Analysis of N-nitrosamines in compounds.
Measuring low levels of N-nitrosamines emitted from and contained in elastomeric compounds has become crucial in light of the German regulation (ref. 1) governing the maximum allowable limits for 12 carcinogenic N-nitrosamines in workplace air. If these regulations are adopted by OSHA, many accelerators currently used in the U.S. rubber industry could become obsolete. With this regulation acting as the catalyst, our laboratory has been inundated with requests for the analysis of elastomers as well as pure chemicals and chemical blends for volatile N-nitrosamines. Currently in our laboratory, samples are routinely analyzed by extraction and headspace techniques at the parts per billion level. The instrument used for these analyses, the GC/TEA (ref. 2) (gas chromatograph/thermal energy analyzer), is specifically designed to detect only N-nitrosamines and to omit the signals produced by other types of compounds that may interfere with the analysis. The TEA detector is available for both liquid and gas chromatographs; the LC/TEA instrument is useful for detecting non-volatile or thermally unstable N-nitrosamines while the GC/TEA is more practical for volatile N-nitrosamines. The volatile N-nitrosamines are of most concern to the rubber industry and are the focus of the German regulation. This article will describe the analytical techniques and data interpretation necessary to perform this low level analysis.
The N-nitrosamine content of elastomers is measured by using the extraction technique. Here, 5 grams of the elastomer is diced into 2 [mm.sub.3] cubes, placed in a thistle tube, covered with cotton and placed in a Soxhlet extractor. Approximately 75 ml of N-nitrosamines free methanol is added to a preweighed 250 ml Florence flask and fitted with the Soxhlet tube containing the sample and extracted for 24 hours; previous time dependance studies (ref. 3) indicate that 95% to 100% of the nitrosamines are extracted in this time period. Methanol was chosen as the extracting solvent to minimize the amount of dissolved polymer in the extract.
The volume of the methanol extract is then reduced to 5-10 ml by rotary evaporation and the 250 ml Florence flask is weighed again; the weight of the extract is obtained by difference. We have found that 40% of the N-nitrosodimethylamine is lost during the solvent evaporation step, this loss is appropriately accounted for in the calculations of the concentrations. All other N-nitrosamines are 95% to 100% recoverable and no recovery factor is used to calculate their concentrations. Using the weight of the reduced extract and the density of methanol, the volume is calculated. We chose this method for the volume measurement to eliminate the sample loss that would occur upon transferring to a volumetric flask. We also make the assumption that the density of the extract is similar to that of pure methanol. The extract is passed through a 0.45 micron filter and then analyzed by the GC/TEA instrument.
The results from the extraction analysis provide a measure of the N-nitrosamine content of the elastomer with the units of parts per billion (nanograms N-nitrosamine/gram of elastomer); note, this is a weight/weight result. We believe that N-nitrosamines are not formed or destroyed during the sample preparation and that this technique reflects an accurate measure of the N-nitrosamine content of the elastomer. Typically, uncured elastomers that contain 1-2 phr of an N-nitrosamine producing ingredient will contain 50-500 ppb of that N-nitrosamine. However, the cured counterparts of these elastomers will usually contain lesser amounts of N-nitrosamines since the heat needed to cure the sample volatilizes most N-nitrosamines causing them to be expelled. The lower limit of detection for the extraction method is approximately 25 ppb.
The analysis of the headspace vapors generated by heating an uncured elastomer sample furnishes insight into the amounts of N-nitrosamines formed and discharged during the curing process. This experiment was originally designed to mimic worker exposure to N-nitrosamines and to provide an in-house basis for comparing various compounding recipes. For this analysis, 5 grams of the elastomer is diced into 2 [mm.sup.3] cubes and placed in a test tube. The test tube is placed in an Envirochem (ref. 4) headspace analyzer, which allows the sample to be heated to any desired temperature from 25 [degrees] C-200 [degrees] C while a gas stream passes over the sample at a rate of 2 liters/minute (figure 1). As the gas exits the test tube containing the sample, it passes through a specifically designed Thermosorb (ref. 5) cartridge which contains a nitrosation inhibitor. The cartridge captures N-nitrosamines and prevents the internal formation of N-nitrosamines. After sampling the air for an allotted time (usually 15 to 18 minutes), the air is turned off and the cartridge is removed. The cartridge is then backflushed with a 75%/25% dichloromethan/methanol solution which removes the N-nitrosamines as well as amines and small amounts of the nitrosation inhibitor. This eluent is then analyzed by GC/TEA.
The headspace results are reported as parts per billion of N-nitrosamines in air (nanoliters N-nitrosamine/liter of air). Note that these are volume/volume results and are not directly comparable to the extraction results. The lower limit of detection for this analysis is approximately 0.05 ppb with typical samples giving results in the 0.1 to 10 ppb range. Normally, uncured elastomers are best suited for headspace analysis since the experiment was designed to simulate the curing process when most of the N-nitrosamines are formed and liberated. Furthermore, cured samples would not normally contain N-nitrosamines as a substantial portion of those that form are discharged during the curing process.
