Anal. Chem. 1992, 64, 2826-2830
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High-Performance Liquid Chromatographic Method for the Determination of Ultratrace Amounts of Iron(I I) in Aerosols, Rainwater, and Seawater Zhen Yi, Guoshun Zhuang,+JPhyllis R. Brown,’ and Robert A. Ducetps Department of Chemistry and Center for Atmospheric Chemistry Studies, Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island 02881
A reversed-phase hlgh performance llquld chromatography method was developed to determlne concentrations of Iron(II)In Chlnese loess, aerosols, rainwater, and seawater. This method was Isocratlc, rapld, sendtive, and reproduclble.Iron(11) f o r m a stable complex Ion wlth the reagent ferrozlne, [Fe(FZ)o]z+ In a pH range of 4-10. By meawrlng the absorbanceof [Fe( FZ)d2+at 254 nm, Iron( II)concentratlons were determined. [Fe(F&I2+ was separated from ferrozlne and was detected In 6 min. Reversed-phase C,, solid-phase extractlon cartrldges, whlch were used for the sample preparatlon, concentrated the [Fe( FZ)#+ and Increased sensltlvlty of the analysis. A detectlon llmlt of 0.1 nM Fe( II) was obtained. Retentlon tlme, addltion of known amount of standard, and peak area r a t b were usedfor peak ldenttfkatlon of [Fe(FZ)s]z+. There was no Interference from F e ( I I I ) , NI(II), Co(II), and Cu(1). The recovery of 104-10-10 M [Fe(FZ)#+ uslng the preconcentratlon step was In the range of 9 2 4 9 % . The Fe(I1) concentratlon In remote marine aerosols has been determlned for the flrst tlme by uslng thls new method.
INTRODUCTION The determination of Fe(I1) is of considerable interest in research in both atmospheric chemistry and oceanography. It has recently been suggested that iron, a micronutrient required by organisms, may be the limiting factor for primary biological productivity in some open ocean regions, including high latitude and equatorial upwelling regions, where other major nutrients (nitrate and phosphate) are abundant.’-4 In clouds, fog, and rainwater the photoreduction of Fe(II1) to Fe(I1) has also been proposed as a major source of aqueous hydroxylradical (OH*),b7which is a key radical in atmospheric photochemistry and plays a central role in the atmosphere in the oxidation of many tropospheric trace gasessand species in solution in atmospheric waters. + Center for Atmospheric Chemistry Studies, Graduate School of Oceanography. f Present address: Environmental Sciences Program, University of Massachusetts a t Boston, 100 Morrissey Blvd., Boston, MA 02125-3393. 0 Present address: Collegeof Geosciencesand Maritime Studies Texas A&M University, College Station, TX 77843. (1) Martin, J. H.; Gordon, R. M. Deep Sea Res. 1988,35,177-196. (2)Martin, J. H.; Fitzwater, S. E. Nature 1988,321,341-343. (3)Martin, J. H.; Gordon, R. M.; Fitzwater, S. E.; Broenkow, W. W. Deep Sea Res. 1989,36,649-680. (4) Martin, J. H.; Gordon, R. M.; Fitzwater, S. E. Nature 1990,345, 156-158. (5)Graedel, T.E.; Weschler, C. J.: Mandich, M. L. Nature 1985.31 . 7., 24C-242. (6) Weschler, C.J.; Mandish, M. L.; Graedel, T. E. J. Geophys. Res. 1986,91,5189-5204. (7)Faust, B. C.; Hoigne, J. Atmos. Enuiron. 1990,24A,79-89. (8) Davis, D.; Niki, H.; Mohnen, V.; Liu, S. Global Tropospheric Chemistry, A Plan for a Action; National Academy Press: Washington, DC, 1984; pp 78-87.
