Determination of Nitrilotriacetate in Environmental Water by Gas Chromatography of the Trimethylsilyl Ester Richard J. Stolzberg‘ and David N. Hume* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02 139
A rapid, simple method for the determlnatlon of trace levels of nitrilotrlacetate (NTA) in aqueous samples Is based on gas chromatography of the trimethylsilyl derivative. The 1-mL sample is evaporated directly in the reaction vial wlth an excess of the ammonium salt of EDTA to mask lnterferlngcatlons and convert NTA to a reactive form. After evaporatlve drylng with methylene chloride, derivatiration Is carried out with bls(trimethylsilyl)trlfluoroacetamlde In dlmethylformamlde. Chromatography on OV-17 columns gives good separatlon from all common anions, organic acids, and amlnopolycarboxylate chelating agents. The method Is suitable for samples contalnlng 1-100 pg/mL NTA without a prior concentration step. A minor modification permits simultaneous determlnation of imlnodiacetate and glycine whlch are produced in the photodegradationof NTA.
The possibility of extensive use of nitrilotriacetate (NTA) in industry and in domestic detergents has resulted in the need for rapid, sensitive, and selective methods for its determination in water and sewage. A number of methods have been developed, principally polarographic and colorimetric. These are based on the chelating properties of NTA and, because of this, there is often interference due to the presence of other chelating agents and complex-forming cations in the sample, resulting in a need for lengthy separations and cleanup procedures. An alternative approach is to treat NTA as an organic acid, rather than a chelating agent, and apply the technique of quantitative gas chromatography to its volatile derivatives. This has been done, often with a considerable degree of success, by a number of authors. Murray and Povoledo ( I ) and Rudling (2) used the trimethyl ester, Aue et al. ( 3 )and Warren and Malec ( 4 ) the tributyl ester, Chau and Fox ( 5 ) the tripropyl ester, and Rudling (6) the tri(2-chloroethyl) ester. None of these methods, however, was ideal, each having one or more of the drawbacks of multiple steps, time consuming operations, interference by other ligands or complexing cations, unfavorable chromatographic characteristics, or incomplete recovery. In the present work we have sought to develop a method which is rapid and simple as well as sensitive and selective. We have chosen to mask interfering cations rather than remove them and have utilized the desirable chromatographic characteristics of the trimethylsilyl (TMS) derivative of NTA. When an active hydrogen is replaced by a trimethylsilyl (TMS) group, the volatility of the compound and its thermal stability tend to be increased. Both organic (7) and inorganic (8) molecules can be determined by gas chromatography after silylation. Salts require prior conversion to the free acids or ammonium form and this may be accomplished by ion exchange (9) or aqueous-phase reaction with an excess of ammonia (10). While gas chromatography of TMS derivatives of moderate amounts of many acids presents little difficulty, the extension of the technique to trace level concentrations Present address, Harold Edgerton Research Laboratory, The New England Aquarium, Boston, Mass. 02210. 374
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
reveals the existence of a number of complications. Gehrke and coworkers ( I I ) in their elegant studies on the analysis of amino acid mixtures have pointed out many of the pitfalls which may be present. In investigating the possibility of using the TMS derivative of NTA for trace level determination of that compound in natural waters (12),we have observed additional factors which are important in derivative formation and gas chromatography. While some of these may be peculiar to NTA and other strong chelating agents, some clearly apply to many compounds when they are determined as TMS derivatives in trace quantities.
