Table V. Observation of Rate Change after Use Raten in pg/min @ 25°C Period of use Tube No. Initial 22-14 1.08 34-4 1.70 54-11 1.25 1.39 38-8 1.09 37-18 1.32 37-19 2.02 48-11 1.30 45-7 1.34 16-1 0.84 39-12 a All values i l % relative.
Final 1.12 1.60 1.26 1.44 1.10 1.31 2.06 1.30 1.34 0.82
in months 18
12 8
C0NCLUS I ON Relative change +4 -6 0
8 8 8
+4
5 5 3 1.5
+2
0 0 0 0 0
not great and within a recommended calibration period of 6 months, the changes would not have been significant. A troublesome problem has been reported concerning a very small number of tubes. This involves t h e appearance of “spikes” in the output when the tube is being used to generate calibration mixtures which are being observed with a continuous monitor. The spikes appear to result from small bursts of nitrogen dioxide released from the device. Three devices have been returned which exhibited such behavior. Two of the three were found t o have a small longitudinal defect in the Teflon sleeve originating a t the lower stainless steel band and extending downward about 3-4 mm. The defect appeared t o be a crack or fissure which either did not reach the surface or which was too small to be detected under moderate magnification. I t was detectable, however, by the manner in which light was refracted through the Teflon. Such defects have not been observed in other devices. T h e cause of the defects is not known but all devices are now screened visually for such defects prior to use. The potential exists that the flaw may appear after a period of use and any evidence of “spiking“ in the output should be cause for careful examination of the Teflon a t the junction with the stainless steel band.
Nitrogen dioxide permeation devices can be prepared which will have long-term stable permeation rates uncertain to about fl % relative. Such devices may be used in the temperature range from 20 to 35 “C to generate concentrations of nitrogen dioxide in air with a n uncertainty osf slightly more than 1% relative. T h e devices respond rapi.dly to small changes in temperatures and reach equilibriuim in conveniently short times. Exposure t o high temperatures may result in permanent rate changes. As a result of t h e laboratory studies and of t h e success achieved in field use, these devices are considered reliable means of generating accurate gas mixtures and are offered as a National Bureau of Standards Standard Reference Material (SRM No. 1629, Nitrogen Dioxide Permeation Device).
ACKNOWLEDGMENT T h e authors thank K. A. Rehme and J. E. Hodgeson for their help in the independent Cali bration of the nitrogen dioxide permeation devices.
LITERATURE CITED (1) A. E. O’Keeffe and G. C. Ortman, Anal. Chem., 38, 760-763 (1966). (2) F. E. Scaringelli, S. A. Frey, and B. E. Saltzman, Am. Ind. Hyg. Assoc. J . , 28, 260-266 (1967). (3) C. Huyman, Atmos. Environ., 5 , 55 (1971). (4) J. R. Jacobson, An? Chem. SOC.,Div. Water, Air Waste Chem., Abstr., 7 (l),232-234 (1967). (5) D. P. Lucero, Anal. Chem., 43, 1744-1749 (1971). (6) F. P. Scaringelli, A. E. O’Keeffe, E,Rosenberg, and J. P. Bell, Anal. Chem., 42, 871-876 (1970). (7) National Bureau of Standards Technical Note 858 “Summary of Activities of Microchemical Analysis Section, July 1970-June 1971”, p 26-31, (January 1972). Available through Superlntendent of Documents, U. S. Government Prining Office, Washington, D.C. 20402. Price 70 cents. (8) A. E. O’Keeffe and G. C. Ortman, Anal. Chem., 39, 1047 (1967). (9) B. E. Saitzman, Environ. Sci. Techno/.. 1 1 , 1121-28 (1971). (10) F. Lindquist and R. W. Lanting, Atmos. Environ., 6 , 943-946 (1972). (11) E. M. Stoddart, J . Chern. SOC., 1459 (1938). (12) E. M. Stoddart. J . Chem. SOC..448 (1’345). (13) Dan Lucero, Monitor Labs., private communication. (14) L. J. Purdue and R. J. Thompson, Anal Chem., 44, 1034 (1972).
RECEIVED for review August 19, 19758. Resubmitted April 7, 1977. Accepted July 27, 1977.
