Anal. Chem. 1995, 67, 2474-2478
Determination of Chromium Species in Environmental Samples Using HighmPressure Liquid Chromatography Direct Injection Nebulization and Inductively Coupled Plasma Mass Spectrometry Mark J. Powell* and David W. Boomer Ontario Ministry of Environment and Energy, 125 Resources Road, Etobicoke, Ontario, Canada MGP 3V6
Daniel R. Wederin Cetac Technologies Inc., 5600 South 42nd Street, Omaha, Nebraska 68107
A sensitive technique for speciation and quantihition of CrOII), C W ) , and total chromium has been developed using high-pressureliquid chromatography (HPLC) combined with direct injection nebulization (DIN) and inductively coupled plasma mass spectrometry (ICP-MS). A characterizationand optimization of a mitxocmlumn specifically designed for chromium speciation has been conducted. The direct injection nebulizer (DIN) was optimized to produce the best possible sensitivity for the system. Parameters affecting the column component of the system such as sample pH, eluent strength, sample size, flow, and pressure characteristics were studied in addition to optimal DIN-ICP-MSparameters. The detection limit obtained was 30, 60, and 180 ng/L for total chromium, Cr(III), and Cr(VI), respectively. Total chromium and speciated chromium were determined in one measurementwith an analysis time of approximately 500 s. Accuracy measurementsfor the two chromium species were within 5%of the certified value for NIST Standard Reference Materials 2108 and 2109. In order to demonstrate the capability of the technique to analyze a true environmental sample matrix, industrial efhent samples were analyzed, and the results were compared with an alternative conventional method.
Other studies have shown the effects of trivalent and hexavalent chromium on various forms of aquatic life2aand indicate that both forms of chromium can be detrimental depending on the aquatic species studied, hardness of the water, and chromium concentration. One of the most popular methods to determine hexavalent chromium include reaction of C r O with diphenyl carbazide and detection by colorimetry and graphite furnace atomic abs0rption.4,~One of the limitations of this technique is that it suffers from potential interferences from organic matter and other colored species [Le., Fe0II) and C U ~ I ) ] Since . the development of this technique, many advances have been made in the area of chromatographic separations of Cr 011) and Cr 0 with detection by atomic a b ~ o r p t i o nand ~ . ~inductively coupled plasma emission spectrophotometry.8.9 Since the advent of ICP-MS and its use as a detection system, our laboratory has conducted work in the area of ion chromatography (IC) with ICP-MS.lO Results obtained with this combination were produced primarily by using the conventional sample introduction system (i.e., concentric nebulization and spray chamber). In this work, we use a direct injection nebulizer (DIN). The conventional system produces broader peak widths due to the sample dispersion in the spray chamber. Since the DIN has no spray chamber, steady state signals are produced with minimal sample dispersion.ll
The Ontario Ministry of Environment and Energy (MOEE) is concerned with toxicity effects of natural and anthropogenic sources of chromium in the environment. The two oxidation states of chromium, Cr(I1I) or trivalent chromium and C r O or hexavelant chromium, have different levels of toxicity to humans, animals, and plants. The major sources of hexavalent chromium are anthropogenic and originate in the electroplating, steel, and textile industries with hexavelant chromium being transferred to the environment through air and water emissions. Hexavalent chromium is designated a carcinogenic substance by the US.EPA and may cause a range of diseases from dermatitis to lung and kidney cancer. In nature, trivalent chromium is most commonly encountered in the elemental form and is widespread at trace levels. In contrast, trivalent chromium in moderate concentrations is nontoxic and an essential nutrient for human health.'
(1) American Public Health Association, American Water Works Association, and Water Pollution Control Federation. Standard Methodsfor the Ezomination of Water and Wastewater, 18th ed.; APHA: Washington, DC, 1971. (2) Dorn, P. B.; Rodgers, J. H.; Jop, K. M. Environ. Tozicol. Chem. 1987,6, 435-444. (3) U S . EPA. Ambient Water Quality Criteriafor Chromium; Technical Report 440/584-029; U S . EPA: Washington, DC, 1984. (4) State of Caliomia. Method 425, Determination of Total and Hexavelant Chromium Emissions from a Stationary Source. 1990. (5) Ministry of Environment and Energy. Method HEXCR-E3056A, Determination of Hexavelant Chromium in Waters, Landfill Leachates and Industrial Effluent. 1993. (6) Fong, W.; Wu, J. C. G. Spectrosc. Lett. 1991,24, 931-944. (7) Wu, 8.; Zhou, L.; h a n g , S.; Zhou, S.; Chen. Q. LihuoJianyan, Huazue Fence 1991,27, 195-197. (8) Giglio, J. J.; Mike, J. H.; Mincey, D. W. Anal. Chim. Acta 1991,254, 109112. (9) Roychowdhury, S. B.; Koropchak, J. A Anal. Chem. 1990,62, 484-489. (10) Boomer, D. W.; Powell, M. J.; Hipfner, J. Talanta 1990,37,127-134. (11) Powell, M. J.; Quan, E. S. IC;Boomer, D. W.; Wiederin, D. R. Anal. Chem. 1992,64, 2253-2257.
