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ANALYTICAL CHEMISTRY, VOL. 51,
NO. 8, JULY 1979
Determination of Aliphatic Isocyanates in Air by a Liquid Chromatographic-Fluorescence Technique S. P. Levine,” J. H. Hoggatt, E. Chladek, G. Jungclaus,’ and J. L. Gerlock2 Ford Motor Company, Engineering and Research Staff- Research, 24500 Glendale Avenue, Redford, Michigan 48239
A liquid chromatographic (LC) method has been developed for the determination of aliphatic isocyanates in air. The Isocyanates are converted to stable urea derivatives with I-naphthalenemethylamine (NMA). These derivatives are analyzed using reverse-phase isocratic LC and detected by a fluorescence detector using an excitation wavelength of 216 nm. The method Is approximately fifty tlmes more sensitive than currenf techniques and has a detection llmlt of 0.5 ng of aliphatic hexamethylene dilsocyanate-biuret trimer (HDI-BT) which is equlvaient to 3 ppt in a 20-L air sample. Stop-flow LC combined with spectral scanning provides a means of posltively identifying peaks as NMA derivatives. Results are compared to those obtained using a previously published LC technique, and the presence of peak doublets is discussed.
Concern for worker safety has prompted us to re-examine analytical techniques developed to monitor worker breathing zone air for trace quantitites of airborne isocyanates such as those likely t o be associated with large scale polyurethane paint spraying operations. We find that most of the analytical techniques now available (1-8) must be used a t maximum sensitivity t o just detect airborne isocyanates in amounts comparable to expected threshold limit values (TLV) (9). The need for improved analysis sensitivity is apparent, and is emphasized by t h e present industry trend of replacing aromatic isocyanates with yellowing-resistant aliphatic isocyanates. The present work addresses the problem of assaying worker breathing zone air for aliphatic isocyanates a t the ppb-ppt level. T h e standard method for the determination of aromatic isocyanates in air (the Marcoli method (1-3)) depends on the formation of intensely colored derivatives when the corresponding aromatic amines (isocyanate hydrolysis products) are diazotized and react with a derivatizing agent. Such a colorimetric analysis is not readily extended to aliphatic isocyanates which do not form stable diazonium salts. Keller e t al. have approached the problem of aliphatic isocyanate analysis by a double derivative thin-layer chromatography (TLC) technique (6, 7). Aliphatic isocyanates are first reacted with N-4-nitrobenzyl-N-n-propylamine (Nitro Reagent, N.R.) t o form ureas which are readily separated from the reaction mixture by T L C and developed by reducing the nitro group t o a n amine followed by diazotization and coupling with N-1-naphthylethylenediamine.Quantitation is achieved by comparing standard “known” plate spots with unknowns. The lower detection limit of the method is claimed to be 400 kg/M3 for Desmodur N (hexamethylene diisocyanate-biuret trimer (HDI-BT)). In a second report, the idea of separating the derivative mixture into components for analysis was upgraded by using liquid chromatography (LC) with UV detection (8). This technique eliminates the need for a second derivatizing Present address, Battelle Memorial Laboratory, Columbus,Ohio. *Present address, Ford Motor Company, Engineering and Research Staff-Research, 20000 Rotunda Drive, Dearborn, Michigan 48121. 0003-2700/79/0351-1106$01 .OO/O
step (diazotization) to produce colored species and eliminates the ambiguities associated with TLC quantitation methods. Individual species produced by the reaction of aliphatic isocyanates with N.R. are identified by their LC retention times and quantified by integration of peak areas. The sensitivity of the analysis is increased to 100 pg/M3 for HDI-BT by using LC instead of TLC. We find that the lower detection limit of LC analysis can be increased by an additional factor of fifty by substituting a fluorescent derivatizing agent, 1-naphthlenemethylamine (NMA), for nitro reagent. This substitution takes advantage of recent improvements in fluorescence detector sensitivity.
