4-Nitro-7-piperazino-2,1,3-benzoxadiazole as a reagent for monitoring of airborne isocyanates by liquid chromatography

Article abstract of DOI:10.1021/ac0260488

4-Nitro-7-piperazino-2,1,3-benzoxadiazole (NBDPZ) is presented as a new reagent for the determination of mono- and diisocyanates in air samples. NBDPZ readily reacts with the airborne analytes, thus yielding the corresponding urea derivatives, which are s

Full text of DOI:10.1021/ac0260488

Anal. Chem. 2002, 74, 6418-6426
4-Nitro-7-piperazino-2,1,3-benzoxadiazole as a
Reagent for Monitoring of Airborne Isocyanates by
Liquid Chromatography
Martin Vogel* and Uwe Karst
Department of Chemical Analysis and MESA+ Research Institute, University of Twente, P.O. Box 217,
7500 AE Enschede, The Netherlands
acute pulmonary edema2 or even chronic asthma.2,3 Therefore,
identification and sensitive quantification of mono- and diisocy-
anates in air is of crucial importance to workplace analysis.
As the analytes readily react with a wide range of interfering
compounds, e.g., water, direct spectroscopic methods are not
applicable to the analysis of individual isocyanates and derivati-
zation has turned out to be inevitable. While first methods were
mainly based on colorimetric techniques possessing only re-
stricted selectivity and sensitivity,4-8 modern analytical approaches
predominantly comprise the derivatization with nucleophilic
compounds such as alcohols or amines, which is followed by
chromatographic separation and, in most cases, photometric or
fluorometric detection. Over the past decades, especially the use
of amine-based reagents has proven to show best results with
respect to the analysis of isocyanates.9-22 Although a number of
different derivatizing agents is currently available, only few of them
offer sufficient sensitivity, e.g., 9-(N-methylaminomethyl)-an-
4 -Nitro-7 -piperazino-2 ,1 ,3 -benzoxadiazole (NBDP Z) is
presented as a new reagent for the determination of mono-
and diisocyanates in air samples. NBDP Z readily reacts
with the airborne analytes, thus yielding the correspond-
ing urea derivatives, which are subsequently separated
by means of reversed-phase liquid chromatography. On
a phenyl-modified stationary phase, excellent baseline
separation for numerous mono- and diisocyanate deriva-
tives is obtained. Both diode array and fluorescence
detection are performed with limits of detection of 1 1 -
3 5 and 5 -9 nmol/ L for the individual derivatives, re-
spectively. In contrast to established derivatizing agents
for the analysis of isocyanates, NBDP Z provides for
increased selectivity due to the favorable detection wave-
lengths in the visible range (UV/ visible, absorption maxi-
mums ∼4 8 0 nm; fluorescence, excitation maximums
∼4 7 0 nm, emission maximums ∼5 3 5 nm). In addition,
the high molar absorptivities of the reagent and the
derivatives provide excellent sensitivity that is superior
to most literature-known methods. Finally, air sampling
methods comprising both the use of impingers and test
tubes are developed and successfully applied to the
determination of isocyanates in gaseous samples. Excel-
lent recovery reaching values of >9 0 % is observed for
each of the two techniques investigated.
(2) Roth, L. Isocyanate: Eigenschaften, Vorkommen, Verwendung, Arbeitsschutz,
Umwelt, Entsorgung, Lagerung, Toxizita¨ t, Diagnostik, Therapie, ecomed
Verlagsgesellschaft: Landsberg, 1997.
(3) Purnell, C. J.; Walker, R. F. Analyst 1 9 8 5 , 110, 893-905.
(4) Buckles, R. E.; Thelen, C. J. Anal. Chem. 1 9 5 0 , 22, 676-678.
(5) Layton, R. F.; Knecht, L. A. Anal. Chem. 1 9 7 1 , 43, 794-797.
(6) Kubitz, K. A. Anal. Chem. 1 9 5 7 , 29, 814-816.
(7) Swann, M. H.; Esposito, G. G. Anal. Chem. 1 9 5 8 , 30, 107-109.
(8) Marcali, K. Anal. Chem. 1 9 5 7 , 29, 552-558.
(9) Dunlap, K. L.; Sandridge, R. L.; Keller, J. Anal. Chem. 1 9 7 6 , 48, 497-499.
(10) Bagon, D. A.; Warwick, C. J.; Brown, R. H. Am. Ind. Hyg. Assoc. J. 1 9 8 4 ,
45, 39-43.
(11) Molander, P.; Haugland, K.; Fladseth, G.; Lundanes, E.; Thorud, S.;
Thomassen, Y.; Greibrokk, T. J. Chromatogr., A 2 0 0 0 , 892, 67-74.
(12) OSHA Analytical Laboratory Method No. 47: Methylene Bisphenyl Isocyanate
(MDI); OSHA: Salt Lake City, 1985.
(13) Sango¨ , C.; Zimerson, E. J. Liq. Chromatogr. 1 9 8 0 , 2, 971-990.
(14) Andersson, K.; Gude´hn, A.; Hallgren, C.; Levin, J.-O.; Nilsson, C.-A. Scand.
J. Work Environ. Health 1 9 8 3 , 9, 497-503.
Today, both mono- and diisocyanates find widespread applica-
tion in the industrial production of pharmaceuticals, pesticides,
and polyurethanes (PUR), representing one of the most important
classes of polymers, the annual global consumption of which has
reached over 6 million tons.1 PUR-based foam materials are mainly
used for insulation or furniture while solid PUR can even be found
in medical implants. Additionally, prepolymers with terminal
isocyanate groups are applied as glues or sealants.
(15) Streicher, R. P.; Arnold, J. E.; Ernst; M. K.; Cooper, C. V. Am. Ind. Hyg.
Assoc. J. 1 9 9 6 , 57, 905-913.
(16) Roh, Y.-M.; Streicher, R. P.; Ernst, M. K. Analyst 2 0 0 0 , 125, 1691-1696.
(17) Karlsson, D.; Dahlin, J.; Skarping, G.; Dalene, M. J. Environ. Monit. 2 0 0 2 ,
4, 216-222.
Due to their high reactivity, isocyanates are significantly toxic
and exposure to these compounds during production or treatment
of PUR materials may cause severe irritations to the mucous
membranes of the respiratory system, thus finally leading to either
(18) Spanne, M.; Tinnerberg, H.; Dalene, M.; Skarping, G. Analyst 1 9 9 6 , 121,
1095-1099.