2474 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
0003-2700/95/0367-2474$9.00/0 0 1995 American Chemical Society
Roehl et al. reported hexavalent chromium determination using ion chromatography and ICP-MS.’* although Cr(Il9 was not profiled and the deteaion limits were reported to be poorer than what we report Good speciation requires development of sampling and treatment procedures that are consistent with the chemistry of the species to be quantitatively determined. It is paramount that the species that are to be separated and measured are maintained at the same proportion of oxidation state as in the original sample. Hence, one of the most challenging problems to overcome in the determination of speciated chromium is the preservation of its original proportion of oxidation state. C r O will be reduced to CrUID in the presence of organics, oxides of nitrogen, and sulfur compounds. Alternately, Cr(IID in a basic solution will oxidize to C r O in the presence of FeUID, oxidized Mn, or dissolved o~ygen.~~-’~ In addition to the development of an analytical technique, this paper will show that using HPLC DIN-ICP-MS enables the researcher to measure the ratio of C r O / C r O over time. This is a valuable tool in determining the appropriate treatment to reduce changes in the oxidation state of a sample.
To Waste I)
By-PBSS LOOP
Micro-Column
To DIN and ICP-US
Flgum 1. Configuration of the rotary valve setup required for simultaneous monitoring of total and speciated chromium. Table 1. Parameter Settings Used To Obtain Best Possible Detection Limits and Separation While Maintaininga Stable Retention Time=
ICP-MS Parameters EXPERIMENTAL SECTION Apparatus. Chromatographic separations were performed
using a Cetac microcolumn designated ANX1606cr. The column packing was a proprietary anion exchange resin. The high. pressure delivery of the liquid sample was supplied by a gas displacement pump (GDP). The column eluent, containing the nitrate anion, was adjusted to an acidic pH. The eluent concentration and pH were varied either by dilution with deionized water or by the addition of nihic acid. The liquid sample and carrier were introduced to the column and ICP-MS via the GDP and rotary valves. The GDP has an operating pressure of up to 500 psi but is usually optimized at a much lower pressure. The rotary valves used are made of PEEK and are designated “metal free”. A 10 pL sample loop was connected to the rotary valves for injection of the sample into the column. A sample bypass tube was used for a total Cr analysis. This tube bypasses the column through the rotary valve system to the DIN) and is switched once a steady state signal has been established. The GDP and DIN incorporated into a unit called the M i m N e b 2MM system and is designed by Cetac Technob pies. Omaha. NE. The effluentfrom the analytical column is then introduced into the plasma ria the DIN using a capillary diameter of 0.060 nun. Figure 1shows a schematic diagram of the valve arrangement to obtain total and speciated measurement of chromium. The ICP-MS used was a Sciex Elan 5ooo with the standard sample inwoduction system removed and replaced with a DIN. Chromium was monitored at atomic masses 52 and 53. O p t i m i tion parameters are listed in Table 1. These parameters were obtained by monitoring total chromium. Chromatogram data was collected using a 100 ms dwell time and monitoring the appropriate mass over a 700 s time period. The resolution of the quadrupole was set at 0.8 Da. Quantitative analysis was conducted using peak area calculations through an algorithm in the Perkin-Elmer soHware. The timing sequence (12) Roehl. R Allorwe. M. M.Afomic Sped-. 1990,II. 211-215. E.J.: Pfaff. J. D.I.f3mmofqr. 1991.546.335-340. (14) W e n . R:James B./. Ewimx. Quof. 1979.8.31. (15) Zatka. V. J. An. h d Hm.Assoc 1985. 46.327. (13) Arar.
plasma flow (Wmin) auxiliary flow O./min) forward power (w) El lens W C ) S2 lens W C ) P lens W C ) bl lens W C )
detector O C )
13.0 0.8 1100
5.07 -10.08 -58.69 -9.89 -3.9
DIN and Column Parameters GDP pump pressure (psi) 7.80 psi eluent concentration 0.25% HNOJ sample loop size (uL) 10pL makeup gas flow (Wmin) 0.225 Wmin DIN capillary diameter (in,) 0.060 nebulizer gas pressure (psi) 120
OThese settings were obtained fromthe optimization and characterization wrfotmed on the svstem.