EXPERIMENTAL Reagents and Apparatus. All chemicals used were of reagent grade except where noted, and distilled water was used throughout. The 1.00 mg/mL stock solution of NTA was prepared by direct weighing of the acid (Baker 99% minimum), dissolution in a minimum amount of the aqueous ammonia, and dilution to volume. The EDTA used as the masking agent was obtained as the disodium salt (assay for NTA “none”) and was recrystalized as the acid three times from hydrochloric acid. Solutions of EDTA in dilute ammonia were stable for 1 to 2 weeks. After this time, a small gas chromatographic peak appeared with a retention time slightly greater than thatof NTA on an OV-17 column. The size of this peak increased with time; hence new solutions were prepared every 2 weeks or whenever samples containing less than 10 pg of NTA were derivatized. A 2 mg/mL octadecane in dimethylformamide (DMF) internal standard was used to adjust for small differences in the final volume of the reaction mixture. Reagent grade methylene chloride and DMF were used with no further drying or purifying, but care was taken to exclude water. Bis(trimethylsily1)trifluoroacetamide (BSTFA) was obtained from Regis Chemical Co. and used without further purification. Silylation reactions were carried out in 6 or 9 mL soft glass vials fitted with screw caps. These were soaked in 0.1 M NazEDTA for 30 min or more to remove leachable cations, thoroughly rinsed with distilled water, and oven-dried before use. The plastic caps were drilled with approximately 2-mm holes, and silicone septa were used to seal the vials while allowing for removal of aliquots of the derivatized mixture with a syringe. Gas chromatographic columns were all packed in the laboratory. The empty ‘/*-inch 0.d. stainless steel columns were cleaned with chloroform, acetone, and methanol, and were then dried with prepurified nitrogen. The ends of the column were plugged with silanized glass wool prepared as follows. Unsilanized glass wool was dried for 2 h at 110 “C, cooled, and immersed in a 15%dimethyldichlorosilane in toluene solution for approximately 1h. The glass wool was rinsed with toluene and methanol, and dried under an infrared lamp for 3 h. The glass wool retained a very low activity when stored in a desiccator containing phosphorus pentoxide. The importance of proper pretreatment of the columns, glass wool, and solid support cannot be overemphasized. Columns of OV-17 prepared with care allowed quantitative passage of a few nanograms of the TMS derivative of NTA on the first or second injection as indicated by a zero intercept and constant relative response over the entire range. One packing material used was 3% SE-30 on Supelcoport (SO/lOO mesh) purchased from Supelco, Inc. The other packing materials were all prepared in this laboratory using Chromosorb WHP and either SE-30 or OV-17 as the liquid phase. Toluene used to dissolve the OV-17 had been distilled from molecular sieve “3A”, and the large middle fraction (109.0-109.8 “C) collected. Chloroform used to dissolve the SE-30 was washed with water to remove the ethanol present, dried over anhydrous CaC12, and distilled from molecular sieve. The first 10% of the distillate was discarded, and the middle fraction (61.1-61.5 “C) was collected and used immediately. Packings of 5% and 12% OV-17 and 7% SE-30 were prepared by the slurry method
(13).Columns were flow conditioned overnight at 290 or 300 "C. Our experiences confirm Gehrke's observations (11) concerning the great importance of excluding moisture from the support materials, liquid phase, and solvents during all stages of column preparation. Helium carrier gas (US.Navy) was dried and purified by passage through a Perkin-Elmer filter dryer assembly, and when that was ins$alled, the portion of the helium delivery tubing downstream from the filter dryer was flamed with a Bunsen burner to desorb water from the inside walls. This operation was done with the chromatograph disconnected from the gas supply and helium flowing slowly. Compressed air for the flame ionization detector was dried by passage through