(19) Tinnerberg, H.; Spanne, M.; Dalene, M.; Skarping, G. Analyst 1 9 9 6 , 121,
1101-1106.
(20) Tinnerberg, H.; Karlsson, D.; Dalene, M.; Skarping, G. J. Liq. Chromatogr.
Relat. Technol. 1 9 9 7 , 20, 2207-2219.
* Corresponding author. Fax: ++31-53-489-4645. E-mail: m.vogel@ct.utwente.
(21) Batlle, R.; Colmsjo¨ , A.; Nilsson, U. Fresenius' J. Anal. Chem. 2 0 0 1 , 369,
524-529.
nl.
(1) Ulrich, H. Chemistry and Technology of Isocyanates; John Wiley & Sons:
(22) Nordqvist, Y.; Melin, J.; Nilsson, U.; Johansson, R.; Colmsjo¨ , A. Fresenius'
Chichester, 1996.
J. Anal. Chem. 2 0 0 1 , 371, 39-43.
6418 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002
10.1021/ac0260488 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/19/2002

thracene (MAMA)13,14 or dibutylamine (DBA).17-22 Applying
MAMA, detection can be performed by means of both UV/ visible
and fluorescence spectroscopy, but owing to absorption maxi-
mums in the short-wavelength range, this may be prone to matrix
interferences. In contrast, DBA, which lacks a chromophoric or
fluorophoric moiety, is mostly restricted to mass spectrometric
detection whichsalthough quite sensitivesrequires a more ex-
pensive instrumental setup than photometric or fluorometric
detectors, which are found in almost any of today’s analytical
laboratories.
Derivatizing agents based on the 2,1,3-benzoxadiazole back-
bone have found widespread application especially for bioanalytical
and environmental purposes.23-32 They are mainly used as tagging
reagents for amino acids,29 carboxylic acids,30 amines,29 alcohols,31
and thiols32 but also for the quantification of aldehydes and ketones
in air samples.25 Although all benzoxadiazoles are likely to be
excellent chromophores that possess UV/ visible absorption
maximums in a range >350 nm in conjunction with high molar
absorptivities, fluorescence is restricted to a limited number of
compounds. The influence of substituents on spectroscopic
properties is thoroughly discussed in the literature.33
This paper describes the quantification of airborne isocyanates
making use of 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBDPZ),
which has formerly been applied only to the determination of
carboxylic acids.30 The analyte derivatives are separated by means
of reversed-phase (RP) high-performance liquid chromatography
(HPLC). Owing to the versatile spectroscopic characteristics that
are introduced with the NBDPZ reagent, detection can be
performed by both UV/ visible and fluorescence spectroscopy.
Regarding selectivity, derivatization of isocyanates applying NB-
DPZ thus allows photometric detection in the visible range of the
electromagnetic spectrum where spectral interferences resulting
from matrix constituents are not quite likely to occur. Additionally,
the possibility of the NBDPZ method to perform fluorescence
spectroscopy leads to a further improvement of sensitivity when
compared to photometry.
obtained from Gru¨ ssing (Filsum, Germany) in the highest quality
available. Acetonitrile (Elution grade) was from Merck Eurolab
(Fontenau S/ Bois, France), and water for chromatography was
purchased from Merck Eurolab (Briare le Canal, France). 4-Chloro-
7-nitro-2,1,3-benzooxadiazole and formic acid were delivered by
Fluka (Neu-Ulm, Germany) in the highest quality available.
Safety Considerations. Dealing with volatile isocyanatess
especially methyl, ethyl, propyl, butyl, pentyl, hexyl, and phenyl
isocyanatesinevitably recommends handling in a hood with
appropriate safety precautions, e.g., gloves, safety goggles, and,
optionally, inhalation precautions.
1
Instrumentation for P roduct Identification. H NMR mea-
surements were performed with an AM 300 spectrometer from
Bruker (Bremen, Germany). The solvent for all NMR measure-
ments was hexadeuterated dimethyl sulfoxide (DMSO-d6). All
peaks are given as δ in ppm and are related to the signal of
remaining nondeuterated DMSO content (2.49 ppm vs TMS). FT-
IR spectral information was obtained for the products in potassium
bromide pellets using an IFS 48 from Bruker. Mass spectra were
recorded on a MAT 212 from Varian (Darmstadt, Germany).
Elemental analyses were performed with a vario EL III CHNOS
analyzer version H from Elementar Analysensysteme GmbH
(Hanau, Germany). Melting points were determined with a Mel-
Temp II from Holliston. The analytical data for the reagent
derivatives are provided as Supporting Information.
P hotometer. UV/ visible spectra were recorded with a HP
8453 diode array spectrophotometer (Hewlett-Packard, Wald-
bronn, Germany) equipped with HP Chem Station 845x-biochemi-
cal UV/ vis system.
Spectrofluorophotometer. A RF 5301-PC spectrofluoropho-
tometer from Shimadzu (Duisburg, Germany) with software
version 1.10 was used to record fluorescence spectra.
Syntheses. (1 ) 4 -Nitro-7 -piperazino-2 ,1 ,3 -benzoxadiaz-
ole. A 5.00-g amount of 4-chloro-7-nitro-2,1,3-benzoxadiazole (25
mmol) in 300 mL of dichloromethane was added dropwise to a
solution of 8.74 g (100 mmol) of piperazine in 400 mL of methanol.
During this procedure, the product precipitated as red crystalline
material, which was filtered off and washed with water and cold
methanol. Subsequently, the product was allowed to dry for 12 h
in a vacuum. The yield was 66%. The purity of the product was
examined by means of HPLC. The product was fully characterized
EXPERIMENTAL SECTION
Chemicals. Methyl isocyanate was obtained from ChemSer-
vice (West Chester, PA). 4,4′-Methylene bis(phenyl isocyanate)
was purchased from Lancaster (Eastgate, White Lund, Morecamb,
U.K.). All other isocyanates used, trifluoroacetic acid (TFA),
piperazine, and ammonium formate were from Aldrich (Steinheim,
Germany). Solvents used for synthesis and spectroscopy were
1
by means of H NMR, IR, UV/ visible, MS, melting point, and
elemental analysis. These data are provided as Supporting
Information.
(23) Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K. Biomed.
Chromatogr. 2 0 0 1 , 15, 295-318.