applied to the rotavalves and data acquisition triggering was dependent on the optimum retention time. Reagents. The acid used for sample and standard preservb tion was Anachemia EnvironmentalGrade nihic acid. AU aqueous solutions used for calibration were made from a chromium NlST standard loo0 mg/L stock. In addition to the NlST stock, standards traceable to NIST were purchased from Cetac Technologies, Omaha, NE. NlST Standard Reference Materials 2108 and 2109 are references for Cr(IID and C r O , respectively. The material was prepared as mshucted in the information sheets provided by NIST. RESULTS AND DISCUSSION
System Optimization. In this work, the only modfiation to the DIN compared to previous work” has been the addition of a third gas port. This allows for an independent adjustment to be made to the total gas flow and to the velocity in the plasma central channel., In addition, the third port provides a sheathing gas around the nebulizer tip to aid cooling. In order to achieve an optimum sensitivity for total and speciated chromium, ICP-MS and DIN parameters were adjusted on the basis of a total chromium signal. A 100 pg/L standard Analytical Chemistry, Vol. 67, No. 14. Jufy 15, 1995 2475
100
.
73 80
%
Stable Sleady Stale Signal 100 ppb Total Cr.
Ln
C
0 ._
60
v)
U
C
22 0
40
20
0 0
300
600
900
1,200
1,500
Time (secs) Figure 2 Dltferencein separation and signal strength between C162
and Cr” isotopes in a mixed standard containing 1OOpqlL chromium. The chromatogram with the larger signal IS the CPz soto ope. while the smaller of the two IS the CP3 isotope. The total chromlum signal IS produced by a sample tube that bypaws the analytical column. Cr(lll) and Cr(VI) s w i e s are measured m the column elfluentvla a 1OpL sample loop. was injected into the bypass tube. The sample then bypassed the column, entered through the DIN. was injected into the
plasma.
This is the most efficient procedure for opthizing an ICP-MS incorporating a DIN and should be performed prior to utilizing a column. F’revious work has shown sbategies for achieving the most sensitive signal using the DIN with ICP-MS.” figure 2 shows two signal profiles of Crs?and Cr” as total and speciated chromium. The signal to backgmund ratio (S/B) difference between CrsZ and C+ is not overwhelming but is substantial enough to produce better detection limits when using C+ with peak integration. Total chromium is determined using peak height measurements once a stable steady state signal has been reached. AU chromatograms used for optimization purposes and graphical representation were produced using Cr‘ since it has a larger signal that aids in the visual interpretation. After the DIN and ICP-MSwere optimized. the analytical column was characterized to the system. The eluent flow rate through the column is a major factor affecting analyte separation. retention time, and signal strength. In order to find the optimum flow rate. the unit pressure was adjusted to produce c h r o m a t o m s representing a Mlying range in steps of 20 psi from 250 to 530 psi. F i r e 3 shows the effects of flow change on three chromatograms representing the en!& pressure range. The lower the flow rate, the broader the peak widths, and hence, greater separation. As well, the peak height decreases at a lower flow rate. This would seem to indicate a limitation on sensitivity by improving separation. However, using peak area measurements compensates for loss in peak height, and for the most part, detection limits are retained. 2476
Analyiical Chemistfy, Vol. 67, No. 14. July 15, 1995
0
100 200 300 400
Time (sec) Figure 3. Three pressure settings show the relative difference in
peak height and separation as opposed to retention time. Eluent strength is another leading factor conhibuting to the overall performance of the chromatographic system. The acidic strength will determine how long the anaiyte is retained since the trivalent chromium is cationic under acidic conditions. This will affect retention time and peak separation of the resulting chromatogram. In order to find the optimum eluent strength for this work, the eluent concentration was varied from 0.15%to 0.8% HN03 in four steps. The resulting chromatograms showed differences as very narrow peak widths for the highest acid concentration to extremely broad peak widths for the lowest acid concentration. From these results, the eluent strength chosen as the optimum was 0.25%. At this eluent skngth, the chromate gram that was produced was well defined with good separation of the peaks. As well, the precision of the retention time with respect to different batches of eluent at the same concentration is quite acceptable. No signiicant change was noticed that would affect the positioning of data acquisition “windows” that were established through data acquisition parameters. The above experiments were conducted using a 10p L sample loop. Experiments in previous work have shown that an increase in sensitivity may be gained by using a larger sample loop.” However, when using a chromatographic column, consideration must be made for column overloading. An experiment was designed to choose the optimum sample loop size for the lowest detection limit and best separation. Various loop sizes were chosen ranging in size from 2 to 50 pL 7he smaller loop sizes showed good separation. but poor signal. The larger loop sizes showed narrowed peak widths with a distorted signal. The latter are caused by an&e overloading of the column. Ultimately, column overloadingwill affect the linear dynamic range and, hence, accuracy. From this experiment, the optimum sample loop size chosen for this work was 10 pL Precision and Detection Limits. m e reproducibility of the entire system is dependent on several variables. Previous work
400
Table 2. Technique Comparison.