Acid Interference in the Determination of Arsenic by Atomic Absorption Spectrometry H a n K. Kang’ and Jane L. Valentine School of Public Health, Center for Health Sciences, University of California at
A study was undertaken to determine the degree of acld ( H2S04,“OB, HCi04) Interference on the measurement of arsenic. This measurement was based on the reduction of arsenic to arsine by NaBH, and subsequent analysis by atomic absorption spectrometry. When the peak height was used to calculate the amount of arsenic, all three aclds interfered with arsenic recovery at higher concentrations. The pattern was not dependent on the total amount of acid present, but rather on its concentrations in the solution. Peak area remained relatively constant irrespective of acid concentrations in the soiutlon up to 2 4 % (v/v). The K I pretreatment did not compensate for the interference. Steps to reduce the amount of residual acid in the samples should be taken, or the measurement of peak area should be employed to correct for the interference posed.
Los Angles, Los Angles, California 90024
The measurement of arsenic based cln its reduction to arsine in acidic solution by NaBH, and subsequent analysis by atomic absorption spectrophotometry (AAS) has been published by several investigators (1-5). The use of NaBH4 as a reductant is favored since it provides many advantages over the previously employed Zn-HC1 reducing system (1). In order to determine arsenic in biological samples, t h e organic matter must be destroyed. ‘This is usually accomplished by wet digestion using H2S04,HC104, or “ 0 3 singularly or in combination. The application of this method, however, requires further investigation into potential interference of excess acids in the sample solution on arsenic measurement. I t is possible that these strong acids in the sample solution may inhibit the reduction of arsenic (As5+) to arsine (ASH,). Methods published to date do not discuss ANALYTICAL CHEMISTRY, VOL. 49, NO. 1 2 OCTOBER 1 9 7 7
1829
or specify maximum acid to remain in sample solution after digestion is finished. This paper is a result of a study which evaluated the degree and the extent of the acid interference on arsenic measurement by AAS using arsine gas. EXPERIMENTAL Apparatus. Measurements were carried out on a Perkin-Elmer Atomic Absorption Spectrophotometer, Model 303, equipped with an electrodeless discharge lamp (EDL), a recorder, and a deuterium background corrector. The apparatus used for the generation and collection of gaseous hydride was purchased from Perkin-Elmer Corp. Reagents. All acids (HC1. "0,. H,SO,. HC10,). KI. Na2HA;O4.7H20, and NaAs02were anal&ical-reaient grade. The stock arsenic standard solution of 1.0 mg of As5+/mL was purchased from the Fisher Scientific Co. The dilute arsenic standard solution was prepared to contain 5 kg of As5+/mL by dilution of the stock solution with 20% (v/v) HC1 solution. Sodium borohydride (NaBH4)pellets inch) having a mean weight and standard deviation of 326 f 8 mg were available from Alpha Products (Beverly, Mass.). Distilled-deionized water was used throughout. To assess acid interference on the recovery of arsenic from samples prepared in distilled-deionized water, 1 gg of arsenic (As") was added to flasks and mixed with appropriate amounts of distilled-deionized water, HC1, and either concentrated H,SO,, HC104, or HN03. The resulting samples had a final volume of either 15, 25, 30, or 40 mL with a constant HCl concentration of 20% (v/v) in each sample. The percentage of strong acid ranged from 4 to 2570 (v/v). The possible compensatory effect of KI pretreatment against acid interference was examined for a 24% (v/v) acid solution. Varying amounts of 20% (wjv) KI solution were added to the acid sample solution, a t room temperature, 1 h before the measurement. The effect of the oxidation state of arsenic in the solution on arsenic measurement was also examined using 1 pg arsenic equivalent of sodium arsenate (As5+)and sodium arsenite (As3+). When KI was used for pretreatment of the sample solution, 1 mL of 2070 (w/v) solution of KI was added, a t room tempeature, 1 h before the measurement. T o check for acid interference on recovery of arsenic added t o biological matrices, samples of blood, hair, urine, and natural water were chosen, and the following sample sizes used: blood, 5 mL; hair, 1 g; urine, 20 mL; and natural water, 10 mL. Flasks containing either a sample with no added arsenic (As5+)or with added arsenic (As5+)were treated with 5 mL each of concentrated HNOBand HC104. This mixture was warmed on a hot plate. After organic matter was destroyed, the volume of each sample was reduced to less than 2 mL and cooled to room temperature. To the cooled sample, 20 mL of distilled-deionized water and 5 mL of concentrated HC1 were added, and the flask was connected to the hydride generation sampling system. The resulting sample