(24) Huang, C. Z.; Santa, T.; Imai, K. Analyst 2 0 0 2 , 127, 741-747.
(25) Bu¨ ldt, A.; Karst, U. Anal. Chem. 1 9 9 9 , 71, 1893-1898.
(26) Bu¨ ldt, A.; Karst, U. Anal. Chem. 1 9 9 9 , 71, 3003-3007.
(27) Vogel, M.; Bu¨ ldt, A.; Karst, U. Fresenius' J. Anal. Chem. 2 0 0 0 , 366, 781-
791.
(28) Meyer, J.; Bu¨ ldt, A.; Vogel, M.; Karst, U. Angew. Chem., Int. Ed. 2 0 0 0 , 39,
1453-1455.
(29) Gosh, P. B.; Whitehouse, M. W. Biochem. J. 1 9 6 8 , 108, 155-156.
(30) Toyo’oka, T.; Ishibashi, M.; Takeda, Y.; Nakashima, K.; Akiyama, S.; Uzu,
S.; Imai, K. J. Chromatogr. 1 9 9 1 , 588, 61-71.
(2 ) Methyl Isocyanate NBDP Z Derivative (MI-NBDP Z).
For the synthesis of MI-NBDPZ, 555 mg (2.23 mmol) of NBDPZ
was dissolved in 140 mL of dichloromethane. Afterward, 500 mg
(8.77 mmol) of methyl isocyanate was rapidly added to the stirred
solution and the mixture was allowed to react for 1 h under
stirring. After 1 min, the MI-NBDPZ precipitated as reddish
crystalline material. To remove excess amounts of methyl isocy-
anate, 2 mL of methanol was added to the mixture. Finally, the
product was filtered off, washed with water and 20 mL of cold
methanol, and dried in a vacuum for at least 12 h. The yield was
77%. The urea derivative was fully characterized by means of 1H
NMR, IR, UV/ visible, MS, melting point, and elemental analysis.
These data are provided as Supporting Information.
(31) Uchiyama, S.; Santa, T.; Suzuki, S.; Yokosu, H.; Imai, K. Anal. Chem. 1 9 9 9 ,
71, 5367-5371.
(32) Toyo’oka, T.; Suzuki, T.; Saito, Y.; Uzu, S.; Imai, K. Analyst 1 9 8 9 , 114,
413-419.
(33) Uchiyama, S.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. J. Chem. Soc.,
Perkin Trans. 1 9 9 8 , 2, 2165-2173.
Analytical Chemistry, Vol. 74, No. 24, December 15, 2002 6419

(3 ) NBDP Z Derivatives of Ethyl, P ropyl, Butyl, P entyl,
Hexyl, and Benzyl Isocyanate (EtI-NBDP Z, P I-NBDP Z,
BI-NBDP Z, P eI-NBDP Z, HI-NBDP Z, Bz-NBDP Z, Re-
spectively). These derivatives were prepared by adding a solution
of 4.00 mmol of the respective isocyanate in 2 mL of dichlo-
romethane to a stirred solution of 250 mg (1.00 mmol) of 4-nitro-
7-piperazino-2,1,3-benzoxadiazole in 70 mL of the same solvent.
The mixture was allowed to react for 2 h. To remove the excess
amount of isocyanate, 2 mL of methanol was added. For the
precipitation of the formed urea derivatives, 70 mL of hexane was
subsequently added in portions. The orange-reddish product was
filtered off, washed with water and cold methanol, and dried in a
vacuum for at least 12 h. If necessary, the product was recrystal-
lized from acetonitrile. Yields ranged from 54% to 90%. All urea
derivatives were fully characterized by means of 1H NMR, IR, UV/
visible, MS, melting point, and elemental analysis. These data are
provided as Supporting Information.
melting point, and elemental analysis. These data are provided
as Supporting Information.
(7 ) 4 ,4 ′-Methylene Bis(phenyl isocyanate) NBDP Z De-
rivative (MDI-NBDP Z). Initially, 1.31 mmol of 4-nitro-7-piper-
azino-2,1,3-benzoxadiazole was dissolved in 90 mL of dichlo-
romethane. To the stirred solution, 0.27 mmol of 4,4′-methylene
bis(phenyl isocyanate) in 10 mL of dichloromethane was added.
The corresponding urea derivative precipitated within a few
seconds and was filtered off after a reaction time of 30 min. The
product was washed with 20 mL of cold methanol and 20 mL of
ethyl acetate. Finally, the crystalline material was additionally
suspended in 20 mL of hot acetonitrile. After the MDI derivative
had been filtered off again, it was washed with cold methanol and
allowed to dry under vacuum for 12 h. The yield was 72%. The
1
product was fully characterized by means of H NMR, IR, UV/
visible, MS, melting point, and elemental analysis.
HP LC Instrumentation and Analysis. All quantitative liquid
chromatographic measurements and reactivity investigations were
performed on a Shimadzu (Duisburg, Germany) HPLC system
comprising the following parts: two LC-10AS pumps, GT-104
degasser unit, SIL-10A autosampler, sample loop with variable
injection volume of up to 50 µL, SUS mixing chamber (0.5 mL),
CTO-10ACvp column oven, SPD-M10Avp diode array detector, RF-
10AXL fluorescence detector, CBM-10A controller unit, and Class
LC-10 software version 1.6. Stability tests and chromatographic
method development were carried out on a Shimadzu HPLC
system consisting of the following: two LC-10AS pumps, GT-104
degasser unit, Rheodyne six-port valve (series 7725i) with 5-µL
sample loop, SUS mixing chamber (0.5 mL), CTO-10ASvp column
oven, SPD-10AVvp UV/ visible detector, CBM-10A controller unit,
and Class LC-10 software version 1.6.
(4 ) NBDP Z Derivatives of P henyl and Naphthyl Isocy-
anate (P hI-NBDP Z and NI-NBDP Z, Respectively). A 4.00-
mmol sample of the isocyanate in 2 mL of dichloromethane was
added to a solution of 250 mmol of 4-nitro-7-piperazino-2,1,3-
benzoxadiazole in 70 mL of dichloromethane. The reaction
mixture was stirred for 2 h. To remove the excess amount of
isocyanate, 2 mL of methanol was added. The urea derivatives
precipitated as orange-reddish crystalline material, which was
subsequently filtered off, washed with water and cold methanol,
and finally dried under vacuum for 12 h. The yield was 61% for
PhI-NBDPZ and 87% for NI-NBDPZ, respectively. The products
1
were fully characterized by means of H NMR, IR, UV/ visible,
MS, melting point, and elemental analysis. These data are provided
as Supporting Information.