Drierite and molecular sieve "5A". A Perkin-Elmer model 990 gas chromatographwas used. The glass injection port liners were periodically cleaned in a hot 2:l sulfuricnitric acid mixture to remove organic residues, then silanized by immersion in a toluene solution of 5% dimethyldichlorosilanefor about 1h, toluene and methanol rinsed,and oven dried before use. A number of temperature programs were used, but for simple samples the oven was held at 150 O C for 1 min and temperature programmed at 8 "C/min. On a 6-ft, 5% OV-17 column the retention time was slightly less than 7 min. The NTA-TMS peak was off the solvent tail even at high detector sensitivity,and it was well separated from the n-octadecane internal standard. Typically the injection port and manifold temperatures were 250 and 285 "C, respectively. A lyophilized culture of mutant bacteria capable of rapidly degrading NTA was used in the biodegradation experiments. The medium used was that described by Wong (14) or a variant of it in which the sodium and potassium concentrations were reduced and the ammonium ion concentration increased. Procedure. The basic procedure for samples containing 1 to 100 pg/mL of NTA in complex aqueous ~olutionscontaining appreciable amounts of dissolved salts is as follows. 1) Pipet a 1.00-mLsample into a precleaned and dried 6-mL screw cap vial and add 1mL of EDTA reagent containing 2 mg/mL of EDTA as the ammonium salt. Evaporatejust to dryness in a 150 "C sandbath under a stream of dry nitrogen, and remove. 2) Add and evaporate under nitrogen flow in a 70 OC sandbath,two 1.5-mLportions of dry methylene Fhloride. 3) Immediatelyafter the second evaporation, close the vial tightly with a screw cap which is fitted with a silicone rubber septum. Moisture should be excluded rigorously from the apparatus and reagents from this point on. Add by syringe a carefully measured 10.0 WLvolume of DMF containing 2 mg/mL of n-octadecane internal standard. Add also by syringe 0.20 mL each of BSTFA and pure DMF, and agitate. 4) Heat thesealed vial in a 70 "C sand bath for 30 min, cool, and inject 1.0 pL aliquots for chromatography.
RESULTS AND DISCUSSION Chromatographic Behavior. Preliminary experiments with milligram quantities of NTA or its ammonium salt showed it to react smoothly with BSTFA t o give a homogeneous solution after heating for 30 min a t 70 "C. The product yielded a single gas chromatographic peak on 3% SE-30, following the solvent and reaction by-product peaks, with a retention of approximately 18.0 methylene units. Identical results were obtained if an aqueous solution of the acid or its ammonium salt was evaporated to dryness before derivatization, but the trisodium salt was relatively insoluble and unreactive. Excellent linearity of peak area was observed over a 100-fold concentration range, but positive concentration-axis intercepts indicated varying losses of up to a few hundred ng. Systematic examination of the phenomenon showed that the injector port temperature, detector temperature, and carrier gas flow-rate were unrelated to the losses over a wide range of operating conditions. T M S derivatives are often very susceptible to hydrolysis, both before and during chromatography. The most likely causes of loss are apt to be decomposition in the hot injection port and decomposition within the column. Repeated injections of silylated samples reduced the losses but this effect was transient. The reversible nature of most of the loss suggested that water introduced in the carrier gas and diffusing into the system during shutdowns might be the primary problem. In extending the method to the measurement of amounts
Table I. Effect of Cation Exchange Removal of Copper on Recovery of NTAa % Cross-linking
12 8 4 1
%Copper % NTA unremoved recovered 2.9 2.9 5.4 17.
% NTA
recovered, copper absent
52. 4.8 9.5
68
2.4
n.d.
58 52
Conditions: Copper-containing samples: 30 mL containing 25 mg NTA and molar equivalent of cupric chloride. Copper-free samples: 30 mL containing 30 mg NTA. All treated at pH 3.2 with 2.1 g of Dowex 50, hydrogen form, for 16 h and analyzed.