EXPERIMENTAL Reagents. Isocyanates. Samples of commercial grade hexamethylene diisocyanate (HDI), hexamethylene diisocyanatebiuret trimer (HDI-BT) and isophorone diisocyanate (IPDI) were obtained from Celanese and Mobay Chemical Companies, and used without further purification. No difference between the samples was noted. Deriuatizing Agents. Reagent grade N-4-nitrobenzyl-N-npropylamine hydrochloride (Nitro Reagent-NR) was obtained from Regis Chemical Co. Samples of technical grade 1naphthlenemethylamine (NMA) were obtained from Aldrich Chemical and Pfaltz and Bauer. All derivatizing agents were used without further purification and no difference between samples was noted. Soluents. Chromatographic solvents consisted of UV grade acetonitrile (Burdick and Jackson), reagent grade absolute ethanol (J.T. Baker),UV grade hexane (Burdick and Jackson), Nanograde hexane (Mallinckrodt), and Milli-Q purified water (Millipore). Other solvents used included UV grade dimethylsulfoxide (Burdick and Jackson), Nanograde toluene (Mallinckrodt), and Nanograde dichloromethane (Mallinckrodt). Apparatus. Air Sampling. DuPont P4000A and Bendix (2115 air pumps were used to pull air through 25-mL Greenburg-Smith midget impingers. Chromatograph. A Waters Associates LC system was used for all separations. This system includes two model 6000 high pressure pumps, a U6K injector, a model 660 solvent programmer, and a model 440 UV absorbance detector. A Schoeffel model FS970 fluorescence detector equipped with a SFA 338/501 wavelength drive and a model MM700 memory module was also utilized. Signal noise level was reduced through the use of a Spectrum model 1021A filter-amplifier. Signals were recorded with either a Weather Measure EPR-2OOA two-channel recorder (integration by peak height times width at half height) or a Hewlett-Packard 3385A printer-plotter electronic integrator. Liquid Chromatographic Procedure. Keller Method. The preparation of Nitro-Reagent, as well as air sampling and analysis procedures has been described in detail in references 6 and 8. It was desirable to modify these conditions to achieve separation of the isocyanate derivatives examined in the present work. Air sampling flow rate was reduced from 2 L/min to 0.5 L/min to minimize NR absorbant solution evaporation. A longer, smaller diameter Whatman HC Pellosil column (3 f t X 2.1 mm i.d.) improved resolution. The peak shape of slower eluting compounds was improved by (a) the addition of a 6-min initial condition hold period (470 solvent/B) followed by (b) an exponential increase in the percent B (program 7 ) as opposed to a linear increase in the percent B (program 6), and (c) raising the final concentration of B from 40% to 52% using a 13-min program time. Solution D 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
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OCN-(CH,),-NCO
fl 0 II OCN-(CH,),-NH-C-N-(CHZ),-NCO
I
c=o I NH I
(CHz), I
NCO
?
CH,NH,
I
/
CH,NHCNHI
HDIBT-NR
Figure 1. Structure of aliphatic isocyanates commonly used in urethane
paints and the NMA derivatization reaction B was 80% Burdick and Jackson UV-grade hexane and 20% Baker Reagent grade ethanol. Solution A was 99% B-J UVhexane and 1% Mallinckrodt Nanograde hexane. NMA-Fluorescent Derivative Method. Concentrated solutions of l-naphthlenemethylamine in toluene, lo-* M, were prepared in advance and diluted to working concentration, M, with toluene as required. For analysis of ppt-level samples, a M absorbing solution should be used. Stock solutions were stable for months when stored in a refrigerator. In a typical test, 10 mL of lo-*M NMA absorbing solution was placed in a 25-mL Greenburg-Smith midget impinger and air was drawn through at 0.5 L/min. All tests were performed in duplicate or triplicate. When sampling was completed, the contents of the impinger were transferred to a 25-mL pear-shaped flask and flash evaporated to dryness over hot (approx. 60 "C) water with a rotary evaporator. The residue was dissolved in 500 pL of dimethylsulfoxide and chromatographed by injecting a 5- or 10-pL sample of the solution onto a Waters Associates 30 cm X 3.9 mm pBondapak CN column and eluting with an isocratic, 55% water/45% acetonitrile, liquid phase at a flow rate of 1.9 mL/min. The WBondapak CN column must have been prepared by Waters Associates using a water- or methanol-containing slurry. If it was manufactured using nonpolar solvents, a short column life will result owing to collapsing of the solid support. Components were detected with a fluorescence detector set at a wavelength of 216-nm excitation operating with a UV 30 emission filter in front of the photomultiplier. Detector operating parameters used were: Multiplier gain, 520; attenuation, 0.1-1.0; and a time constant of 7 s. The possibility that the toluene solvent might be incompletely removed was recognized. Possible interferences from that source were tested by performing complete LC-fluorescence detection analyses on toluene as well as on a complete system blank. No interferences were found from this source. Excitation spectra of individual peaks were obtained as follows. An excitation scan from 200 to 700 nm is performed using the source wavelength drive. The results of this scan are entered in the background memory of the Memory Module. The sample of interest is then injected into the LC and the LC pumps are stopped just before the NMA-derivative peak maximum, allowing residual pressure to bring the chromatogram to the peak maximum. The wavelength scan is then performed again and the background signal is automatically subtracted by the Memory Module and recorded using the printer-plotter.