(5 ) NBDP Z Derivatives of 2 ,6 - and 2 ,4 -Toluene Diisocy-
anate (2 ,6 -TDI-NBDP Z and 2 ,4 -TDI-NBDP Z, Respec-
tively). An amount of 1.00 mmol of 4-nitro-7-piperazino-2,1,3-
benzoxadiazole was dissolved in 60 mL of dichloromethane.
Afterward, a solution of 0.26 mmol of the respective diisocyanate
in 2 mL of the same solvent was added to the stirred reagent
solution. After a few seconds, the product precipitated as orange-
reddish crystalline material. The latter was filtered off, washed
with cold methanol, and dried under vacuum for 12 h. The yield
was 87% for the 2,6-TDI derivative and 81% for the 2,4-TDI
compound, respectively. The ureas were fully characterized by
means of 1H NMR, IR, UV/ visible, MS, melting point, and
elemental analysis. These data are provided as Supporting
Information.
For liquid chromatographic analysis, the following columns
were used:
(Column 1) ProntoSIL 120-5-Phenyl (Bischoff Chromatography,
Leonberg, Germany); particle size 5 µm; pore size 120 Å; column
dimensions 250 mm × 3 mm; guard column 10 mm × 3 mm.
(Column 2) Discovery RP18 (Supelco, Deisenhofen, Germany);
particle size 5 µm; pore size 200 Å; column dimensions 150 mm
× 3 mm; guard column 20 mm × 3 mm. (Column 3) ProntoSIL
120-5-C18-ace-EPS (Bischoff Chromatography); particle size 5
µm; pore size 120 Å; column dimensions 150 mm × 3 mm; guard
column 20 mm × 3 mm. (Column 4) LiChrospher RP-18ec in
ChromCart cartridges (Macherey-Nagel, Du¨ ren, Germany); par-
ticle size 5 µm; pore size 100 Å; column dimensions 250 mm × 3
mm; guard column 8 mm × 3 mm. (Column 5) Discovery RP 18
(Supelco, Deisenhofen, Germany); particle size 5 µm; pore size
200 Å; column dimensions 20 mm × 3 mm.
(6 ) 1 ,6 -Hexamethylene Diisocyanate NBDP Z Derivative
(HDI-NBDP Z) and Isophorone Diisocyanate NBDP Z De-
rivative (IP DI-NBDP Z). First, 1.00 mmol of 4-nitro-7-piperazino-
2,1,3-benzoxadiazole was dissolved in 60 mL of dichloromethane.
Subsequently, 0.25 mmol of the respective diisocyanate in 2 mL
of dichloromethane was added to the stirred solution. The mixture
was allowed to react for 2 h. To precipitate the urea derivatives,
40 mL of n-hexane was added in 10-mL portions. The solids were
then filtered off, washed with water and cold methanol, and dried
under vacuum overnight. If necessary, the products were recrys-
tallized from acetonitrile. The yield was 74% for HDI-NBDPZ and
45% for IPDI-NBDPZ, respectively. The urea compounds were
For separation, binary gradients with the profiles shown in
Table 1 were chosen.
Reactivity Measurements. (1) Reactivity of NBDP Z toward
2 ,6 -TDI. For the investigation of the reactivity of NBDPZ toward
2,6-toluene diisocyanate, 46.4 µL of a 5.0 × 10-2 mol/ L solution
of 2,6-TDI in acetonitrile was added to 100 mL of a 2.0 × 10-4
mol/ L solution of NBDPZ in acetonitrile. After thorough mixing,
the reaction solution was immediately analyzed by liquid chro-
matography using column 5 and applying gradient C. Detection
was performed by means of fluorescence spectroscopy (excitation
wavelength λ ) 470 nm; emission wavelength λ ) 538 nm).
1
fully characterized by means of H NMR, IR, UV/ visible, MS,
6420 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

temperature-supported evaporation of defined analyte amounts was
performed. In series, either two reagent solution-filled impingers
(sampling and backup) or two reagent-coated cartridges (sampling
and backup) were placed. All connections were Teflon tubing and
were kept as short as possible to avoid analyte loss owing to
adsorption phenomena. The simulated air sampling with a defined
amount of the analyte(s) was performed by pipetting the respective
volume of an acetonitrile solution containing a known concentra-
tion of isocyanate(s) into the heatable flask. Afterward, a constant
air stream was pumped through the setup for a defined period of
time. After sampling, the test tubes were eluted with 10 mL of
acetonitrile. A 10-µL portion of this solution was injected into the
HPLC system. After the impinger sampling procedure was
finished, the volume was made up to the calibration mark and
subsequently, 10 µL of the solution was injected into the HPLC
system. For separation, column 2 and gradient E were used.
Detection was performed photometrically at 478 nm.
Table 1. Profiles of Binary Gradients
Gradient A: flow, 0.65 mL/ min; T, 45 °C;
(A) acetonitrile (0.05% (v/ v) TFA),
(B) water (0.05% (v/ v) TFA)
time (min)
A (%)
0
27.5 29.0 30.0 32.0 35.0 (stop)
c
20 60 100 100 20 20
Gradient B: flow, 0.60 mL/ min; T, ambient;
(A) acetonitrile, (B) buffer pH 3.5 (1000 mL of water,
60 µL of formic acid, 265 mg of ammonium formate)
time (min)
A (%)
0
1.0
15.0 16.0 18.0 19.0
60 100 100 35
21.0 (stop)
35
c
35 35
Gradient C: flow, 2.00 mL/ min; T, ambient;
(A) acetonitrile, (B) water (0.05% (v/ v) TFA)
time (min)
A (%)
0
0.6
1.0
100
1.5
100
1.8
15
2.5 (stop)
15
c
15 15
Gradient D: flow, 2.00 mL/ min; T, ambient;
(A) acetonitrile, (B) water (0.05% (v/ v) TFA)
time (min)
A (%)
0
1.0
1.5
100
2.0
100
2.3
50
3.0 (stop)
50
c
50 50
Gradient E: flow, 2.00 mL/ min; T, ambient;
(A) acetonitrile, (B) water (0.05% (v/ v) TFA)
RESULTS AND DISCUSSION
time (min)
A (%)
0
1.0
15
60
17
100
18
100
20
30
23 (stop)
30
The aim of the present study was to develop a reagent for the
c
30 30
derivatization of mono- and diisocyanates in air samples serving
for both UV/ visible and fluorescence spectroscopic detection.