contained no more than 8% (v/v) of HCIOl and no significant amount of HN03. Arsenic values were calculated from the calibration curve established with the arsenic standard solution. Generation a n d Measurement of Arsine. Sodium borohydride was added to the flask via the dosing stopcock to initiate the reaction. The generated arsine and excess hydrogen were introduced to a nitrogen-hydrogen-entrained air flame by the method of gas collection or continuous flow. In the gas collection method, the generated gases were stored in an expandable balloon reservoir attached to the flask during the reaction. When the reaction had subsided, the four-way stopcock was opened, allowing the balloon to deflate, forcing the collected hydrogen and arsine into the flame. The atomic absorption signal of arsenic was recorded on a recorder as peak height. For the continuous flow method, the generated gases were continuously introduced to the flame. The resulting signal of arsenic was recorded as peak height and peak area. Calculations of peak area were made by peak height X peak width at 'I2 of peak height. Instrumental operating conditions are shown in Table I. R E S U L T S A N D DISCUSSION A typical calibration curve for the different methods of 1830
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
Table I. Instrumental Operating Conditions Source Electrodeless discharge lamp (EDL) Wavelength 193.7 nm Source power 8W Slit width 1.0 mm Spectral band width 0.7 nm Gas flow rate N,: 7.1 L/min H,: 3.5 L/min Table 11. Precision Summary of Aqueous Standard Solution Containing 1pg of As Amount of Re1 std NaBH,, pellet dev, %"
Methods of measurement Continuous flow, peak height Continuous flow, peak area Gas collection, peak height Gas collection, peak area
1
2.58
1
2.53
1
2.95
2
2.81
a Relative standard deviation based on 1 0 replicate de terminations.
w
0
z
U
m
E
3'/
I
2
5m:
U
ARSENIC i A:+l,pg
Figure 1. Arsenic calibration curve by different method of measurement. (A)Peak area, continuous flow, 1 pellet of NaBH4. (0)Peak height, continuous flow, 1 pellet of NaBH,. (0)Peak height, gas collection, 1 pellet of NaBH,. ( X ) Peak height, gas collection, 2 pellets of NaBH,
measurement is shown in Figure 1. A more linear calibration for arsenic (As") was obtained when peak height and peak area were measured by continuous flow of gases rather than measuring peak height with gases collected in the reservoir balloon. Peak area measurement of collected gases was not done because, once the stopcock was open, the collected gases flowed into the flame almost instantaneously. Precision for the different methods employed above was determined on 30 mL of 20% (v/v) HC1 solution containing 1pg of m e n i c Relative standard deviation ranged from 2.5% to 3.0% (Table 11). T h e presence of the acids, "03, H2S04,and HCIOI was found to produce no difference on the precision. All three acids were found to interfere with arsenic recovery a t higher concentrations (Figures 2-4) when peak height measurement was employed. H 2 S 0 4 showed the greatest and inhibition among the three acids, followed by "03 HC104 in successive order. The interference by these acids was not totally unexpected realizing that they are oxidizing reagents and would therefore react with the reductant along with the arsenic in the solution. H2S04also has the greatest potential of causing interference since in practice it remains in solution long after both H N 0 3 and HCIOl have been boiled away. The oxidizing character of HC104 is known t o become negligible in diluted solution a t room temperature (6). Figure 4 tends t o verify this. Employing peak area measurement again provided an advantage over peak height with respect to minimizing acid
E
W 0
s
601c
4c
20
20;
'
-
0
3
5
10
5
ri,SO, CONCENT?ATIONS,
Figure 2. Nitric acid interference on arsenic (As5+)recovery from 30 mL of 2 0 % (v/v) HCI solution when 1 pellet of NaBH, was used. (A) Peak area measurement, continuous flow. (0)Peak height measurement, continuous flow. (0)Peak height measurement, gas collection method
EO
25
(V/V)
Figure 3. H2S04interference on arsenic (As5+)recovery from 30 mL of 20 % ( v h ) HCI solution when 1 pellet of NaBH4 was used. (A)Peak area measurement, continuous flow. (0)Peak height measurement, continuous flow. (0)Peak height measurement, gas collection method
Table 111. Arsenic Signal'" with Respect to Its Oxidation State Continuous Gas collection, OxidaKI flow height, abs tion pre- state treat- Height, Area, 1 2 of As mentb abs arbitC Pellet Pellets ASj+
NO Yes No Yes
0.295 0.301 0,232 0.298
235 249 257 242
0.275 0.273 0.255 0.279
0.303 0.309 0.319 0.315
a Values represent mean of three measurements with relative standard deviation less than 5%. 1 mL of 20% (wiv) KI solution was added t o the arsenic solution 1 h prior t o the measurement. Peak area was calculated by peak height x peak width a t of peak height in arbitrary scale.