delayed analysis diphenyl LC-DIN carbazide ICP-MS method 0.003 0.042 0.077 0.182 0.240
ndb 0.043 0.079 0.179 0.257
a
immediate analysis
actual spike value
LC-DIN
ICP-MS
diphenyl carbazide method
0.010 0.050 0.100 0.200 0.300
0.009 0.054 0.092 0.191 0.312
nd 0.040 0.086 0.188 0.273
“This table indicates that better spike recovery is achieved by analyzing the sample immediately after spiking. The first two columns show an analysis comparison of a spiked aqueous solution made 3 weeks prior to analysis. The last two columns show a analysis comparison of a spiked aqueous solution analyzed immediately after spiking. nd, not detected.
-
300
0
8
/
v)
C
.-0
v v)
U
200
C
$ 3 0
Cr 6+ 107000
100
has shown that the precision of the DIN as an introduction device has proven to be equal or better than the conventional sample introduction system with ICP-MS. The limitation on precision of the HPLC DIN-ICP-MS system as a whole is related to the reproducibility of sample injection, the precision of the valve movement, and the analytical column. As well, the retention time must remain stable to maintain good result precision when using software for automated data collection. To demonstrate the system short-term precision, five injections of a 100 pg/L mixed chromium standard were analyzed sequentially. The resultant short-term precision is 8%,7%,and 5%RSD for total, Cr(IJl), and C r O , respectively. The data were gathered using peak height measurements. Detection limits for total and speciated chromium were determined using 3x standard deviation of a blank signal after calibration with a 100 pg/L mixed standard. Two isotopes of chromium were individually used for this calculation to determine which isotope produced the lower detection limits. Crj2and Cr53 produced detection limits for Cr(I1I) at 180 and 60 ng/L, respectively. The peak area mode of the Elan software was used to perform realtime integration of peaks. Data acquisition parameters such as valve timing and “integration windows” were set up experimentally. Direct concentration readings calculated by using the Elan software was possible after a calibration curve was determined. To confirm the validity of the counting statistics of the Elan integration software, an alternate integration program was utilized. Raw data in ASCII form were imported into a Lotus spreadsheet where a program was written for peak integration. Using Crj3, the C r O detection limit was 180 ng/L, and total chromium detection limits were determined to be 30 ng/L after performing blank and standard measurements on the steady state signal. Interferences and Accuracy. With respect to chromium, spectral interferences on the isotopes monitored in aqueous matrices are limited to oxide formation in the plasma. C15* suffers interference from M60l6.This is the reason for the higher than normal background readings when monitoring a blank solution. Cfi3 also has oxide interference, but to a much lower extent. Although W6017is the oxide interference on this mass, the isotope abundance of 017is much lower than 0I6, thus a lower background count occurs with Crj3with an attendant decrease in background variability.
0 0
100 200 300 400 500 600 700 Time (sec)
Cr 6+ 170000
200
100
0. 0
I
I
t
I
I
100 200 300 400 500 600 700
Time (sec) Figure 4. (a) Response after spiking an industrial waste sample with lOOpg/L Cr(VI). This chromatogram was produced directly after the sample was spiked. (b) After 3 weeks, the chromatogram shows a measurable presence of Cr(lll) much higher than in the original spike. The change in ratio of Cr(lll)/Cr(VI) represents the extent of oxidation or reduction change.