of NTA injected in the nanogram range, great care was taken to remove and exclude all moisture from the support materials, liquid phases, and solvents during preparation of the columns, to bake out the system and prevent the introduction of moisture with the carrier gas. With these precautions, the use of the commercial helium dryer and careful flaming of the inlet lines reduced the NTA losses to below the limit of detectability obtainable with the flame ionization detector using the OV-17 columns. The SE-30 columns, however, still showed residual losses of 10-20 ng per sample and therefore were not used for the lowest-level samples where this would be significant. Carrier gas flow was maintained a t all times except when changing helium tanks. Effective silylation requires both the essentially complete removal of water and the removal or masking of interfering substances. T o have a truly practical analytical method, the number of operations and manipulations should be kept to a minimum. Accordingly we have attempted to eliminate any unnecessary transfers or phase separations. Fortunately it is possible to remove the bulk constituent, water, by evaporation in the vial in which the silylation is to take place. A residue of salts and adventitious organic material tends to hold some moisture. This is conveniently removed by addition and evaporation a t 70 "C of two portions of dry methylene chloride which volatilizes the residual moisture as an azeotropic mixture boiling a t 38.8 O C . No detectable loss of NTA occurs under these conditions. The importance of complete removal is seen by the fact that 1 mg of water can consume approximately 20 mg of BSTFA reagent. A further desirable condition for effective silylation is solubility of the reactants in the reaction mixture. Although NTA and its ammonium salt dissolve readily in BSTFA, many residues do not and the help of a compatible solvent is needed. Dimethylformamide added in an amount equal to the BSTFA was found to be a very satisfactory solvent and extractant for residues from the evaporation step. Treatment with the resulting mixture was found to give complete aerivatization a t 70 "C in 30 min with minimal solvent loss by volatilization and negligible thermal decomposition. Characteristically there was no change up to 3 h of further heating. Pyridine and acetonitrile appeared to be satisfactory alternatives but dimethylsulfoxide tended to give a two-phase system and hexane was an inferior solvent. A large excess of BSTFA, a t least 40:l on a molar basis is used to ensure complete silylation of NTA and allow for side reactions with other active hydrogen compounds which may be present. Cationic Interferences. NTA is a strong chelating agent, and it was apparent that it did not undergo normal silylation when tightly bound to metal ions. Exploratory experiments to test the feasability of using ion exchange for remokal of interfering metals were not encouraging. Results with copper present in amounts equivalent to the NTA are shown in Table I. Not only was the removal of copper incomplete but very ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
375
Table 11. Conditional Stability Constantsu of NTA and EDTA Complexes of Cu2+,Cd2+,and Ca2+ Metal
pH
logK’MeNTA
log K’ MeEDTA
cu
0 1
-1.8
-2.7
1.3 7.3
1.4
8 Cd Ca
0 1 8 0 1
-6.4 -1.9
8
4.6
7.8
-8.0 -5.0
12.9 -7.0 -1.5 13.7 -10.7 -6.7
8.4
a Conditions: Acidification to pH 0 or 1 with HCI. Total concentration of ammonia 0.1 M at pH 8.
Table 111. Recovery of 10.0-gg Portions of NTA in the Presence of Excess Metal Ionsu Metal Na(I) K(U MgW) Ca(I1) Ba(I1) Cu(I1) Zn(I1) Pb(I1) Co(I1) Al(II1) Cr(II1) Fe(II1) a
Reagent aloneb 8.3 jlg 10.5
7.9 10.5
9.0 0.0 6.5 6.3
n.d. 8.3 n.d. 3.8
Reagent and DMF‘ 10.0 jlg 10.0
9.8 9.8,9.7 9.0 10.8, 11.2 10.0,9.9 10.0, 9.9 10.6 9.7,9.7 9.9 10.0,g.g
Conditions: All test solutions contained a 40-fold molar excess
of the metal as chloride; 1-mL samples were mixed with 1mL of 2 mg/mL EDTA reagent, evaporated, and derivatized. Derivatized with 0.2 mL of BSTFA. Derivatized with 0.2 mL of BSTFA and 0.2 mL of DMF solvent. Each result is the average of two or more injections of the same sample.