Furthermore, the new method should additionally provide for good
chromatographic separation as well as for sufficient reactivity
toward the analytes. Although a number of derivatizing agents
have already been described in the literature, each one having
its individual advantage, none of them fulfills all needs, which are
directed to a modern derivatization method. Within this work,
NBDPZ is first described for the derivatization of mono- and
diisocyanates. The reagent has been applied earlier to the
determination of carboxylic acids.30 NBDPZ can easily be syn-
thesized in a nucleophilic substitution reaction of piperazine with
4-chloro-7-nitro-2,1,3-benzoxadiazole (NBDCl).30
(2 ) Reactivity of MAMA toward 2 ,6 -TDI. For the investiga-
tion of the reactivity of 9-(N-methylaminomethyl)anthracene
toward 2,6-toluene diisocyanate, 61.7 µL of a 5.1 × 10-2 mol/ L
solution of 2,6-TDI in acetonitrile was added to 100 mL of a 3.0 ×
10-4 mol/ L solution of MAMA in acetonitrile. After thorough
mixing, the reaction solution was rapidly analyzed by means of
liquid chromatography using column 5 and applying gradient D.
Detection was performed by means of fluorescence spectroscopy
(excitation wavelength λ ) 254 nm; emission wavelength λ ) 412
nm).
P reparation of NBDP Z Sampling Solution and NBDP Z
Sampling Tubes. Preparation of a NBDPZ reagent solution for
impinger sampling: In a 250-mL volumetric flask, 31.2 mg of
4-nitro-7-piperazino-2,1,3-benzoxadiazole was suspended in 100 mL
of acetonitrile and the resultant mixture was treated in an
ultrasonic bath for 1 min. Afterward, the flask was filled to the
calibration mark.
Preparation of NBDPZ sampling tubes: 6-mL glass cartridges
with Teflon frit (Supelco) were packed with 500 mg of Supelclean
LC-18 material (particle size 45 µm, pore size 60 Å), also from
Supelco. The packed sorbent was topped with a glass wool layer.
These test tubes were conditioned first with 1 mL of acetonitrile,
and afterward, the packing material was coated with NBDPZ by
allowing 10 mL of a 2.3 × 10-3 mol/ L reagent solution to seep
through the tubes. Subsequently, the cartridges were dried in a
gentle stream of nitrogen.
Isocyanates readily react with the NBDPZ reagent, thus
forming the corresponding urea derivatives:
Air Sampling P rocedure and Analysis. Air sampling was
performed using a two-channel air sampler pump (model 1067)
from Supelco. Flow rates were determined applying a 1000-mL
bubble flowmeter from Supelco. The generation of simulated
isocyanate atmospheres was performed in thoroughly dried glass
apparatus to avoid hydrolysis of the reactive analytes. The
instrumental setup used in this work is described in the follow-
ing: Initially, air is pumped through a 100-mL impinger filled with
50 mL of concentrated sulfuric acid, which is connected with a
second impinger serving to avoid contamination of the rest of the
setup. The latter was followed by a heatable flask in which the
All NBDPZ ureas show attractive chromophoric properties,
e.g., absorption maximums in the range of 480 nm and high molar
Analytical Chemistry, Vol. 74, No. 24, December 15, 2002 6421

Figure 3. UV/visible spectrum of 4-nitro-7-piperazino-2,1,3-ben-
Figure 1. UV/visible spectra of selected NBDPZ isocyanate deriva-
tives dissolved in acetonitrile/dimethyl sulfoxide 20:1 (v/v): methyl
isocyanate (dotted), 2,6-toluene diisocyanate (dashed), methylene bis-
(phenyl isocyanate) (short dotted), and isophorone diisocyanate
(solid). Concentrations range from 1.2 to 1.6 × 10-5 mol/L.
zoxadiazole (NBDPZ, dotted) compared to that of 9-(N-methylami-
nomethyl)anthracene (MAMA, solid). Solvent, acetonitrile; cNBDPZ
)
1.1 × 10-4 mol/L; cMAMA ) 1.9 × 10-5 mol/L.
Figure 4. UV/visible spectrum of NBDPZ (dotted) compared to that
of 1-(2-methoxyphenyl)piperazine (MPP, solid). Solvent, acetonitrile;
cNBDPZ ) 1.1 × 10-4 mol/L; cMPP ) 1.1 × 10-4 mol/L.
Figure 2. Fluorescence spectrum of methyl isocyanate NBDPZ
(MI-NBDPZ) in acetonitrile/dimethyl sulfoxide 20:1 (v/v). cMI-NBDPZ
) 1.3 × 10-5 mol/L; λexc ) 470 nm; λem ) 537 nm.
by means of liquid chromatographic separation followed by
photometry and fluorometry, respectively. As for fluorescence
detection, LOD range from 5.1 × 10-9 mol/ L for benzyl isocyanate
(BzI) to 8.3 × 10-9 mol/ L for isophorone diisocyanate (IPDI).
LOD for photometry range from 1.1 × 10-8 mol/ L for 2,4-toluene
diisocyanate (2,4-TDI) to 3.5 × 10-8 mol/ L for 1-naphthyl isocy-
anate (NI). Photometric LOD are in good accordance with the
molar absorptivities stated above because diisocyanate derivatives
can be detected more sensitively due to the presence of two NBD
moieties. Although fluorescence spectroscopy yields significantly
lower LOD than UV/ visible detectionsup to 6.2 times lower as
observed for BzIsthere is no decreasing effect on LOD for
bifunctional analytes in fluorescence spectroscopy. Unexpectedly,
detection limits are even slightly worse than for monofunctional
analytes. However, the applicability of fluorescence spectroscopy
especially provides higher selectivity when isocyanates in complex
matrixes are determined. Linear dynamic ranges for all NBDPZ
derivatives were determined and comprise 3 orders of magnitude
for fluorescence and four decades for UV/ visible detection.
