Determination of Nitrilotriacetic Acid by High-speed Ion Exchange Chromatography J . E. Longbottom Environmental Protection Agency, AnaIytical Quality Control Laboratory, I014 Broadway, Cincinnati, Ohio 45202
BECAUSE OF THE ENVIRONMENTAL effects of phosphorus, the detergent industry has sought to find a substitute in washing compounds. Nitrilotriacetic acid (NTA), until recently, was a compound considered as a substitute for phosphorus. Some test-marketed formulations contained as much as 10% nitrilotriacetic acid as efforts were made to meet phosphate elimination guidelines proposed by regulating authorities. However, in December 1970, the Surgeon General of the US. Public Health Service, Dept. of Health, Education, and Welfare, on the basis of preliminary reports that the toxicity of mercury and cadmium are considerably increased when they are chelated with NTA, announced a moratorium (I) on the use of NTA until further, more conclusive studies were completed. Meanwhile, work has continued on methods to treat NTA in domestic wastes. Elaborate toxicity studies are continuing on NTA (and other chelates) in all types of environments (2). For these studies, a reliable analytical method is needed that will detect and measure NTA in a sample when it is present in an ionized, acid, or chelated form. The method should also determine NTA in the presence of metals and other chelates. Of the methods in current use, the zinc-Zincon procedure (3) has been most widely used for the analysis of NTA in lake and river waters. This method is sensitive to 0.5 mg/l. and can be adapted to automatic analyzer equipment (3, 4). It suffers, however, from interference from metals, notably Feaf, which are more likely than zinc to chelate with NTA. In addition, the method is not accurate in the presence of other chelates or high chloride ion concentration. Polarographic techniques ( 5 , 6 ) have also been proposed for the determination of low levels of NTA in aqueous solution. Sensitivity to 0.1 mg/l. or less has been reported for these methods, but they also suffer from the inability to measure all chelated forms of NTA. Like the zinc-Zincon procedure, polarography is nonspecific for NTA in the presence of other chelating agents. Thin-layer chromatography and derivativegas chromatographic techniques (7-9) can be used to separate various chelates from each other but have inherent shortcomings that preclude accurate and reproducible quantitative measurement. High efficiency ion-exchange chromatography has been used to detect various amine derivatives (IO). Accordingly, anion (1) “Statement on NTA,” Press Release, December 18, 1970. (2) C. M. Tarzwell, “Quarterly Report of Research, Jan.-March, 1971,” National Marine Water Quality Laboratory, West Kingston, R.I. (3) J. E. Thompson and J. R. Duthie, J . Water Pollut. Contr. Fed., 40, 306 (1968). (4) “WQO Methods for Chemical Analysis of Water and Wastes,” Water Quality Office, Environmental Protection Agency, Cincinnati, Ohio, April 1971, p 209. (5) J. P. Haberman, ANAL.CHEM., 43, 63 (1971). (6) P. D. Goulden, Enciron. Sci. Technol., in press. (7) E. Heinerth, Fette, Seifen, Anstrichm., 70, 495 (1968). (8) Y.K. Chau and M. E. Fox, J. Chromatogr. Sci., 9, 271 (1971). (9) J. M. Goldberg, Hampshire Chemical Division Report, W. R. Grace and Co., Nashua, N.H., 1970. (10) C. D. Scott, Clin. Chem., 14, 521 (1968). 418
Table I. List of Chelates Chromatographed Monoamines Diethanol glycine (DEG) Ethanol diglycine (EDG) Iminodiacetic acid (IDA) Nitrilotriacetic acid (NTA) Diamines Ethylene diamine diacetic acid (EDDA) Hydroxyethylethylene diamine triacetic acid (HEEDTA) Ethylene diamine tetraacetic acid (EDTA) Triamine Diethylene triamine pentaacetic acid (DETPA)
exchange chromatography was investigated as a way of separating NTA from other amino-acid chelates. By this method NTA was measured over a wide range of sample pH values. Possible interferences from metallic ions were overcome by converting all metal-NTA chelates to the analyzable ferric chelate. The method has been applied to the analysis of sewage samples and solutions of detergent formulations with a sensitivity of 1.O mg/l. EXPERIMENTAL
Apparatus. A DuPont 820 Liquid Chromatograph, operated at room temperature, was used for this work. A pressure of 1000 psi was required to maintain a flow rate of 0.5 ml per minute. The flow was monitored with a UV photometric detector that measured the absorbance of unfiltered light emitted at 254 nm by a low pressure mercury lamp. in. 0.d. The separating column was 1 meter long by stainless steel packed by DuPont with SAX, a strong anion exchange resin coated onto Zipax (11). The column was used regularly for several months with no significant loss of efficiency. Reagents. The mobile phase was 0.02M NazBaO,.10HzO. The pH of this buffer was approximately 9.0. A solution of 0.2M F ~ ( N O Swas ) ~ prepared for use in the treatment of samples with possible metal interferences. Procedure. Sewage samples were first filtered through filter paper and treated by the addition of 50 p1 of 0.2M Fe(N03h to 100 ml of sample. One hundred microliter injections of the samples were made onto the column after momentarily stopping the flow. Sensitivity was limited to 1.0 mg/l. when making direct injections in this manner. To determine the level of NTA in detergent formulations, 2.00-gram portions of the detergents were dissolved in a liter of distilled water. The samples, if not completely in solution, were filtered through paper and direct injections of 2 to 5 p1 of each sample were made with a 10-pl syringe. To determine if closely related chelating agents interfered with the detection of NTA, the chelating agents listed in Table I were chromatographed., The possibility of metal interferences was checked by testing a series of solutions containing 0.001M NTA in 0.1M metallic salt. Similar mixtures of 0.001M NTA in 0.1M NaOH and 0.1M “ 0 8 were also tested. (11) J. J, Kirkland, J . Chromatogr. Sci., 8, 72 (1970).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
