Determination of Isocyanates in Working Atmospheres by High Speed Liquid Chromatography K.
L. Dunlap, R. L. Sandridge,"
and Jurgen Keller'
'
Mobay Chemical Corporation, New Martinsville, W.,Va. 26 755
A high speed liquid chromatographic (HSLC) method is presented for the determination of isocyanates in working atmospheres. A reagent containing N-4-nitrobenzyl- N-npropylamine is used to convert the isocyanates to stable urea derivatives. An internal standard is added, if desired, and an aliquot is chromatographed using a hexane-ethanol solvent program, a UV detector, and a commercial pellicular silica packing. The method is applicable to both aliphatic and aromatic isocyanates at levels of interest in industrial hygiene situations. Amines do not normally interfere. Under selected conditions, levels of TDI equivalent to 0.2 ppb in a 20-1. air sample can be detected.
T h e extensive use of isocyanates in industrial applications coupled with t h e general concern over industrial chemical exposures place additional demands on existing methods for determining t h e isocyanate content of working atmospheres. Presently, t h e threshold limit value for toluene diisocyanate is 0.02 ppm (0.14 mg/m3) for a n 8-hr work-day and 40-hr work-week. T h e Marcali method ( 2 ) has been adopted for the determination of toluene diisocyanate in air b u t required modifications ( 2 ) t o be able t o meet t h e increasingly stringent requirements. Of the methods currently available, only the thin-layer chromatographic (TLC) method of Keller, Dunlap, and Sandridge ( 3 ) can be used to determine both aliphatic and aromatic isocyanates in air. In addition to isocyanate monomers, isocyanate resins with free isocyanate groups can also be quantitatively determined. This is of particular importance in spraying applications where both free isocyanate monomers and isocyanate resin aerosols with free isocyanate groups may be present, and it is desired to differentiate between isocyanate monomers and resin aerosols. However, there are some limitations with t h e TLC method, For quantitative determinations where a visual comparison of sample and standard spots is used, the results are dependent upon the subjective judgment of t h e analyst. T h e detection limit for toluene diisocyanate (TDI) with the TLC method is 0.04 mg/m3 assuming a 20-1. air sample. With these limitations in mind, a high speed liquid chromatographic (HSLC) method was developed to improve t h e sensitivity a n d precision of t h e analysis and eliminate analyst bias in t h e interpretatibn of t h e TLC results. T h e HSLC method which was developed for t h e analysis of isocyanates in t h e working atmosphere is based on t h e determination of the ureas formed from t h e reaction of t h e isocyanates with N-4-nitrobenzyl-N-n-propylamine(nitro reagent).
C H-
I CH>--S-C-XH-
EXPERIMENTAL Reagents. Nitro Reagent Absorber Solution. The synthesis of the nitro reagent has previously been described ( 3 ) .The nitro reagent is stored as its hydrochloride salt for reasons of stability. To prepare a fresh reagent solution, dissolve approximately 120 mg of nitro reagent hydrochloride in about 10 ml of distilled water; add 13 ml of 1 N NaOH to precipitate the free amine, and extract the free nitro reagent with toluene. Dry the toluene extraction with anhydrous NaZS04, and dilute the extraction to 250 ml, yielding a 2 X M solution. Dilute this solution tenfold to obtain the 2 X lo-* absorber solution. Store nitro reagent solutions in the dark, and do not use the solutions after more than three weeks. Nitro Reagent Solution f o r Standard Solutions. Standard solutions of the urea derivatives of isocyanates are prepared in a 2 X M solution of nitro reagent in methylene chloride. This nitro reagent solution is prepared as outlined for the toluene nitro reagent solution, except that methylene chloride is used in place of toluene. Isocyanates The isocyanates used in the study are commercially available from Mobay Chemical Corporation. They are: Mondur TD-80, 80% 2,4-TDI and 20% 2,6-TDI; Multrathane M, 4,4'-diphenylmethane diisocyanate, MDI; Mondur HX, 1,6-hexamethylene diisocyanate, HDI; Desmondur N-75, a high molecular weight biuret of hexamethylene diisocyanate. Apparatus. Air Sampling. A portable air sampling pump (Bendix Environmental Science Division Type C115 or its equivalent) is used in conjunction with a midget gas impinger (30-ml capacity) containing the nitro reagent absorber solution. HSLC Analysis. A Waters Associates liquid chromatograph, Model ALC/GPC-202/401 (or its equivalent with an ultraviolet detector) is used for the analysis of the air samples. In this study, the liquid chromatograph was equipped with a 254-nm ultraviolet detector, refractive index detector, a U6K Universal