As a selective analysis of isocyanates by means of derivatization
requires an excellent separation of reagent and derivatives, a
absorptivities. Figure 1 presents UV/ visible spectra of selected
isocyanate derivatives. Obviously, the spectra are mainly charac-
terized by the NBD backbone independent of the derivatized
analyte. In accordance with a derivatization at both functionalities,
molar absorptivities of diisocyanates are ∼2 times higher than
those of monoisocyanate derivatives. Additionally, all NBDPZ
compounds show good fluorescence characteristics with excitation
wavelengths at ∼470 nm and emission wavelengths at ∼535 nm.
Figure 2 presents the excitation and emission spectra of MI-
NBDPZ, which is characteristic for the other isocyanate derivatives
discussed within this paper. Compared to all established amine
reagents as MAMA,13,14 DBA,17-22 or MPP,10,11 NBDPZ provides
for the most red-shifted absorption maximums (Figure 3, Figure
4), thus guaranteeing spectroscopic detection with limited matrix
interferences and going along with highest selectivity. With
respect to molar absorptivities, NBDPZ is by far superior to most
established amine reagents apart from MAMA (Figure 3). This
results in excellent limits of detection (LOD). Table 2 summarizes
the instrumental detection limits of several NBDPZ ureas obtained
6422 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

Table 2. Instrumental Limits of Detection (LOD) for the
HPLC Determination of NBDPZ Derivatives Performing
UV/Visible and Fluorescence Spectroscopya
fluorescence detection
λex ) 470 nm;
UV/ visible detection
λem ) 538 nm
λ ) 480 nm
LOD (10-9
mol/ L)
LOD (pg
of analyte)
LOD (10-8
mol/ L)
LOD (pg
of analyte)
NBDPZ
MI
EtI
PI
BI
PhI
BzI
HDI
2,4-TDI
NI
7.8
7.2
6.8
6.0
5.6
5.1
6.4
7.2
5.7
8.3
4
5
6
6
7
2.5
3.0
3.1
3.1
3.2
3.2
1.5
1.1
3.5
1.3
14
21
26
31
38
43
25
19
59
29
7
11
13
10
19
IPDI
Figure 6. Stability of NBDPZ dissolved in acetonitrile applying three
different storage conditions. cNBDPZ ) 5.5 × 10-6 mol/L; HPLC
analysis was performed using column 1 and gradient A; detection
wavelength was 478 nm.
a
These data were obtained using column 3 and gradient B.
eluent modification. To overcome the coelution problem, a phenyl-
modified reversed-phase column turned out to show the best
selectivity for the given separation problem (Figure 5, chromato-
gram A). Here, the crucial pair of peaks (8/ 9) is well separated,
and analogous to the separation on ODS columns, two peaks for
the IPDI derivative are observed again. This is due to the fact
that the commercially available IPDI contains both the cis and
the trans isomer whose derivatives chromatographically behave
differently.
Figure 5. (A) LC separation of a series of mono- and diisocyanate
NBDPZ derivatives on a phenyl-modified stationary phase (column
1, gradient A). (B) LC separation of the same compounds on a RP-
18 column (column 4, gradient A). Peaks: NBDPZ (1), methyl
isocyanate (2), ethyl isocyanate (3), propyl isocyanate (4), butyl
isocyanate (5), pentyl isocyanate (6), phenyl isocyanate (7), 2,6-
toluene diisocyanate (8), 1,6-hexamethylene diisocyanate (9), 2,4-
toluene diisocyanate (10), cis-/trans-isophorone diisocyanate (11/11′),
and methylene bis(phenyl isocyanate) (12).
Compared to the MAMA method, NBDPZ provides for a better
liquid chromatographic separation, which is due to the smaller
NBD backbone. Therefore, structural differences of the derivatized
analytes are more likely to influence the separation process. For
MAMA derivatives, steric demands of the anthracene moiety
dominate, thus showing worse separation of structurally related
compounds.
The performance of analytical derivatization methods is strongly
dependent on the stability of the reagent used as well as on the
stability of the formed analyte derivatives. Therefore, storage
stability of NBDPZ and its derivatives dissolved in acetonitrile was
investigated by applying different conditions: An amount of the
sample was stored at room temperature and under the influence
of daylight, another part was also stored at room temperature but
kept in the darkness, and a last sample was stored in the
refrigerator at 5 °C. All solutions were regularly monitored by
means of HPLC with subsequent UV/ visible detection at 478 nm.
Figure 6 summarizes the results that were obtained for NBDPZ.
When stored in a cooled environment, the reagent solution turned
out to be stable for at least 3 days. Over a period of two weeks in
number of reversed-phase columns for liquid chromatography
were tested with respect to their applicability to the NBDPZ
method. Performing a binary gradient that consisted of an aqueous
buffer and acetonitrile, most commercially available RP-18 columns
revealed good results for the separation of the reagent and its
corresponding analyte derivatives. Long-wavelength detection at
∼480 nm is advantageous as the UV chromatographic baseline is
quite flat even when running strong gradients. The observed
fronting of peaks 2-4 in chromatogram B (Figure 5) was because
the sample injected was dissolved in 100% of acetonitrile while
the gradient applied started with an amount of only 20% of the
same solvent. Although a baseline separation for the 2,4-TDI and
the 2,6-TDI urea was obtained on all octadecyl-modified silica
(ODS) columns, coelution of 2,6-TDI and HDI (Figure 5, chro-
matogram B) could not be avoided even by means of gradient or
Analytical Chemistry, Vol. 74, No. 24, December 15, 2002 6423

the refrigerator, the peak area decreased down to ∼80% of its initial
value. In contrast, both storage experiments performed at ambient
temperature revealed a significantly larger decrease in peak area,
which went down to 41% (room temperature, darkness) and 27%
(room temperature, daylight), respectively. As for HPLC analysis,
this was accompanied by the occurrence of a new peak that eluted
significantly later in the chromatogram, thus indicating the
formation of a more nonpolar compound. First assumptions that
this might result from an noncovalent adduct formation of two
NBDPZ molecules could be falsified by adding an excess of
isocyanate to the sample. This showed that the peak area of the
unknown product was not influenced by this experiment. In
contrast, all NBDPZ derivatives turned out to be stable under the
given storage conditions. This leads to the conclusion that the
NBD backbone does not undergo any kind of degradation reaction
but that the secondary amine function of the piperazine group
seems to be responsible for the formation of a new compound,
the identity of which has not been elucidated yet. Stability
problems of amine-based agents are not restricted to NBDPZ and
have already been described in the literature. For example, MAMA
was reported to be not very stable and was recommended to be
freshly prepared from its hydrochloride prior to use.3 Although
the NBDPZ reagent revealed only limited stability when stored
in organic solution, storage as a solid in the refrigerator yielded
no degradation over a period of at least six months. With respect
to the application of NBDPZ in sampling solutions, the results
observed are not significantly crucial as the reagent can freshly
be dissolved in organic media prior to use. Furthermore, the good
stability of the reagent when stored as a solid offers the op-
portunity to develop sampling methods based on NBDPZ-coated
test tubes.