RESULTS AND DISCUSSION Keller HPLC Method. The use of nitro reagent (NR) for the derivatization of aliphatic isocyanates with subsequent analysis by T L C or H P L C is a widely applied technique (6, 8). T h e structures of aliphatic diisocyanates commonly used in paints are given in Figure 1. We found that it was necessary to modify Keller's procedure slightly to improve separation of mixtures of IPDI, HDI, and
Figure 2. Liquid chromatogram of NR derivative of HDI-BT at and near the lower limit of detection
HDI-BT derivatives. A different solvent program (see Experimental) and a longer column improved sensitivity. Base-line drift problems associated with UV-absorbing impurities in solution B (hexane-ethanol) were nullified by adding traces of UV-absorbing hexane to solution A (hexane). These changes increased the lower detection limit of the method for HDI-BT from 100 pg/m3 to 12 pg/m3. Typical chromatograms for samples containing (a) 140 ng and (b) 30 ng of HDI-BT, respectively, are shown in Figure 2. Assuming an average HBI-BT molecular weight of 478, these samples were equivalent to a n airborne isocyanate concentration of 4 ppb and 1 ppb, respectively. All sensitivity calculations throughout this paper are based on 20-L air samples and 100-pL injection volumes unless otherwise indicated. T h e colored components of polyurethane paints may pose problems when derivative mixtures are analyzed by UV absorption. In one case, a red pigment was found to obscure a peak assigned to the N R derivative of IPDI. NMA-Fluorescent Derivative Method. The primary amine, 1-naphthlenemethylamine (NMA), reacts with aliphatic isocyanates to yield urea derivatives which fluoresce when irradiated with broad band 200-700 n m light. T h e excitation spectra of these derivatives (as determined on this instrument) are featureless with a maximum emission obtained when an excitation wavelength of 216 nm was used. This maximum can be used to distinguish NMA-isocyanate derivatives from interferences. The possibility that the actual absorption maximum of these derivatives might not be a t 216 nm was not explored since maximum sensitivity was achieved with this detector when excitation at this wavelength was utilized. No interference from paint pigments has yet been observed. Fluorescence intensity is proportional t o concentration for HDIBT-NMA over the concentration range lo4 t o lo-' M although significant scatter develops a t lower concentrations when 10-pL sample injection volumes are used (Table I). Because of the sensitivity limitations of the fluorescence detector-memory module combination, excitation spectra can only be obtained for NMA-derivative peaks that are present in the range 10-4-10" molar concentration greater (Figure 3). The use of fluorescent derivatives could, a t least in theory, enhance the detection of LC separated derivative mixtures by several orders of magnitude compared to the UV detection
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
Table I. Calibration Curve for HDIBT-NMA Standard [HDIBT-NMA] detector response, (molar) countsa 10-4 M 42 000 10-5M 4 150 M 414 10-7M 48b a Calibration performed using 5-10 rL injection. a signal/noise ratio of approx. 3 / 1 .