interference. Peak area remained relatively constant irrespective of acid concentrations in the solution for all three acids up to 24% (v/v). I t appears t h a t H N 0 3 , H2S04,and HCIOl in the solution slow down the production of arsine after NaE3H4 addition. Peak height was decreased and peak width was increased proportional to the acid concentrations while peak area remained constant. T h e gas collection method was expected to show no acid interference, and thus results similar to peak area measurement, since the reservoir balloon would contain all the generated gases however long the generation time. On the contrary this method demonstrated a different pattern of acid interference on arsenic recovery. Fiorino et al. ( 4 ) contended that residuals of H N 0 3 and HC104 from the sample oxidation did not interfere with arsenic determination when peak height
20
0l 0
1
5
10
u15
2C
25
HCI O4 CONCENTQATIONS,%(V// Figure 4. HC104 interference on arsenic (As") recovery from 30 mL of 20 % ( v h ) HCI solution when 1 pellet of NaBH, was used. (A) Peak area measurement, continuous flow. (0)Peak height measurement, continuous flow. (0) Peak height measurement, gas collection method
was measured. No statement was made on the effect of residual H 2 S 0 4 . However, it should be pointed out t h a t maximum possible concentrations of H,SO, and HC104 in their sample solution were 5% (v/v) for each acid. Residual of HNO;, in the solution would be almost negligible. In the light of present findings, such low concentrations would not be expected to cause interference. Acid interference in four different final volumes, 15, 25, 30, and 40 mL, were measured by the gas collection method using 2 pellets of NaBH4. It was found t h a t data from 15 and 25 mL were closely matched as were data from 30 and 40 mL. The pattern of acid interference was found not to be dependent on the total amount of acid present, but rather on its concentration (Figure 5 ) .
Table IV. Recoverya of Arsenic Added to Sample Sample Hair, 1 g Blood, 5 m L Urine, 20 mL Water. 1 0 mL D ist ilieddeionized water, 1 0 mL
As in Sample,
As added, pug
Total as found, p g
Recovery,
Pg
0.12
0.25
0.36
1.0
1.10
96 98
0.25
0.25
100
0.50 0.25
0.51
102 96 96 96 97
Below detection limit 0.38 0.25 Below detection limit
0.62
0.50
0.86
0.50
0.73
1.0
0.97
70
'" Values represent average of duplicate measurement of samples after digestion with HNO, and HCIO,. Detection limit was 0.02 p g and relative standard deviation was not more than 5%.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
1831
CONCLUSIONS
100-
>
60-
> 0
,
W ti
ar
4c-
0
0
L
-
5
L 10
15
ACID CONCENTRATIONS,
20
-25
%(V./V'
Figure 5. Comparison of acid interference on arsenic (As") recovery when 2 pellets of NaBH, were used. (A)H2S04.( 0 )NH03, (0) HC104 in 15 mL of 20 % (v/v) HCI solution. (A)H2S04,(e)HN03, (H) HCIO, in 30 mL of 2 0 % (v/v) HCI solution
T h e KI pretreatment did not compensate for the acid interference. Up t o 5 mL of 20% (w/v) K I solution added t o t h e acid solution did not improve t h e peak height. Peak area was not measured because of multiple peaks. The effect of t h e oxidation state of arsenic in the solution on arsenic measurement is shown in Table 111. The solution containing 1 Fg of As" produced a smaller peak than t h a t containing As3+. However, prior treatment of the solution containing 1 pg of As5+ with 1 mL of 20% (w/v) K I increased the peak height to that of As3+. When two pellets of N&H4 were added and the gas collection method was used, the two arsenic species (As3+,As") produced similar peak heights. This indicates t h a t prereduction of As5+to As3+by KI is not necessary when the above method is used. Peak area was not affected by the oxidation state of arsenic in the solution. A similar observation has been reported by others ( 4 ) . Biological samples were analyzed by the gas collection method using 2 pellets of NaBH, with the effect of acid interference on recovery of arsenic in mind. Over 95% recovery of added arsenic was found in all samples when the amount of residual acids was carefully controlled prior t o NaBH, addition (Table IV).