To study the reactivity toward the analytes, the reaction of
NBDPZ with isocyanates was investigated by means of fast HPLC.
For these measurements, an amount of an isocyanate was added
to an excess of the reagent dissolved in acetonitrile. Immediately
after additon, samples were injected into the HPLC system at
constant intervals. For separation, a short gradient program was
applied, and the peak area of the derivative was observed with
time. Conditions for reactivity measurements are described in
detail in the Experimental Section. The reaction of 4-nitro-7-
piperazino-2,1,3-benzoxadiazole with 2,6-toluene diisocyanate was
complete within less than 10 min, and the recovery was ∼95%.
Compared to NBDPZ, the reaction of the MAMA reagent with
2,6-TDI was obviously slower with the reaction being complete
after ∼25 min. Although the reaction monitoring carried out within
this work is much too slow to effectively monitor relative reaction
rates as described in the literature,34 the experiments performed
indicate that the rate enhancement of NBDPZ relative to MAMA
is consistent with that reported by Streicher et al. for the two
piperazine reagents 1-(9-anthracenylmethyl)piperazine (MAP) and
MPP versus MAMA.15 Therefore, NBDPZ can also be considered
as a good alternative to MAMA with regard to reactivity.
Table 3. Recovery Rates for the Investigation of
Simulated 2,4-TDI Atmospheres Obtained by Means of
Impinger Samplinga
n2,4-TDI-NBDPZ
c2,4-TDI
recovery
(%)
no. of
samples
sample
(mol)
(µg/ m3)b
1
2
3
4
2.3 × 10-7
1.2 × 10-7
5.8 × 10-8
2.9 × 10-8
1830
916
458
229
82 ( 15
95 ( 10
86 ( 5
86 ( 5
3
3
3
3
a
Flow rate was determined to be 369 mL/ min; sampling time was
60 min. The evaporation flask was heated to 130 °C. b c is calculated
on the idealized assumption that the analyte is continuously evaporating.
Table 4. Recovery Rates for the Simultaneous
Determination of Propyl (PI) and Phenyl Isocyanate
(PhI) Obtained by Impinger Samplinga
sample/
analyte
nanalyte
(mol)
canalyte
recovery breakthrough
no. of
samples
(µg/ m3)b
(%)
(%)
1 (PI)
4.0 × 10-7
2.0 × 10-7
5.0 × 10-8
1520
1820
761
909
190
71 ( 2
88 ( 1
77 ( 4
91 ( 2
71 ( 2
79 ( 3
10 ( 1
3
3
3
3
3
3
1 (PhI) 3.4 × 10-7
2 (PI)
9 ( 4
2 (PhI) 1.7 × 10-7
3 (PI)
11 ( 1
3 (PhI) 4.2 × 10-8
227
a
Flow rate was 369 mL/ min; sampling time was 60 min. The
evaporation flask was heated to 130 °C. b c is calculated on the idealized
assumption that the analyte is continuously evaporating.
compound was tested. As summarized in Table 3, recovery rates
for 2,4-toluene diisocyanate range from 82% to 95%. For all
measurements, no breakthrough into the backup impinger was
observed. The large standard deviations from 5% to 15% may result
from technical circumstances that occurred during sampling
procedure. Based on these promising results, impinger sampling
was subsequently performed with respect to the simultaneous
determination of PI and PhI, the results of which are presented
in Table 4. On one hand, good results were obtained for the
aromatic analyte showing recovery from 79% up to 91% without
any breakthrough into the backup impinger. On the other hand,
recovery for the aliphatic propyl isocyanate was significantly lower,
and additionally, breakthroughs occurred. This can be explained
on the basis of the lower reactivity of aliphatic isocyanates toward
nucleophiles compared to the aromatic representatives. If the
recoveries from the sampling and the backup impinger are
summed up, total recovery of propyl isocyanate of >80% is
obtained.
In addition to air sampling by means of impingers, a method
for the determination of airborne isocyanates using test tubes was
developed. These were filled with a supporting material that was
coated with the reagent prior to use. In contrast to impinger
sampling, the application of test tubes is less laborous and can
be performed by nonexperienced personal. As for method
development, a number of commercially available solid-phase
extraction (SPE) tubes filled with ODS material were tested.
Although the material could be easily coated with NBDPZ, thus
yielding a sufficient reagent excess, recovery turned out to be far
below 50% of the calculated value. This was due to adsorption of
the airborne analytes on the tube walls, which were made out of
polypropylene. Further method development was performed using
In the following, NBDPZ was used to develop active (pumped)
sampling methods for the determination of isocyanates in the gas
phase. The generation of analyte atmospheres was performed by
introducing a robust setup that is described in the Experimental
Section. Initially, air sampling by means of impingers for a single
(34) Kuck, M.; Balle´, G.; Slawyk, W. Analyst 1 9 9 9 , 124, 933-939.
6424 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002

aerosol is more difficult for reagent-coated test tubes than TDI
vapor because diffusion is much slower for the aerosol. This is
often likely to result in breakthroughs. However, those could not
be observed during the analyses described. Although the calcula-
tion of the concentration determined was based on the assumption
of a continuous evaporation of the analytes and despite the fact
that a rather concentrated MDI atmosphere was generated within
the 250-mL evaporation flask, the results obtained clearly dem-
onstrate the risk resulting from heated PUR materials containing
residual amounts of monomeric isocyanates.
Table 5. Recovery Rates for the Investigation of
Simulated 2,4-TDI Atmospheres Obtained by Means of
Glass Cartridges Filled with NBDPZ-Coated ODS
Materiala
n2,4-TDI-NBDPZ
c2,4-TDI
recovery
(%)
no. of
samples
sample
(mol)
(µg/ m3)b
1
2
3
1.0 × 10-7
5.0 × 10-8
2.5 × 10-8
878
439
220
89 ( 4
99 ( 7
86 ( 14
3
3
3
a
Flow rate was 330 mL/ min; sampling time was 60 min. The
evaporation flask was heated to 150 °C. b c is calculated on the idealized
assumption that the analyte is continuously evaporating.