significant losses of NTA were observed, even in the absence of copper. It has long been known that cation exchangers containing sulfonate groups in the free acid form retain amino acids (15). Experiments using Dowex 50 in the sodium and ammonium forms at pH values near 7 likewise did not remove copper satisfactorily, in agreement with the observations of Longman (16). Masking of interfering cations with a competitive chelating agent appeared to offer a better approach to eliminating their effect on NTA recovery. Conditional stability constants for the formation of NTA and EDTA complexes with Caz+,Cd2+, and Cu2+,were calculated at three pH values by Ringbom’s method of side reaction coefficients (17) using data from the literature (18).The side reactions considered were the protonation of the ligands at all pH values, the formation of chloro complexes at pH 0 and 1(from the hydrochloric acid) and the formation of ammine complexes in 0.1 M total ammonia at pH 8. The results, given in Table I1 imply that on acidification of a solution containing NTA and any of these metals to a pH of 0 or 1,the NTA should be protonated. In slightly ammoniacal solutions, the metals should be in the form of EDTA complexes if equivalent amounts of EDTA were present. Stability constant data for alkaline earth and transition metal complexes of NTA and EDTA indicate that the EDTA complexes are generally stronger by several orders of magnitude. If the metal-ligand speciation distribution does not change dramatically as the solvent is removed, the NTA should be 376
ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
Table IV. Effect of Vial Pretreatment and Mode of Sample Introduction: pg of NTA Recovered from 10.0 gg Samples Introduction techniquea Vial pretreatment
A
B
C
D
E
None Water washed 1M HC1 soak 0.1 M EDTA soak
0.0 0.3 1.7 2.7
0.0 6.0
1.9
7.1
10.0
2.7
9.9
6.9
6.0
0.0
2.9
8.8 8.9 9.3
7.7 10.0
a Conditions:A, sample added as 1.0 mL of 10 jlg/mL NTA. B, 1mL HzO evaporated in vial, then 10 pL of 1+g/mL NTA added. C, 1 mL 10 pg/mL NTA 1 drop concd HCl. D, 10 pL 1jlg/pL NTA. E, 1mL 10 jlg/mL NTA 1 mL 150 jlg/mL EDTA.
+
+
present as the derivatizable acid or ammonium salt after evaporation and drying. The applicability of the masking technique was tested as follows. Solutions containing 10 pg/mL of NTA and metal chlorides equivalent to a 40-fold molar excess were equilibrated overnight. Duplicate 1.00-mL samples were taken, 1.0 mL of ammoniacal 2 mg/mL EDTA reagent added to each and evaporated in the normal fashion. One set of samples was derivatized with and the other without DMF as an auxiliary solvent. The results, summarized in Table I11 demonstrate the success of the technique and the importance of the DMF. I t is interesting to note that neither the cobalt nor the chromium, which might be expected to undergo slow ligand exchange, interfered with the derivatization. Simple acidification of the sample before evaporation was found to be ineffective, giving satisfactory results only when sodium and potassium were the competing cations. Effects of Vial Pretreatment. The importance of the EDTA masking treatment, even when no interfering cations are anticipated, is illustrated by the effects introduced through the derivatization vials. Results obtained while working with milligram quantities of NTA indicated that pretreatment of the vials was unnecessary and accordingly they were us“ed directly from the packing box or after a simple distilled water rinse and drying. With microgram amounts of NTA and in the absence of EDTA masking, extremely erratic and variable results were obtained which seemed to be linked to the volume of aqueous sample and therefore the wetted surface area. Believing leaching of alkali and alkaline earth metal ions from the glass to be causing incomplete derivatization, we designed an experiment to determine the effects of vial pretreatment, sample size, and sample treatment. The vials with their samples were allowed to stand for 0.5 h, the contents evaporated and dried in the usual way and then derivatized. No additional EDTA was added as a masking agent. The results, summarized in Table IV confirm that interference increases with increased area of exposure of the container to the sample, decreases with treatments which remove foreign cations before exposure to the sample, and decreases as EDTA is added to provide masking. It seems possible that the glass surface may have three types of reactive cations at the surface: water leachable, acid leachable, and chelator leachable, all of which are to some extent removed by NTA. In working with very dilute solutions, even in the supposed absence of interfering cations, it is prudent both t o pre-leach the reaction vials and to include the ammoniacal EDTA masking reagent in the procedure. Anionic Interferences. The effects of fifteen common anions that might interfere with the derivatization or produce overlapping chromatographic peaks were studied. Each anion was tested individually and two levels of EDTA were added for each anion tested. Each sample contained 20.0 pg of NTA
Table V. Recovery of 20-pg Portions of NTA in the Presence of I mg of Foreign Anionsa Anion Chloride Bromide Iodide Thiocyanate Nitrate Bisulfate Sulfate Peroxydisulfate Carbonate Oxalate Hydrogen phosphate Acetate Benzene sulfonate Citrate Tartrate
Rbcovery using 0.1 mg EDTA
Recovery using 2 mg EDTA
18.8 18.8 25.6 17.4 15.2 24.8 23.8 17.0 17.6 21.6 16.4 19.6
18.6 19.4 19.6 19.4 19.8 22.6 20.4 17.2 20.6 18.6 18.4 20.6 20.2 18.6 20.0
n.d. 14.2 19.6
Conditions: 1.00-mL samples containing 20.0 WglmL NTA evaporated with either 0.1 mg of 2 mg EDTA as masking agent, dried and derivatized with BSTFA and DMF in the standard procedure. Results are averages of 2 or more injections.