septumless injector valve, two Model 6000 high pressure pumps, and a Model 660 solvent programmer. A 2-foot X va-inch 0.d. (yapinch i.d.) stainless steel column was packed with HC Pellosil, a commercially available pellicular silica packing (Reeve Angel and Co., Inc.). Procedure. Air Sampling A midget impinger is filled with 10 ml of the nitro reagent absorber solution, and the required volume of air is drawn through the impinger at a rate of 1-2 l./min. In an investigation of the collection efficiency of TDI vapor in two midge t impingers (connected in series), each filled with 10 ml of absorbing solution, no TDI was detected in the second impinger at a flow rate of 2 l./min by the TLC method ( 3 ) .Therefore, it is felt that one impinger can be used to collect the air sample. If long sampling times are employed, and the volume of nitro reagent solution has decreased significantly because of evaporation, more toluene can be added to the impinger to restore the solution to its original volume. After the air sample is taken, the solution is carefully evaporated to dryness in a vacuum oven set at 35 ,"C, and the residue is dissolved in 1 ml of methylene chloride. This solution is then submitted for analysis by HSLC. Preparation of Standard Solutions Standard solutions are prepared by adding a weighed amount of the isocyanate of interest to the methylene chloride nitro reagent solution. Normally, a stock solution of the urea derivative of the isocyanate is prepared and then diluted to obtain standard solutions in the concentration range of interest. Store these solutions in the dark, and do not use them after more than three weeks. Analysis of the Ureas of Isocyanates. A 90-kl aliquot of the solution to be analyzed is injected into the liquid chromatograph.
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Present address, Farbenfabriken Bayer GmbH, OC-Produktion, Organic Analytical Laboratory, 509 Leverkusen-Bayerwerk, West Germany (to whom correspondence from Europe should be addressed). ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
497
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4
;ir-~;
Figure 1. Chromatogram of nitro reagent and urea derivatives of TDI, MDI, HDI, and Desmodur N Attenuations indicated on chromatogram. Isocyanate concentrations: 2.0 pg TDlIml, 2.9 pg MDlIml, 4.0pg HDlIml, and 19 pg Desmodur N-75lml. NR = excess nitro reagent. (- - -) Solvent program (0.80/0 absolute ethanol in hexane to 8% absolute ethanol in hexane), by refractive index detector, attenuation 64
With a flow rate of 2 ml/min, a linear solvent program (curve 6 on the 660 solvent programmer) is used to separate the excess nitro reagent and the urea derivatives of isocyanates. The initial solvent composition is 0.8% (by volume) absolute ethanol in hexane, and the final composition obtained after 10 min is 8% (by volume) absolute ethanol in hexane (600 solvent programmer conditions: Curve 6, initial 4% B, final 40% B, 10-min program, solvent A-hexane, solvent B-20% absolute ethanol in hexane by volume). Liquid pressure at a flow rate of 2 ml/min is approximately 300 psig; therefore, it is necessary to use the high sensitivity noise filter on the Waters Associates pumps to minimize the base-line noise due to the low flow rate and pressure. The ultraviolet detector output is recorded on a 10-mV strip chart recorder at 0.5 inchedminute. Peak areas for the urea derivatives of the isocyanates in the chromatograms are determined manually or electronically. Calibration curves for each of the isocyanates of interest are prepared by plotting peak area vs. isocyanate concentration of the standard solutions. The concentrations of isocyanates in the sample solutions are determined from their respective areas and calibration curves. Each concentration may then be related to the concentration of isocyanate in the atmosphere by the volume of air sampled.
RESULTS AND DISCUSSION With this HSLC method, isocyanates can be qualitatively identified by their elution times and quantitatively determined by peak areas. A chromatogram of the urea derivatives of TDI, MDI, HDI, 2nd Desmodur N-75 is given in Figure 1. T o obtain an adequate separation of these derivatives and t h e excess nitro reagent, it was necessary to utilize solvent programming. Because of t h e lack of uniformity of solvent programming between different manufacturers' instruments, the solvent program used is also shown in Fig-
Table I. Detection Limits for Several Isocyanates= Detection limit
w/m3
PPb
(VP)
TDI 0.005 0.70 MDI 0.005 0.50 HDI 0.010 1.5 Desmodur N 0.10 b a Detection levels are based o n a 20-1. air sample and a 90pl sample size. bThe molecular weight of Desmodur Nis not well defined; therefore it is unsuitable t o express the concentration in ppb (viv).