CONCLUSIONS
NBDPZ has been applied as a new reagent for the determi-
nation of mono- and diisocyanates in air samples, thus revealing
numerous advantages compared to established derivatization
methods. A series of NBDPZ isocyanate derivatives, all of which
are new compounds, has been synthesized and fully characterized
by means of melting point, infrared spectroscopy, 1H NMR, mass
spectrometry, and UV/ visible and fluorescence spectroscopy. Both
the reagent and its urea derivatives, which are formed with the
isocyanates, are characterized by the most red-shifted absorption
maximums of all established amine reagents as well as by high
molar absorptivities (ꢀ). Compared to the MPP reagent, ꢀ for
NBDPZ compounds are ∼4 times higher. In addition, all ureas
show good fluorescence, which is characterized by emission
wavelengths in the visible range of the electromagnetic spectrum.
Owing to the excellent spectroscopic properties, selectivity of this
method is higher than for any other reagent described, and the
limits of detection are superior to most known derivatizing agents.
Stability in organic solution and as a solid has been thoroughly
tested and has turned out to be very good for the ureas dissolved
in acetonitrile as well as for the solid compounds, while reagent
solutions were stable for at least 3 days only when stored in a
refrigerator. NBDPZ and the corresponding derivatives can be
separated on a number of octadecyl-modified silica HPLC columns
while highest chromatographic selectivity is obtained on a phenyl-
modified phase. Thus, chromatographic resolution of the new
method is quite advantageous, and it is superior to that of the
MAMA method, where coelution of isocyanate derivatives is more
likely to occur. The reactivity of NBDPZ toward the analytes has
been investigated in organic media and has turned out to be faster
than that of MAMA. Regarding sampling procedures, active
(pumped) methods based on impingers and reagent-coated test
tubes were developed and successfully applied to the analysis of
isocyanate-containing air samples.
Table 6. MDI Concentrations during the Thermal
Treatment of a Commercially Available Polyurethane
(PUR) Foam Determined on the Basis of the NBDPZ
Method Performing Test Tube Samplinga
UV/ vis detection
λ ) 478 nm
amount of
foam (g)
sample
n (10-7mol)
c (mg/ m3)b
1 (RT)
2 (130 °C)
3 (150 °C)
10
13
17
2.5 ( 0.1
8.0 ( 0.3
3.2 ( 0.1
10.1 ( 0.3
a
Flow rate was set to 330 mL/ min; sampling time was 60 min. The
PUR sample was investigated under three different temperatures. b c
is calculated on the idealized assumption that the analyte is continu-
ously evaporating.
glass cartridges that were manually packed with the respective
filling material. Table 5 summarizes recovery experiments for 2,4-
toluene diisocyanate using ODS-filled glass tubes for sampling
experiments. Recoveries ranged from 86% up to 99% and break-
through of the analyte into the backup tube was not observed.
Good recovery within the lower concentration range thus suggests
that workplace concentrations can be effectively monitored, e.g.,
the German maximum workplace concentration (MAK) of 70 µg/
m3.35 To sum up, with the introduction of glass tubes filled with
NBDPZ-coated ODS material, a new sampling setup for individual
workplace monitoring of isocyanates that is easy to use has been
established.
To investigate the applicability of the NBDPZ test tube method
for the determination of real samples, emissions resulting from a
PUR foam based on 4,4′-methylene bis(phenyl isocyanate) (MDI)
were investigated. The foam, which is mainly used for insulation
purposes, was freshly sprayed into a flask. Immediately afterward,
emissions from three PUR samples were analyzed: The first
sample was kept at room temperature, and the second and the
third samples were heated to 130 and 150 °C, respectively. The
amounts of MDI that were determined on the basis of the NBDPZ
test tube method are presented in Table 6. For the nonheated
PUR foam, no MDI emission was detected, while the thermally
treated samples revealed significant release of residual monomeric
MDI that was higher than the German MAK value for MDI of 50
µg/ m3.2 With respect to MDI, the thermal treatment of freshly
sprayed PUR foam is prone to yield an aerosol. Collection of an
ACKNOWLEDGMENT
Financial support of this work by the Fonds der Chemischen
Industrie (Frankfurt am Main, Germany) is gratefully acknowl-
edged.
SUPPORTING INFORMATION AVAILABLE
Text presenting melting point, IR, 1H NMR, MS and elemental
analysis data for methyl isocyanate NBDPZ urea derivative (MI-
NBDPZ), ethyl isocyanate NBDPZ urea derivative (EtI-NBDPZ),
propyl isocyanate NBDPZ urea derivative (PI-NBDPZ), butyl
isocyanate NBDPZ urea derivative (BI-NBDPZ), pentyl isocyanate
NBDPZ urea derivative (PI-NBDPZ), hexyl isocyanate NBDPZ
urea derivative (HI-NBDPZ), phenyl isocyanate NBDPZ urea
(35) Deutsche Forschungsgemeinschaft MAK- und BAT-Werte-Liste der Senatsko-
mmission zur Pru¨ fung gesundheitsscha¨ dlicher Arbeitsstoffe; VCH: Weinheim,
1996.
Analytical Chemistry, Vol. 74, No. 24, December 15, 2002 6425

derivative (PhI-NBDPZ), benzyl isocyanate NBDPZ urea derivative
(BzI-NBDPZ), 1-naphthyl isocyanate NBDPZ urea derivative (NI-
NBDPZ), 1,6-hexamethylene diisocyanate NBDPZ urea derivative
(HDI-NBDPZ), 2,6-toluene diisocyanate NBDPZ urea derivative
(2,6-TDI-NBDPZ), 2,4-toluene diisocyanate NBDPZ urea derivative
(2,4-TDI-NBDPZ), 4,4′-methylene bis(phenylisocyanate) NBDPZ
urea derivative (MDI-NBDPZ), isophorone diisocyanate NBDPZ
urea derivative (IPDI-NBDPZ), and 4-nitro-7-piperazino-2,1,3-
benzoxadiazole (NBDPZ). This material is available free of charge
via the Internet at http:/ / pubs.acs.org
Received for review August 14, 2002. Accepted October
23, 2002.
AC0260488
6426 Analytical Chemistry, Vol. 74, No. 24, December 15, 2002