At
216nm 200nm
280nm
n
v I'lk:: 200nm
280nm 200nm
EXCITATION SPECTRUM UNCORRECTED
INCLUDING B A C K G R O U N D FROM I N S T R U M E N T ANDSOLVENT
CORRECTED EXCITATION SPECTRA
Figure 3. Excitation spectra of peaks from HDIBT-NMA and IPDI-NMA standards
of the corresponding NR derivatives. In practice, we find an enhancement factor of 40 to 50 for HDIBT-NMA compared to HDIBT-NR and it is clear that there is substantial room for further improvement. (The use of a quartz, rather than a glass, photomultiplier window may result in an additional gain in sensitivity.) The sensitivity improvement observed reduces the lower detection limit for HDIBT to 0.25 pg/m3 (10 ppt) per 20-L air sample or approximately 0.5 ng per 1O-rL sample injection volume (Figure 4). This improvement in sensitivity provides a reasonable safety margin when determining airborne isocyanates at levels comparable to present threshold limit values. At higher isocyanate concentration levels, the improved sensitivity allows air sampling times to be decreased, or in the case of volatile absorbing solution solvents like toluene, allows the air sampling flow rate to be reduced. In addition to the sensitivity increase afforded by NMA derivatives, NMA appears to be more reactive toward isocyanates than NR. A responsive derivatizing agent is desirable to ensure that air samples correctly represent actual isocyanate concentration levels. For example, airborne isocyanates can occur as aerosols dispersed in a mixture with prepolymers and catalysts (10) such t h a t polymerization can continue in the absorbing test solution. One hour or more is required for the reaction of NR with isocyanates to approach completion. Semiquantitative studies in our laboratory indicate that the reaction of NMA with aliphatic isocyanates is 95% complete within 5 min. Therefore, although it is probable that minor side reactions may occur when using a primary amine (NMA) derivatization reagent instead of a secondary amine (NR), the higher derivatization reaction rate is a significant advantage. I t is probable that these side reactions are minimized by the use of dilute (10-4-10-5 M) reagent solutions. IPDI-NMA standard solution and air samples obtained from paint overspray containing IPDI yield chromatograms with two strongly overlapping peaks (Figure 5). If these peaks were due to secondary reaction products, such as biurets, then they should be easily separable from the primary reaction product. Since these peaks are strongly overlapped, the
Figure 4. Liquid chromatogram of NMA derivative of solution at and near the lower limit of detection
HDIBT
standard
IPDI-NMA DOUBLET67 ng
-
Figure 5. Liquid chromatograms of NMA derivative of solution illustrating the doublet peaks
IPDI
standard
difference between these species is probably due to a subtle change, such as that which could be attributed to IPDI-NMA conformers. Therefore, the sum of the areas under both has been used to calculate isocyanate concentrations. T h e chromatogram of a standard initially consists of the earlier eluting IPDI-NMA peak with a trace of the second peak. Within approximately two days (depending on storage conditions) the second peak in the doublet appears and attains an area approximately equal to the first peak. The sum of the areas of the peaks is constant and may be used to yield the IPDI value. The chromatogram of a sample from paint overspray consists of the overlapped peak doublet right from the beginning, possibly because the evaporation/heating step in the sample preparation may result in an equilibrium mixture of conformers. As stated previously, the presence of two peaks may also be attributed to two closely related reaction products. However, the relative merits of these hypotheses have not been tested.
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
IMPURITY
!I
7 8 ng H D I B T - N M A
, , lA
/\I(
0
1
a
I
IN PAINT
TIME
MIN I
-
37
1
42
Figure 6. Liquid chromatogram of NMA derivative of HDIBT obtained from paint overspray air sample illustrating the doublet peaks and the
position of the excess NMA peak
Figure 6 shows the presence of a similarly overlapped doublet peak in the chromatogram of the HDIBT-NMA derivative. This result is from an air sample collected from actual paint overspray. The presence of this doublet peak, seen only in air samples, is not easily explained. Efforts to prepare a standard containing HDIBT-NMA with double chromatographic peaks have been unsuccessful. Standards prepared from HDI-BT reagent, clear components of the paint containing HDI-BT, mixtures of clear and color components, and mixtures added to the NMA solution in an impinger with subsequent air bubbling, all result in single peaks as shown in Figure 4. Time limitations have prevented the exploration of the possibility of the HDI-BT peak doublet being caused by