498
12
Figure 2. Chromatogram of pnitrophenol and urea derivatives of TDI, MDI, and HDI
- , -1.8c .- (MINI
Isocyanate
6 TIN:
* ANALYTICAL CHEMISTRY, VOL.
48, NO. 3, MARCH 1976
Attenuation 16. Solvent program as in Figure 1. NP = pnitrophenoi, 30
Nml
ure 1. As seen in the chromatogram, t h e 2,4- and 2,6-isomers of T D I are not completely resolved. Since the UV absorptivities of the urea derivatives of these isomers are essentially t h e same, it was felt desirable not to separate the isomers and obtain a lower detection limit for total TDI content. T h e detection limits of t h e method are given in Table I for several of the isocyanates of current industrial interest. T h e detection levels for the three isocyanate monomers are all considerably lower than t h e accepted time-weighted average of 0.02 ppm for TDI. In some situations i t may be necessary t o determine isocyanate levels lower than t h e detection limits given in Table I. This may be accomplished by increasing the volume of t h e aliquot injected into t h e liquid chromatograph. Injection volumes of 300 ~1 were used to lower the detection limits to 0.2 ppb (v/v) for TDI. T h e sensitivity of the method may also be increased by sampling a larger volume of air. p-Nitrophenol can be used as a n internal standard with t h e method if desired. As shown in Figure 2, this compound is eluted immediately before TDI. T h e p-nitrophenol must be added to the solutions to be analyzed after t h e isocyanates have completely reacted with the nitro reagent, viz., 1 hr or more. A calibration curve can be prepared by plotting t h e ratio of peak areas of p-nitrophenol and t h e isocyanate vs. isocyanate concentration. An important consideration in t h e choice of a method for the determination of isocyanates in air is the conversion of the isocyanate to a stable derivative t o eliminate the possibility of undesirable reactions of t h e collected isocyanate. With this method, t h e isocyanates are reacted with t h e nitro reagent to form stable urea derivatives. One limitation is t h a t it cannot be used in atmospheres which decompose the nitro reagent by oxidation or reduction. As with the T L C method, this method can be used t o determine aliphatic and aromatic isocyanate monomers or resins with free isocyanate groups. Aromatic isocyanates have lower detection limits than aliphatic isocyanates because of the contribution of the aromatic isocyanate t o the UV absorptivity of the urea derivative. In contrast to t h e T L C method, the HSLC method offers a much simpler analysis procedure. T h e use of a UV detector has eliminated the need for several chemical reactions (reduction of t h e nitro group, diazotization, and coupling) involved in the visualization of the nitro reagent and urea derivatives. As with any chromatographic procedure, the possibility of interferences should be considered. An interfering material must have a n elution time similar to t h e material of in-
terest and must absorb in the UV region in which the detector is sensitive. Although unlikely, if a problem is suspected, then minor changes in the solvent program should be investigated in an a t t e m p t to adequately resolve the peaks. Alternately, another chromatographic technique such as TLC (3) may be necessary for those samples which contain interfering materials which cannot be satisfactorily resolved. In conclusion, the HSLC method presented in this paper offers improved precision, greater simplicity, general appli-
cability, and a significantly lower limit of detection for the determination of isocyanates in the working atmosphere,
LITERATURE CITED (1) Kalman Marcali, Anal. Chern., 29, 552 (1957). (2) K. E. Grim and A. L. Linch, Am. Ind. Hyg. Assoc. J., 25, 285 (1964). (3) Jurgen Keller. K . L. Dunlap, and R. L. Sandridge. Anal. Chern., 46, 1845 (1974).
RECEIVEDfor review August 27, 1975. Accepted November 3 , 1975.
High Performance Liquid Chromatographic Determination of Prostaglandins F2a, E2, and D2 from In Vitro Enzyme Incubations F. A. Fitzpatrick Research Laboratories, The Upjohn Company, Kalamazoo, Mich. 4900 1
The high performance liquid chromatographic separation of prostaglandins F2a, El, and D2 as p-bromophenacyl esters on a microparticulate, bonded, reversed phase column is described. Detection and simultaneous quantitation of less than 3 pg of each prostaglandin is possible. The method has been applied to monitor prostaglandins produced from sheep seminal vesicle enzyme preparations and to evaluate antiinflammatory drugs on the basis of their inhibition of the synthetase enzyme. Advantages of this technique over other inhibitor screening techniques are discussed.