Matrix interference is also an important consideration when using ICP-MS. Roehl et al. reported that defhite interference may occur on monitored chromium isotopes from samples containing significant concentrations of carbon, sulfur, and chloride. C15* will be interfered by AP0CI2and S36016. W3will suffer interference from C137016.As well, Gjerde et al. have studied the effects Analytical Chemistty, Vol. 67,No. 14,July 15, 1995
2477
of sulfate interference on this particular column and have found that a matrix with a concentration up to 500 ppm is tolerated without affecting retention times.I6 In this paper, several industrial waste effluentsweregnalyzed and compared to an existing method utilizing a diphenyl carbazide reaction and detection of C r O by colorimetry. By conducting this experiment, a preliminary indication of technique versatility to a more complex sample matrix was obtained. To confirm this, representative industrial waste samples were spiked with C r O at different concentrations which ranged from 0.010 to 0.400 pg/L. The samples were analyzed by the diphenyl carbazide technique prior to spiking to see if any background Cr existed in the samples. No hexavalant chromium was detected. The result comparison is listed in Table 2. In the first column of this table, labelled “delayed analysis”, are analytical results that are comparable. This indicates that both techniques are relatively precise. However, the spike recovery is poor; therefore, an interference of some kind was indicated between the two techniques affecting accuracy. Investigations into the cause of the loss in spike recovery suggested that preservation of the sample was not sufkient to maintain the stability of the oxidation state. The C r O standard spiked in the sample was being reduced to Cr(III) over time. This hypothesis was deduced by determining the change in ratio of the Cr(I1I) and C r O species. This deduction was logical because the samples were analyzed by both techniques on the same day but were spiked with the standard approximately 3 weeks earlier. Since the HPLC DIN-ICP-MS produces chromatograms of total chromium, Cr (III) , and C r O , changes in ratio of Cr(II1) to C r O can be measured. These changes in ratio can reflect the extent of oxidation or reduction in the chromium species over time. In Table 2, a second column of results labeled “immediate analysis” shows a set of results that were analyzed on the same day of spiking. A more accurate comparison to the spike value was obtained. To support this theory, an experiment was carried out to monitor the change in Cr(III)/CrO ratios over a 3-week period. Figure 4, panels a and b, show the difference in Cr(III)/ C r O ratio of a 100 pg/L CrW) spike in an industrial waste sample. The chromatogram shows that at time zero, very little Cr(I1I) exists; however, after a 3 week period, some C r O has been reduced to Cr(I1I). In fact, this represents a ratio change of approximately 30%,which will ultimately cause erroneous results. Arar et al. has stated that sample pH will affect oxidation state stability. In our work, an experiment was designed to find an “optimum working range” of pH for sample preservation. Replicate 100 pg/L mixed chromium standards were adjusted to a varying range of pH between 1and 12 using HN03 and NaOH. The samples were analyzed and an optimum range established. This range showed a minimal matrix effect to Cr(I1I) and C r O (16) Gjerde, D. T.; Wiederin, D. R;Smith, F. G.; Mattson, B. M.]. Chromatogr. 1993,640,73-78,
2478 Analytical Chemistty, Vol. 67,No. 14, July 15, 1995
on the column and through the ICP-MS. The “working range” was established between pH 4 and pH 9 for both valence forms of chromium.It should be noted that these measurements were made using aqueous standards and that higher concentrations of analyte and the chemical complexity of field samples (depending on application) may change the oxidation stability range. Accuracy was established by comparison of “in house” and supplied standards from Cetac to NIST reference standards for Cr(I1I) and C r O . The NIST reference standards are of an aqueous matrix. The 2108 Cr(I1I) standard is preserved with 1% HC1. The 2109 CrW) standard is not preserved. Precision and accuracy measurements of Cr(I1I) and Cr 0 to the NIST reference gave good recovery (96%-102%) for both techniques. Cr(III) was not measured by the diphenyl carbazide method. CONCLUSIONS
Use of the HPLC DIN-ICP-MS technique has shown to be viable in the study and analysis of total and speciated chromium. An efficient means of optimizing the performance of the system has been achieved. A reproducible retention time was obtained allowing accurate peak area measurements for quantitative analysis. The detection limits obtained by the HPLC DIN-ICP-MS system were at least an order of magnitude better than the alternative diphenyl carbazide technique. This would indicate system versatility to perform low-level speciation studies in environmental applications. In addition to the speciation studies possible with the microcolumn in the system, there are several advantages in having a DIN in the system as well. One is the small sample size. This will reduce effects from high dissolved solids matrices in the ICP-MS and enable the system to handle more complex matrices. The other is the stable state signal achieved. This allows for the total chromium and speciated Cr(I1I) and C r O chromatograms to be obtained in one analysis. This latter advantage is particularly useful in obtaining information on oxidation state stability of a sample by studying ratio data. Comparative results from industrial waste samples with an alternative technique have been shown to be precise, although the accuracy of both techniques is limited to the stability of the oxidation state. It is recommended that until investigations are conducted into developing a means of stabilizing the oxidation state over time, samples should be analyzed immediately. ACKNOWLEDGMENT
Many thanks to Lian Shan Liu, George Wood, and other staff of the Laboratory Services Branch of Ministry of the Environment and Energy for their invaluable help in making this publication possible. Received for review February 8, 1995. Accepted April 27, 1995.@ AC950143H @
Abstract published in Advance ACS Abstracts, June 1, 1995.
