UDMH and MMH

It is of interest to compare the reactivity of the different aromatic dicarbaldehydes with the structurally similar hydraz- ines, e.g. , MMH and UDMH. The addition of one or two methyl groups to Hz (see Fig. 1) has a dramatic impact on its reactivity with the derivatizing reagents, as well as on the fluorescence properties of the final derivative products.

Fig. 6 shows the pH dependence of the rate of fluorescent product formation in the reaction of NDA with Hz, MMH, and UDMH. Focusing first on the reaction between NDA and MMH, the optimal pH for fluorescence detection shifts from a pH of 2.5 for the NDA-Hz derivative, to a pH of 9 for the NDA-MMH derivative. In addition, the reaction rate for MMH is similar to reaction rates seen previously for the reactions between ADA and hydrazine (see Fig. 3), and is nearly three orders of magnitude larger than the reaction rate measured between NDA and Hz at pH 2.5.

By analogy to the reaction sequence discussed previously for hydrazine, the reaction between NDA and MMH results in the formation of a positively charged complex under neutral to basic pH conditions (see Scheme 2). In the second condensa- tion step, 2 + 3, the absence of a second, available proton on the MMH molecule, due to its replacement by a methyl group, results in a formal charge on the derivative. Mass spec- trometry (NH3CI) was used to confirm the identity of 3 (Scheme 2) (CI3HllN2: mlz = 195). In order to verify the presence of a formal charge on the NDA-MMH reaction product, FAB was used as an alternative and corroborating soft ionization technique. Owing to the similarity in structure between the NDA-MMH and NDA-Hz derivatives, we are confident that we can extend the spectroscopic interpretation described earlier to explain the formation of a fluorescent, NDA-MMH derivative at pH 3 7. The formal charge associated with molecule 3 (Scheme 2) is expected to make the

I 1.0~10~

1

0 -1 3 5 7 9 1 1

PH Fig. 6 Dependence on pH of the formation rates for the fluorescent derivatives of NDA with A , Hz, 0, MMH, and H, UDMH.

H

-H,O +H+

0

2 Scheme 2

H

H

3 1

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online

1912 Analyst, August 1994, Vol. 11 9

lowest excited singlet state for this molecule, the s,+ state. The absence of intersystem crossing from this state to the lower energy triplet electronic state, t,,*, results in a strongly fluorescent product. The reaction shown in Scheme 2 should be optimized at a pH near the pK, for MMH, where the majority of MMH in solution is unprotonated. Additionally, because a positively charged NDA-MMH derivative is formed directly at a pH of 9, the rate of the reaction is comparable to the apparent reaction rate for the formation of 2,3-diazaanthracene (5, Scheme 1) andor the fluorescent ADA-Hz derivative, 2,3-diazanaphthacene.

Referring once again to Fig. 6, the reaction between NDA and UDMH forms a fluorescent derivative optimally at a pH of 9, the same pH observed in the derivatization of MMH by NDA. Although the reaction rate for UDMH is similar to that of MMH, the sensitivity is substantially lower, with a detection limit that is several orders of magnitude above that found for either MMH or Hz (see under Analytical Response Charac- teristics for NDA and ADA).

Scheme 3 shows the proposed mechanism for the formation of the weakly fluorescent derivative formed in the reaction between NDA and UDMH. The initial step, 1 + 2, is the same condensation reaction postulated for the other hydrazines. The two methyl groups associated with UDMH prevent the loss of a second water molecule. The fluorescence emission evident at 500 nm is probably attributable to an equilibrium concentration of the positively charged derivative, 3, in solution, arising from the cyclization of the hydrazone. For the same reasoning applied to MMH, the fluorescent detection of UDMH is optimal at a pH of 9. It is unclear whether the lower sensitivity for the detection of UDMH is attributable to a small fluorescence quantum efficiency for molecule 3 (Scheme 3), to a small equilibrium constant for the formation of this product, or possibly to the formation of another, fluorescent side product.

Monomethylhydrazine and UDMH do not form fluorescent products on reaction with ADA. We can extend the spectro- scopic explanations provided earlier, by assuming that MMH and UDMH react with ADA to form positively charged complexes the lowest excited singlet states of which are s,,* in character. Intersystem crossing from these states to the lower energy, triplet state, tnn*, accounts for the lack of fluorescence in these systems. From an analytical standpoint, the critical factor is that ADA serves as a highly selective reagent for Hz in the presence of structurally similar hydrazines.