partial hydrolysis of the isocyanate prior to derivatization, or by partial oxidation of the derivative in the impinger. A t this time, the available evidence makes it appear likely that the peak doublet is the result of a reaction of isocyanate species in the paint spray system. It is therefore advisable to add the area of this peak doublet and consider it all to be HDI-BT. The accuracy of the NMA technique compared to the NR technique for monitoring aliphatic diisocyanate concentrations in paint overspray has been assessed during a limited number of pilot spray trials. The problems associated with the generation of standard streams of these isocyanates include adsorption, reaction with moisture, and the vapor/aerosol nature of these species. This is an especially difficult problem in the case of HDI-BT (the major isocyanate species of these urethane paints), which does not have a significant vapor pressure a t ambient temperature and may cross-link with the paint aerosol spray. In order to assess the relative results obtained with the NMA and NR analysis techniques, 2 to 4 midget impingers containing either NMA or NR reagent were taped together with their inlets within 2 inches of each other. The data obtained from these tests could not be treated statistically, since the inhomogeneity of the automotive paint spray gun pattern yielded nonreproducible results, even for the same derivatizing reagent. The number of such tests was too small to overcome the scatter observed in these results. However,
1109
results obtained from the NR and NMA impingers were generally within better than 50% of each other. Results obkained individually with either method (NR or NMA) showed the same scatter as that seen when the results of the two methods were compared. The use of an airstream mixing device to deliver a homogeneous airstream to the impingers was considered. I t was ruled out because of the probability that paint particles would stick to the device, thus causing a preferential loss of the nonvolatile HDI-BT. This inability to generate uniform, reproducible standard vapor/particulate isocyanate streams is a serious drawback to the validation of this technique, or of any technique used for the analysis of this class of compounds. This is no different than problems encountered by others who have studied isocyanates in paint overspray ( 6 , 8 ) . Thus, it is evident that further work in this area is necessary before a method can be chosen that is classified by the National Institute of Occupational Safety and Health (NIOSH) as Class A (Recommended) or Class B (Accepted) ( 3 ) .
CONCLUSIONS A new method has been developed that is based on the derivatization of aliphatic diisocyanates with a fluorophore, 1-naphthalenemethylamine (NMA), and subsequent LCfluorescent analysis. This method is more sensitive than those previously available resulting in a more flexible air sampling experiment design and permitting a lower air sampling flow rate. A high reaction rate for the derivatization reaction has been achieved. Positive identification of NMA peaks has been achieved using stopped-flow LC with excitation scanning. Certain isocyanate derivatives show strongly overlapped double peaks for either standards or samples. The reasons for this have not been elucidated. Despite the doublet peaks, the high sensitivity of this NMA method commends it for further evaluation in parallel with the Nitro Reagent method, and as a test method for samples of low concentration. ACKNOWLEDGMENT We thank R. J. Caloia and D. Schuetzle for expert technical assistance in this project. We would also like to thank L. C. Hernandez (Mobay Chemical Co.) for correcting the peak assignments for excess NMA reagent and impurities (Figure 6). LITERATURE CITED (1) Marcali, K. Anal. Chern. 1957, 29, 552. (2) Grim, K. E., Linch, A. L. Am. Ind. Hyg. ASSOC.J . 1964, 25, 285. (3) "NIOSH Manual of Ana&4ical Methods",HEW (NIOSH) publication #75-121, PCAM #141,142 (1974). (4) Siggia, S. "Quantlative Organic Analysis v$ Functional Groups", 3rd ed.; John Wiley and Sons: New York, 1963. (5) Veba-Chemie #22-E0170-1-21 (1976), and Ashiand Chemical Co. #DK-10-75 private communications, 1975 (6) Keiler, J.; Dunlap, L. L.; Sandridge, R. L. Anal. Chem. 1974, 46, 1845. (7) Dunlap. K. L.; Sandridge, R. L. Nat. Paint and Coating ASSOC.round robin results, private communications, Feb. 13, 1975. (8) Dunlap, K. L.; Sandridge, R. L.; Keller, J. Anal. Chem. 1976, 4 8 , 497. (9) OSHA Bulletin #2248, Toluene Diisocyanate (Dec. 1975), and Table Z-1 (29 CFR 1910.1000). (10) Levine, S. P.; Hoggatt, J. H.; Caolia, R. J. Presented at the Am. Ind. Hyg. Assoc. Conference, paper #319, May, 1978.
RECEIVED for review October 10, 1978. Accepted March 29, 1979. This paper was presented in part a t the American Industrial Hygiene Association Conference, May 1978.