The experiments performed allow the following conclusions. (I). Calibration curves for As5+, obtained using the continuous flow method, were more linear than those constructed using the gas collection method. (2). When peak height was used t o calculate arsenic, all three acids (H2S04,"OB, HC10,) interfered with arsenic recovery a t higher concentrations. The pattern was found not t o be dependent on the total amount of acid present, b u t rather on its concentration. Peak area remained relatively constant irrespective of acid concentrations in t h e solution u p t o 24% (v/v). An attempt to overcome this acid interference by K I addition was not successful. (3). T h e two arsenic species (As3+,As5+)produced similar peak areas while peak heights from As5+were lower than those of As3+. Pretreatment of As5+ solution with 1 mL of 20% (w/v) K I solution compensated for this variance. (4). By controlling t h e amount of residual acid prior to NaBH4 addition, according t o the above findings over 95% recovery of added arsenic (As"+)was achieved in all biological samples. In summary, t h e overall conclusion to be made is t h a t residual acids in the samples do interfere with arsine generation. Steps t o reduce t h e amount of residual acid in samples should be taken, or the measurement of peak area can be employed t o correct for interferences posed.
ACKNOWLEDGMENT The authors thank Richard Harnish of UCLA for his technical assistance.
LITERATURE CITED (1) (2) (3) (4) (5) (6)
F. J. Fernandez, At. Absorppr. News/., 12 (4) 93 (1973). A. E. Smith, Analyst (London), 100, 300 (1975). T. Matura and G. Sudoh, Anal. Chim. Acta, 7 7 , 37 (1975). J. A. Fiorino. J. W. Jones, and S. G. Capar, Anal. Chem., 48, 120 (1976). F. D. Pierce and H. R. Brown, Anal. Chem., 48, 693 (1976). F. A . Cotton and G. Wilkinson. Adv. Inorg. Chem., 572 (1966).
RECEIVED for review March 14, 1977. Accepted July 22, 1977. This investigation was supported by research grant R803798-01, U. S. Environmental Protection Agency.
Isotopic Assay of Nanomole Amounts of Nitrogen- 15 Labeled Amino Acids by Collision-Induced Dissociation Mass Spectrometry James H. McReynolds' and Michael Anbar' Mass Spectrometry Research Center, Stanford Research Institute, Menlo Park, California 94025
A method of measuring the a-"N content of amino acids by field ionization and collision-induced dissociation mass spectrometry is described. Molecular ions produced by field ionization are subjected to collisional dissociation to produce nitrogen-containing fragments that can be used to measure the I5Ncontent of 0.15 atom % enrichment on 50 nmol of an amino acid with an accuracy and precislon of 10% of the absolute I5N content. P r e s e n t address, D e p a r t m e n t of B i o p h y s i c a l Sciences, State U n i v e r s i t y of New Y o r k at Buffalo, 4234 R i d g e L e a Road, A m h e r s t ,
N.Y. 14226. 1832
ANALYTICAL CHEMISTRY, VOL. 49, NO. 12, OCTOBER 1977
A number of different methods have been developed for the analysis of lSN in metabolites. The traditional methods involve the careful separation of the compounds of interest from other nitrogen-containing materials and Kjeldahl digestion to convert the sample nitrogen to ammonia and a final oxidation of t h e NH3 to N1 with hypohromide. T h e I5N content of the nitrogen may be determined by using either precision isotope ratio mass spectrometry or emission spectrometry ( 1 , 2 ) . These methods, besides being unable to determine the number or location of the "N atoms in the original molecule, require several milligrams of sample t o obtain t h e amount of pure nitrogen needed for an accurate