and 1 mg of the anion as its ammonium salt. The masking agent was added either as 0.1 mL of 1mg/mL EDTA or 1.0 mL of 2 mg/mL EDTA. The samples were run in the normal manner and 1-pL aliquots taken for injection. The cesults, shown in Table V, indicate satisfactory derivatization in the presence of 2 mg of EDTA but considerably more variable results with only 0.1 mg EDTA present. Iodide and, to a much smaller extent, sulfate and bisulfate tend to give unexplainably high results unless the larger quantity of EDTA is present. Some of the anions tested reacted with BSTFA to form volatile derivatives, but all eluted considerably more quickly than the NTA. EDTA elutes much later than NTA. Citrate has been mentioned as a possible interfering substance because of the near-coincidence of retention values for butyl esters of that compound and NTA ( 3 ) .Using a 6-ft 5% OV-17 column with an oven temperature of 150 OC held for 2 min, then programmed at 8 OC/min, the peaks were well separated with the NTA peak barely overlapping the citrate tail. A typical chromatogram is shown in Figure 1. The methylene unit (MU) values of citrate and NTA are 18.2 and 18.6, respectively. In the series of TMS derivatives of myristic, pentadecanoic, palmitic, and oleic acids, NTA was found to elute between C14 and CIScompounds on the same 5% OV-17 column. MU values of the C14 and CISTMS acids were 18.3 and 19.5. The NTA peak was clearly separated and no qualitative or quantitative interference was observed. Common derivatizable inorganic anions such as phosphate, sulfate, and carbonate have retention times much shorter than that of NTA. Glycinate and iminodiacetate were of special interest as possible biodegradation and photodegradation products of NTA; IDA particularly because it could be a precursor of the possibly carcinogenic N-nitrosoiminodiacetic acid. On derivatization, these compounds give peaks which were well separated from NTA and from each other, and it was found that both could be quantitatively determined in aqueous samples using the same basic procedure as for NTA but with a different column. A small unidentified peak with retention characteristics very similar to those of the IDA-TMS derivative was found when samples known to be free of IDA were chromatographed on OV-17. A 7% SE-30 on Chromosorb W-HP column gave no positive interference for IDA or gly-
0.6
VI
E 0.4 0
0
mC VI Cl
;0.2
0
2
150
150
4 166
6 182
8 min. 198 O C
Figure 1. Chromatogram of IDA, citrate and NTA Column: 6-ft X '/*-in. 0.d. 5 % OV-17 on Chrornosorb WHP in stainless steel. Conditions: Injection port 250 OC, manifold 250 OC,oven 150 OC, held for 2 min after injection than programmed to 8 OC/min to 200 O C . Helium flow rate 20 cm3/min, chart speed 0.5 in./min, FID amplifier sensitivity 3.2 X lo-" Alcrn. Sample injected: 1 pL of TMS derivatives of 1.0 ,pg IDA, 0.5 Kg citrate, and 0.5 fius NTA
cine, and was satisfactory for the determination of these compounds in the presence of NTA. The SE-30 column, however, despite extensive conditioning persistently showed on-column losses of the order of 1 0 ng for NTA, IDA, and glycine, precluding its use for derivatized samples containing less than 10-15 ng/pL in the injection mixture. N-Methyl glycine could also be determined but its retention time was only a few seconds greater than that of glycine. The two were therefore not readily distinguishable unless the glycine were forced to tris(TMS)glycineby reaction with the silylation mixture for more than 3 h. The tris(TMS)glycine peak elutes on the steep section of the tail of