0.6
0.5 0.4
0.3
0.2
0.1
0 .o
0
1
2
ELUTION VOLUME [ml]
WAVELENGTH (nm]
Figure 2. Chromatogram of monoamine chelating agents, 1.0 kg each; (1) DEG; (2) EDG; (3) IDA; (4)NTA
Figure 1. Ultraviolet absorbance scans of 0.1% NTA in water adjusted to the following pH values: (1) 8.4; (2) 8.8; (3) 9.2; (4)10.2 DISCUSSION
Monitoring the column effluent only at 254 nm was found to be somewhat restrictive due to the UV absorbance characteristics of NTA. Figure 1 shows ultraviolet absorbance curves for buffered solutions at various pH values. As can be seen, at 254 nm the absorbance does not become significant unless the environmental pH is quite high. The degree of ionization of NTA is dependent upon pH and ionic strength. The relationship in Figure 1 reflects the formation of the NTA3-ion, which uniquely absorbs UV light at 254 nm. If the pH of the buffer is raised to pH 10 or above, the coating of quaternary ammonia substituted methacrylate polymer will hydrolyze rapidly and serious damage to the column can occur (12). Fortunately, it was possible to find a buffer system at pH 9.0 that permitted the separation of NTA from the other chelates, and still gave sufficient NTA response to be useful. The response of the system to NTA was linear through the range of 0.1-1.5 pg. Beyond 1.5 pg, the exchange capacity of the column was exceeded and the NTA eluted early. All the monoamine chelates gave unique skewed chromatograph tracings (Figure 2). At lower levels this resulted in broad peaks that were difficult to measure. The diamines, on the other hand, gave typical Gaussian curves (Figure 3) that extended their level of detection below 0.1 pg. DETPA eluted much later. None of these compounds interfered with the determination of NTA. When the solutions of metallic salts and NTA were tested, the effects of the formation of chelates on the NTA response (12) E. I. DuPont de Nemours, SAX Technical Data Report 820 M.I., Wilmington, Del., 1971.
1
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2
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v, W
a a
W
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a 0
0
2
W
a
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ELUTION VOLUME (ml) Figure 3. Chromatogram of diamine chelating agents, 1.0 kg each; (1) EDDA; (2) HEEDTA; (3)EDTA
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
419
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Table 11. Metallic Salt Solutions and Their Effect on NTA Response before Sample Treatment Effect of NTA chromatogram None None None None None Low response None None No NTA response No NTA response L o w response L o w response None
m
0
2
1
ELUTION VOLUME [ml] Figure 4. Chromatogram of 100 ~1of a filtered sewage sample dosed with 3.0 mg/l. NTA: (1) Flow stopped for injection; (2) Flow restored; (3) Unidentified material; (4) NTA I
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ELUTION VOLUME [ml] Figure 5. Chromatogram of 3.5 pl of 0.2% aqueous solution of a detergent known to contain NTA: (1) “solvent peak”; (2) NTA were observed as listed in Table 11. The metals that form relatively weak chelates with NTA, listed first in Table 11, are probably separated from NTAa- by the ion exchange column. The separation of the stronger metal chelates depends upon a favorable side reaction. In the case of the Fea+chelate, pre420
sumably the precipitation of Fe(OH)3 releases the NTAaanion for detection. Since NTA preferentially chelates with ferric ions when available (13), and since the method successfully reseparates NTAa- from Fe*+, all metal interferences were overcome by introducing an amount of a ferric salt in excess of the NTA to samples before injection. After the addition of the iron, all the metal-NTA mixtures gave 100% of the theoretical NTA response. Cobalt was replaced (in the above) in less than 1 minute after addition of the iron, but nickel chelates required 10 to 15 minutes to be converted to their ferric equivalents. A sample of raw sewage from a metropolitan area was filtered and then dosed with 3.0 mg/l. NTA. The chromatogram of this sample is shown in Figure 4. Other UV absorbing materials in the sample eluted under the conditions of the test but none of these interfered with the NTA peak. Six commercial detergents were tested using this method. Surprisingly, only those known to contain NTA showed any response at all on the recorder. A typical chromatogram of an NTA-containing detergent sample is shown in Figure 5 . Because of the skewed elution pattern of NTA, precision was poor near the detection limit. At the 1.0-pg level, the deviation was j~0.02pg, but at 0.2 pg the standard deviation was ~k0.05pg. The utility of the method was clearly demonstrated when it became necessary to measure and identify a chelating material used in a specific formulation. To quantify the amount of NTA in a proprietary formulation, the method common to the industry (14) requires a 12-hour Soxlet extraction, a three-hour hydrolysis, and colorimetric measurement following formation of a copper chelate. This method measures, but does not identify the chelate. For positive identification, the hydrolyzed chelate had to be further purified and confirmed by both infrared and gas chromatographic technique. Since these methods involve additional sample preparation such as micro pellet preparation for IR and derivative preparation for GC, a number of days would be required. ACKNOWLEDGMENT
The author is grateful to the Hampshire Chemical Division of W. R. Grace, Inc., for supplying the chelating agents used in this work. RECEIVED for review July 26, 1971. Accepted September 22, 1971. (1 3) Hampshire NTA Technical Bulletin, Hampshire Chemical Division of W. R . Grace and Co., Nashua, N. H. (14) “Determination of Nitrilotriacetic Acid in Detergents,” Hampshire Chemical Division of W. R. Grace and Co., Nashua,
N. H.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