An alternate experiment can be run where the sampling air is replaced with an air/N[O.sub.2] mixture (100 ppm N[O.sub.2], balance air). This experiment affords insight into the N-nitrosamine forming potential of a sample. As the secondary amines are volatilized, they are nitrosated and trapped in the Thermosorb cartridge. The cartridges are backflushed as described above and the eluent is analyzed by GC/TEA. Table 1 shows the results of a typical headspace analysis of an uncured elastomer that contains TMTD and a morpholine based accelerator using pure air and air/N[O.sub.2] as the sampling gas. From these results, we observe that each gram of the sample produces 1.1 ppb of N-nitrosodimethylamine (NDMA) and 0.9 ppb N-nitrosomorpholine (NMOR) when air is used as the sampling gas. However, when air/N[O.sub.2] is used as the sampling gas, these values increase to 32 ppb/gram for NDMA and 114 ppb/gram for NMOR. Thus the sample has much more of a potential for making N-nitrosamines than are actually formed. This illustrates the N-nitrosamine forming potential of these samples.
The TEA is a chemiluminescent detector which responds to the photons released upon on the relaxation of electronically excited N[O.sub.2]. After separation on a megabore capillary column, the N-nitrosamines are pyrolyzed at 500 [degrees] C to form nitrosyl radicals (equation 1); this temperature is too low (1) [Mathematical Expression Omitted] to pyrolize nitrosyl radicals from C-nitrosamines. The eluent stream then passes through a filter which removes most of the unwanted by-products of pyrolysis while the nitrosyl radicals are allowed to traverse intact. The radicals are then introduced into a reaction chamber filled with ozone (2) [Mathematical Expression Omitted] (3) [Mathematical Expression Omitted] where electronically excited N[O.sub.2]* is formed (equation 2). As the N[O.sub.2]* relaxes to its ground state, it emits a 600 nm photon (equation 3) which is detected by a photomultiplier tube that forms one side of the reaction chamber. This is the basis for the N-nitrosamine selectivity of the TEA instrument. The lower limit of detection for the instrument is 10 picograms at a signal to noise ratio of 3 to [1.sup.6] and the response is linear over four orders of magnitude (figure 2) (ref. 6). The chromatographic peaks are electronically integrated and all subsequent calculations are performed by computer. The N-nitrosamines are quantified by an external standard method; a 1 microliter injection of the standard mixture described in table 2 produced the chromatogram shown in figure 3. Retention times are matched for identification and peak areas are compared to quantify the N-nitros-amines. Figure 4 depicts a typical chromatogram obtained from our system for an extract sample showing the presence of NMOR.
In many cases the response is very near the lower limit of detection of the instrument, making quantitation difficult. Other samples can be even more problematic. Figure 5 shows the completely uncharacteristic chromatogram of an extracted sample of an elastomeric compound. The substantial peak near six minutes was identified by GC/MS to be a large quantity of dimethylformamide (DMF), neither a nitroso nor nitro compound while the second peak in the chromatogram was never successfully identified. We can only theorize as to why DMF produced a response; perhaps a small amount of the DMF contained a nitrosated impurity that was not detected by GC/MS and coeluted with the DMF. Another theory purports that a complex series of steps occurs in the pyrolyzer leading to the formation of the nitrosyl radical, causing the response. This chromatogram illustrates that the data generated by the TEA can be quite difficult to analyze and is sometimes subject to conjecture.
The GC/TEA is the instrument of choice for performing low level N-nitrosamine analyses in elastomeric compounds. Its sensitivity and selectivity toward N-nitroso compounds is unmatched by any other analytical instrument. The extraction technique provides a measure of the N-nitrosamine content of an elastomeric compound. Extraction results are reported as parts per billion (ppb) with respect to the sample; this is a weight/weight calculation. The headspace technique evaluates the amount of N-nitrosamines that are issued from the sample during the curing process; headspace results are reported as parts per billion with respect to air. Two types of headspace experiments can be performed using either air or an air/N[O.sub.2] mixture. Using air, a workplace environment is simulated while using an air/N[O.sub.2] mixture, the N-nitrosamine forming potential of the sample is measured. The analysis of the data can be challenging to interpret and must be scrutinized carefully to glean useful information.
ppb N-nitrosamines Sample weight (g) Air Air/N[O.sub.2] 5.0242 NDMA : 5.6 - NMOR : 4.5 - 5.0348 - NDMA : 32 - NMOR : 114
Table 2 - standard mixture of N-nitrosamines
Name Symbol Concentration (ug/ml) N-Nitrosodimethylamine NDMA 0.485 N-Nltrosodiethylamine NDEA 0.525 N-Nitrosomorpholine NMOR 1.000 N-Nitrosodipropylamine NDPA 0.520 N-Nitroesodibutylamine NDBA 0.515 N-Nitrosodibenzylamine NDBzA 2.000
[Figures 1 to 5 Omitted]
Technische Regeln fur Gefahrstoffe 552, Bundesarbeitsblatt (9, 1988). TEA is a registered trademark of ThermedeTec Inc. 470 Wildwood Street, Woburn, MA 01888; all experiments use the TEA model 610 detector. Schuster, R.H., Casselmann, H., Foller, F., Kautschuk & Gummi Kunstsoffe, Vol. 41 10, 1988, pgs. 971-973. Envirochem, Inc. Route 896, Box 180, Kemblesville, PA 19347.  Thermosorb is a registered trademark of ThermedeTec Inc. Instrument specifications obtained from ThermedeTec Inc. Figures 2, 3 and 4 were obtained from the ThermedeTec instrument manuals that accompany the Model 610 TEA analyzer and the Thermosorb cartridges.