the TMS-phosphate peak on the SE-30 column, however it is cleanly separated from all major peaks on the OV-17 column and can be quantified on it. Applications. This method has proved to be applicable in a number of studies of NTA degradation. Charles River (Cambridge, Mass.) water samples spiked with 1-50 ppm of NTA and incubated a t room temperature under normal fluorescent lighting were analyzed every few days. We observed what has been reported as characteristic behavior for biodegradation of NTA in river water ( 4 ) : the concentration of NTA remained constant for 1 to 3 days, then dropped abruptly to undetectable levels within a week. During the period of rapid degradation, analysis showed no detectable IDA, glycine, or methylglycine. This was in interesting contrast to our study of the photodegradation of iron-containing NTA solutions in which rapid and essentially quantitative conversion to IDA, followed by slow conversion to glycine was observed using the same methods (19). Experiments were performed with a mutant strain of bacteria in a medium using NTA as the only energy and carbon source. Normal growth curves paralleling those previously reported (14) were observed but a t no time were IDA, glycine, or methylglycine detectable. The totally depleted growth medium left a t the end of the experiment provided a opportunity of testing recovery under demanding conditions. The solution originally contained, per liter, 1.6 g KHzP04, 2.6 g K2HP04,l.O g MgS04,0.2 g (NH4)&04,0.5 g NaSNTA, and ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
377
micromolar amounts of Ca2+,Co2+, Fez+, Mn2+, and Zn2+. Application of the procedure to 0.5-mL portions of the spent medium showed no detectable NTA remaining. Recovery of 10 pg additions of NTA (20 ppm) was quantitative within normal experimental reproducibility and no interfering effects from the medium or the unremoved bacteria were observed.
ACKNOWLEDGMENT The gift of the bacterial mutant from P. T. S. Wong of the Canada Center for Inland Waters, Burlington, Ontario, Canada, is gratefully acknowledged.
LITERATURE CITED (1) D. Murray and D. Povoledo, J. Fish. Res. Board. Can., 28, 1043 (1971). (2)Lars Rudiing, WaterRes., 6, 871 (1972). (3)W. Aue, C. Hastings. K. Gerhardt, J. Pierce, M. Hill, and R. Moseman, J. Chromatogr, 72, 259 (1972). (4)C. B. Warren and E.J. Malec, J. Chromatogr., 64,219 (1972). (5) Y. K. Chau and M. E. Fox, J. Chromatogr. Sci., 9,271 (1971). (6)Lars Rudling, Water Res., 5, 831 (1971).
(7)A. E. Pierce, “Silylation of Organic Compounds”, Pierce Chemical Co., Rockford, Iil., 1968. (8)W. C. Butts and W. T. Rainey, Anal. Chem., 43,538 (1971). (9)W. C. Butts, Anal. Lett., 3, 29 (1970). (IO)T. Hashizume and Y. Sasaki, Anal. Biochem., 21, 316 (1967). (11) C. W. Gehrke, H. Nakamoto, and R. W. Zumwalt, J. Chromatogr., 45,24 (1969). (12)R. J. Stolzberg and D. N. Hume, Anal. Left., 6, 829 (1973). (13)W. R. Supina, ”The Packed Column in Gas Chromatography”, Supeico Inc., Beliefonte, Pa., 1974,pp 91-94. (14)P. T. S.Wong, D. Liu, and B. J. Dutka, Water Res., 6, 1577 (1972). (15) K. Freudenberg, H. Molter, and H. Walch, Naturwissenschaften, 30, 87 (1942). (16)G. F. Longman, M. J. Stiff, and 13. K. Gardiner, Water Res., 5, 1171 (1971). (17)Anders Ringbom, “Complexation in Analytical Chemistry”, interscience Publishers, New York, N.Y., 1963. (18)L. G.SilOn arid A. E. Martell, “Stability Constants of Metal Ion Complexes”, The Chemical Society, London, Special Publication No. 17 (1964),No. 25 (1971). (19)R. J. Stolzberg and D. N. Hume, Environ. Sci. Techno/.,9,654 (1975).