Analytical Response Characteristics for NDA and ADA

The fluorescence response of the NDA reagent following the sequential addition of Hz from 100 ng 1-1 to 500 pg 1-1 was examined as a function of time, and a portion of these data is shown in Fig. 7. Note that the response time necessary to give 90% of a full-scale response is <2 min. With each subsequent addition of hydrazine, the fluorescence at 500 nm increases linearly. The inset is a plot of the fluorescence changes as a function of the concentration of Hz added. The NDA reagent gives an excellent linear response to Hz at these low levels, as well as at significantly higher Hz concentrations. A linear

0

concentration dependence is observed over the dynamic range 50 ng 1-1-500 pg 1-1 of Hz ( r > 0.999), with a signal-to-noise ratio of 3 : 1 for 50 ng 1-1 of Hz. The response of the ADA reagent is similar, with the exception that its time response is significantly more rapid (less than 30 s), and its detection limit is higher (150 ng 1-1).

The selectivity of NDA and ADA for the fluorescent detection of hydrazine and/or MMH is an additional attractive feature of this derivatization scheme. The fluorescence response of the NDA reagent was examined relative to several likely interferents, including NH3, Freon 113, isopropyl alcohol, ethanol, and methyl ethyl ketone. In all instances, there was no measurable or detectable response at he, = 500 nm for concentrations of the interferents at twice their TLVs. Furthermore, by careful control of the pH, it is possible to differentiate quantitatively among the hydrazine, MMH, and UDMH levels in a given sample. Anthracene-2,3-dicarbal- dehyde, for example, reacts to form a strongly fluorescent derivative with Hz (hem = 549 nm), while forming a non-fluorescent derivative (at every pH) with both MMH and UDMH. On the other hand, NDA forms an intensely fluorescent derivative (hem = 500 nm) with both Hz and MMH, giving detection limits of 50 and 120 ng 1-1, respec- tively. The derivative formed with UDMH is nearly 1000 times less intense, bearing a detection limit of 40 pg 1-1. The rate of formation for each of these derivatives is highly pH depen- dent, as indicated by Figs. 3 and 6. This makes it possible to quantify the Hz, MMH, and UDMH levels in a given sample by making the following measurements: (i) the concentration of Hz in solution can be determined by using the selective reagent, ADA, at a pH of 11 and monitoring the fluorescence response at 549 nm; (ii) the concentration of MMH + UDMH in solution can be evaluated using NDA at a pH of 8 (hem = 500 nm), the fluorescent response of the NDA-UDMH derivative is negligible at UDMH concentrations below 40 pg 11'; and (iii) the total concentration of Hz + MMH in solution can be determined simply by lowering the pH of the solution described in (ii) and measuring the fluorescence at he, = 500 nm.

In order to evaluate the accuracy of this methodology, a mixture of 22.5 pg 1-1 Hz, 22.5 pg 1-1 UDMH, and 33.0 pg 1-1 MMH was analysed to determine the Hz and MMH levels. (i) the mixture was first examined using ADA at pH 11; the fluorescent response gave a value of 23.6 k 0.8 pg 1-1 Hz in solution. (ii) The concentration of MMH in solution was determined by analysing the fluorescent response following the addition of NDA at a pH of 8. The UDMH concentration was sufficiently low to prevent any interference. The value obtained for the MMH concentration was 35.2 k 1.4 pg 1-1. (iii) Finally, the Hz concentration was determined once again, by using the NDA reagent to determine the MMH + Hz concentration at pH 2. These results and those obtained at pH 8 with the NDA reagent were used to calculate the Hz concentration in solution at 22.3 k 1.1 pg 1-l.

Conclusion The fluorescent tagging of Hz and MMH using NDA and ADA is a highly sensitive and selective method for the

H

II -H20 0

It 0

H

H OH

3 1 2 Scheme 3

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online

Analyst, August 1994, Vol. 119 1913

detection of these hydrazines. Efforts are currently underway to design an inexpensive, fluorescence monitoring system for Hz, capable of determining the vapour levels of Hz in a given working environment. The simplicity of the derivatization scheme described in this paper has prompted us to examine the applicability of different chemical derivatization schemes. The basic derivatization scheme shown in Scheme 1 for the derivatization of hydrazine can be easily adapted to a number of different aromatic compounds the structures of which include a pair of adjacent carbonyl groups. Reaction with hydrazine is expected to extend the aromatic framework, resulting in a red-shifting of the fluorescence emission. Examples may include the addition of the 2,3-dicarbaldehyde substituents to naphthacene, pentacene, phenanthrene, pyrene, chrysene, and fluoranthrene. By synthesizing and examining the reactivity of these different structures, it may be possible to optimize a given analytical parameter of interest (e.g., he,, hem, response time, selectivity, sensitivity, lifetime, and dynamic range).