High performance liquid chromatography (HPLC) has only recently been applied to the problems of prostaglandin separation and analysis. Mikes et al. ( I ) compared H P L C and TLC separations of epimeric prostaglandins. Morozowich (2) resolved prostaglandins Az and Bz on a triethylaminoethyl cellulose ion exchange column and studied the base catalyzed conversion of prostaglandin E2 to Bz. Andersen and Leovey (3) separated closely related prostaglandins on a pellicular silica support and determined the degree of epimerization of prostaglandin Fz, using a refractive index detector. Except for a comparative study ( 4 ) of prostaglandin Ez levels in r a t kidney papillas by H P L C and bioassay, little work has been reported on the development of quantitative high performance liquid chromatographic assays for prostaglandins in biological specimens. Lack of sensitivity with a fixed wavelength detector has been the major obstacle in this area; however, recently reported work by Morozowich and Douglas ( 5 ) on the formation and separation of prostaglandins as p-nitrophenacyl esters has provided a solution to this problem. H P L C appeared to offer several advantages for monitoring in vitro systems containing enzymes which actively synthesize prostaglandins from precursor fatty acids. Current methods of assessing prostaglandin synthetase activity include polarographic measurement of oxygen uptake (6), spectrophotometric measurement of prostaglandin Bz ( 7 ) , and radiometric thin layer chromatography (8). T h e primary drawbacks to the first two approaches are the lack of specificity and sensitivity, except in the most enzymatically active tissues. Radiometric thin layer chromatography is tedious, and accurate quantitation is difficult. In contrast
to these methods, HPLC is rapid and can supply accurate quantitative results on several compounds in a single chromatographic run. Described here are the reversed phase liquid chromatographic separation of several closely related prostaglandins as their p-bromophenacyl esters and the simultaneous analysis of micromolar levels of prostaglandins F 2 a , El, and DZformed from in vitro synthetase incubations.
EXPERIMENTAL Apparatus. A Chromatec Model 5100 (Tracor Instruments, Austin, Texas) high performance liquid chromatograph with a pneumatic drive, positive displacement pump, and a single wavelength (254 nm) ultraviolet detector was used. Chromatographic columns were operated at ambient temperatures (25 3 "C), and injections were made into a stopped-flow injection port with a Hamilton Model 701 10-pl syringe. Chromatograms were recorded on a Varian A-25 single-pen recorder, 0 to 1mV full-scale. Chromatographic Columns. Corasil C18 and Phenylcorasil (Waters Associates, Milford, Mass.j superficially porous, reversed phase column materials were packed into 1-m by 2-mm internal diameter (i.d.) stainless steel columns. A 2-m by 2-mm i.d. column of Permaphase ODS (Du Pont Instruments, Wilmington, Del.) was prepared by coupling two 1-m columns via a minimum dead volume fitting. A pre-packed, pre-tested, microparticulate, reversed phase column ( M Bondapak C18, Waters Associates) was used as received. Low dead volume fittings were easily constructed to adapt this column to the stopped-flow injection port of the chromatograph used. Reagents. Distilled-in-glass acetonitrile, methanol, and methylene chloride (Burdick and Jackson, Muskegon, Mich.) were.used as received. 2,4'-Dibromoacetophenone and p-nitrophenacylbromide (Eastman Kodak, Rochester, N.Y.) were used as received. Diisopropylethylamine (Aldrich) was redistilled (b.p. 127 "C) before use. All prostaglandins were supplied by the Experimental Chemistry Laboratories of The Upjohn Company. Arachidonic acid, 99% pure (NuChek Prep, Elysium, Ill.) was examined by TLC before use. Procedure. Deriuatization. Prostaglandin p-bromophenacyl and p -nitrophenacyl esters were prepared according to Morozowich and Douglas ( 5 ) .Briefly, the prostaglandin (500 Mg or greater) is dissolved in 1.0 ml of anhydrous acetonitrile containing a threefold molar excess of p-bromo- or p-nitrophenacylbromide. Two pl of diisopropylethylamine are added to catalyze the reaction, which is >95% complete in 1 h at 25 "C. For 1 to 100 pg of prostaglandin, the volume of acetonitrile should be reduced to 0.1-0.2 ml, and the . amount of diisopropylethylamine should be reduced to 0.5 ~ 1 A threefold molar excess of reagent must be maintained. ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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