RECEIVEDfor review September 16,1976. Accepted December 9, 1976. This work was supported, in part, by the U.S. National Science Foundation under Grant GP33950.
Chemical and Physical Considerations in the Use of Atomic Absorption Detectors Coupled with a Gas Chromatograph for Determination of Trace Organometallic Gases G. E. Parris,’’ W. R. Blair, and F. E. Brinckman Inorganic Chemistry Section, National Bureau of Standards, Washington, D.C. 20234
A -crommerclal atomic absorption spectrophotometer with a heated graphite-tube furnace atomizer (HGA) was adapted as a detector for a gas-llquld chromatograph. The combined system was applled to the determinationof elements (i.e., As, Se, Sn) known to be methylated by mlcroorganlsms. The system was optimized by assessing the effects of varying the atomlzatlon temperature, the Inner surface of the furnace (Le., fused slllca, alumina, bare graphlte and pyrolytic carbon surfaces) and the carrler gas (Le., pure argon and argon wlth hydrogen). Using conservative, statlstically-based numerical techniques, the system detection limits for arsenlc, selenium, and tin (introduced as trimethylarsine,dimethylselenlum and tetramethyltln gas solutions with nitrogen dlluent) were found to be 5 ng As, 7 ng Se, and 12 ng Sn. To obtaln these Ilmlts, the bare graphite furnace was run contlnuously at about 1800 “C while the compounds were eluted from the chromatograph wlth argon to which 10% hydrogen was added. Optlmlzatlon of the furnace conditions requires an understanding of the thermodynamics and kinetics associated with thermal and chemical decomposition of the analyte compounds.
Ubiquitous biogenesis of labile organometallic compounds containing a variety of toxic heavy metals covalently bound to methyl groups is now apparent ( I , 2 ) . The need for analytical techniques combining chemical separation and highsensitivity, element-specific detection in environmental studies relevant to metal transport was outlined in previous publications (3-5). In the case of microbial transformations of metals, particularly in those situations in which organoM
Present address, EPA Office o f Toxic Substances, W H - 5 5 7 , 4 0 1 Street, S.W., Washington, D.C. 20460.
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977
metallic metabolites (e.g., (CH3)2Hg, (CH&As, etc.) volatilize across water-air or lipid-air interfaces, a consideration of instrumental capabilities and limitations led to the conclusion that combined gas chromatography-atomic absorption (GC-AA) techniques best fulfilled the requirements for speciation at nanogram levels. Several GC-AA systems have been described in the literature. Some of these systems can be indirectly employed for speciation of trace organometals following a separate preconcentration step (6-9) while others permit direct determination of biogenic metal products during the growth of microorganisms. One system exemplifying the latter approach was applied to examination of bacterial respirant atmospheres ( 5 ) .This system employed reductive combustion of organomercurials in a flame ionization detector (FID) of a conventional GC followed by cold vapor atomic absorption detection of resultant elemental mercury gas. This technique (IO) offers a simple GC-AA system but since it depends on two unique features of elemental mercury (i.e., high volatility and monomeric vapor), the system cannot be considered as a general application of GC-AA technology. Several other GC-AA systems offer more promise as a general analytical tool. Segar ( 1 1 ) described the use of an AA detector in which the GC effluent was directed via a tungsten transfer tube into a high-temperature flameless atomic absorption furnace. There, decomposition of the effluent chemical compounds, and volatilization and atomization of the transported analyte element M, are essentially simultaneous and can probably be regarded as a single process: C,H,M,
-2700 “ C
--+x C + y H + z M
Another interesting development is the application by Chau and co-workers (6-8) of an electrically-heated, hydrogen-air diffusion flame, silica-tube furnace. The actual mechanism