We acknowledge helpful conversations with Dave Kidwell, and technical assistance and advice from John Callahan for the mass spectrometric analysis of the different derivatives. This research was supported by NASAKennedy Space Center (DL-ESS-24, CC-82360A).

0.90

!! 0.70

e! - 2 0.50

Q

7 l f l 16.2 pg I-’

I 5 10 15 20 I I OHycirazine concentration/pg t1

0.10 0 500 lo00 1500

Time/s Fig. 7 Fluorescence response of the NDA reagent following repeated additions of hydrazine. The inset is a plot of these fluorescence changes as a function of the concentration of Hz added.

4

5

6 7

8 9

10

11

12

13 14 15

16

17

18 19 20

21

22

23 24

25

References Schmidt, E. W., Hydrazine and its Derivatives: Preparation, Properties, Applications, Wiley, New York, 1984. Schiessl, H. W., Encyclopedia of Chemical Technology, ed. Othmer, K., Wiley, New York, 3rd edn., 1980, vol. 12, p. 734. Vernot, E. H., MacEwen, J. D., Bruner, R. H., Haun, C. C., Kinkead, E. R., Prentice, D. E., Hall, A., 111, Schmidt, R. E., Eason, R. L., Hubbard, G. B., and Young, J. T., Fundament. Appl. Toxicol., 1985,5, 1050. Wald, N., Boreham, J., Doll, R., and Bonsall, J., Br. J. Znd. Med., 1984, 41, 31. American Conference of Governmental Industrial Hygienists: Documentation of the Threshold Limit Values and Biological Exposure Indices, 6th edn., Cincinnati, OH, 1991. Olsen, E. C., Anal. Chem., 1960,38,487. Stetter, J. R., Blurton, K. F., Valentine, A. M., and Tellefsen, K. A,, J. Electrochem. SOC., 1978, 125, 1804. Malone, H. E., Anal. Chem., 1961,33, 575. George, G. D., and Stewart, J. T., Anal. Lett., 1990, 23(8), 1417. Holtzclaw, J. R., Rose, S. L., Wyatt, J. R., and Hawkins, C. M., US Pat., 4780282, 1988. Taffe, P. A., and Rose-Pehrsson, S. L., US Pat., 4900681, 1990. Weeks, R. W., Jr., Yasuda, S. K., and Dean, B. K., Anal. Chem., 1976,48, 159. Danielson, N. D., and Conroy, C. M., Talanta, 1982, 29,401. Knox, B. E., and McHale, E. J., Anal. Chem., 1966, 38, 487. Leasure, C. S., and Eiceman, G. A., Anal. Chem., 1985, 57, 1890. Holtzclaw, J. R., Rose, S. L., Wyatt, J. R.. Rounbehler, D. P., and Fine, D. H., Anal. Chem., 1984,56,2952. Ravichandran, K., and Baldwin, R. P., Anal. Chem., 1983,55, 1782. Roth, M., Anal. Chem., 1971,43,880. Lunte, S . M., and Wong, 0. S., LC.GC Znt., 1989,7(11), 908. Kwakman, P. J. M., Koeleweijn, H., Kool, I., Brinkman, U. A. Th., and de Jong, G. J., J . Chromatogr., 1990,511, 155. Collins, G. E., and Rose-Pehrsson, S. L., Anal. Chim. Acta, 1993, 2434,207. Alvarez, V. L., and Hadley, S. G., J. Phys. Chem., 1972, 76, 3937. Grabowska, A., and Waluk, J., J. Lumin., 1979, 18/19, 201. Krasovitskii, B. M., and Bolotin, B. M., Organic Luminescent Materials, ed. Vopian, V. G., VCH, Weinheim, 1988, p. 8. Dewey, H., and Hadley, S. G., Chem. Phys. Lett., 1971,12,57.

Paper 31055786 Received September 16, 1993 Accepted December 15, 1993

Dow

nloa

ded

by U

nive

rsity

of

Gue

lph

on 0

9 M

ay 2

012

Publ

ishe

d on

01

Janu

ary

1994

on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/A

N99

4190

1907

View Online