Although there are a number of papers that deal with the total iron in the ocean and in the atmosphere, only a few have reported the concentration of Fe(II).*13 In investigations of Fe(I1) in the ocean and atmosphere a major obstacle in the analytical methods used has been the detection limits. The total dissolved iron concentration in the open ocean is generally less than 1n m ~ l / k g . ~ J , ~Thus, J ~ J ~methods for the determination of Fe(I1)in samples from the open ocean require very low detection limits. Recently, a technique of flow injection analysis with chemiluminescence detection was reported for the determination of subnanomolar levels of Fe(I1) in seawater.I6 Since there was no separation of Fe(I1) and Fe(1II)in the analyses,the measured Fe(I1)concentrations must be corrected for the chemiluminescence produced by the Fe(II1). Waite and Morel’s reported a controlled potential coulometric procedure combined with a masking technique to determine the Fe(I1) in seawater at the nanomol level. A colorimetric method using ferrozine (FZ) (3-(2-pyridyl)-5,6diphenyl-l,2,4-triazine-p,p’-disulfonic acid) has been used for measuring Fe(II) since 1970.11J2J7-20The colorimetricreagent FZ forms a stable complex ion, [Fe(FZ)3I2+,with Fe(I1) in a pH range of 4-10 but not withFe(II1). However, the detection limits of these reported methods are not satisfactory for the measurement of Fe(I1) in the open ocean. Recently King et al.21 reported a colorimetric methods for the determination Fe(I1) in seawater at the nanomol level using FZ immobilized on a reversed-phase Cla Sep-Pak cartridge. This method is only marginally satisfactory for the measurement of Fe(I1) at the less than 1-nmol level. FZ has a significant absorbance at the wavelength that was used for determination of Fe(I1). In previous methods in which FZ was used as a reagent, the excess FZ in the reaction mixture was not separated from the [Fe(FZ)#+ complex ions that were formed, and the absorbance from the FZ itself and from other constituents in the samples (e.g., seawater) was measured by a blank determination. Since it is impossible to determinethe concentration of FZ that is not complexed in the unknown sample solutions, the excess FZ can cause considerable error in a system with very low Fe(I1) concentrations. To date there has been no (9)Behra, P.; Sigg, L. Nature 1990,344,419-421. (10)Landing, W. M.; Westerlund, S. Mar. Chem. 1988,23,329-343. (11)Hong, H. S.;Kester, D. R. Limnol. Oceanogr. 1986,31,512-524. (12)Hong, H. S.;Kester, D. R. Coastal Shelf Sci. 1985,21,449-459. (13)Waite, T. D.; Morel, F. M. Anal. Chem. 1984,56,787-792. (14)Landing, W. M.; Bruland, K. W. Geochim. Cosmochim.Acta 1987, 51,29-43. (15)Symes, J. L.; Kester, D. R. Mar. Chem. 1985,17,57-74. (16)Elrod, V. A.; Johnson, K. S.;Coale, K. H. Anal. Chem. 1991,63, 893-898. (17)Stookey, L.C. Anal. Chem. 1970,42,779-781. (18)Gibbs, M. M. Water Res. 1979,13,295-297. (19)Gibbs, M. M. Anal. Chem. 1976,48,1197-1201. (20)Boyle, E. A.; Edmond, J. M.; Sholkovitz, E. R. Geochim. Cosmochim. Acta 1977,41,1313-1324. (21)King, D. W.;Lin,J.; Kester, D. R. Anal. Chim.Acta 1991,247, 125-132.
0003-2700/92/0364-2826$03.00/0 0 1992 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
method reported for the measurement of Fe(I1) using highperformance liquid chromatography (HPLC). Here, we report a method for measurement of Fe(I1)in environmental samples by derivatizating the Fe(I1)with ferrozine and isolating the Fe(I1)-FZ complex by reversed-phase HPLC.
EXPERIMENTAL SECTION Apparatus. A Perkin-Elmer Series3B liquid chromatograph (Perkin-ElmerCorp., Norwalk, CT) was used. All analyses were performed on a 250- X 4.6-mm, 5-pm Supelco LC-18 column (Supelco Inc., Bellefonte, PA). Detection was performed by means of a Perkin-Elmer LC-95 UV/visible spectrophotometer detector. Injectionswere made via a Waters Model U6K injector (Waters Chromatography Division of Millipore Corp., Milford, MA). Peak heights were recorded with a strip chart Omniscribe recorder (Houston Instruments, Austin, TX). Peak areas were electronicallyintegratedwitha Hewlett-Packard 3390Areporting integrator. Chemicals and Supplies. The chromatographic standard was ferrous ammonium sulfate hexahydrate (Fe(NH4)2(S04)y 6H20)obtained from Sigma Chemical Co. (St.Louis, MO). The monosodium salt of ferrozine, sodium dodecyl sulfate (SDS), and tetrabutylammonium phosphate (TBA) were also obtained from Sigma. Sodium chloride (99.999%) was obtained from Aldrich Chemical Co. (Milwaukee,WI). Sep-Pak C18 cartridges were obtained from Waters. Methanol was purchased from Fisher Scientific (Fair Lawn, NJ). Mobile phases which were prepared with doubly distilled deionized water (DDH20) were filtered through a0.45-pm Nylon 66 membrane filter (AlltechAssociates Inc., Deerfield, IL). All standard and sample solutionswere stored in Teflon vials. Chromatographic Conditions. The mobile phase was 400 mL of an aqueous solution containing 0.0500 g of SDS, 0.5000 g of NaC1,0.0100 g of TBA, and 100mL of methanol. A flow rate of 1.0 mL/min was used. The injected volume of each sample was 50 pL. The temperature was 20 A 1 OC. Sample Preparation. The sample and standard preparation was performed in Class 100 clean laboratories at the University of Rhode Island. I. Standard Solutions. A 0.0392-g sample of Fe(NH4)z(S01)2.6H20 was dissolved in 100 mL of DDH20, and the M Fe2+was concentration of Fe2+was 10-3 M; 0.100 mL of immediately added to 0.400 mL of 10-3 M FZ and diluted to 10 mL. The concentration of [Fe(FZ)3I2+was 10" M. The 10" M [Fe(FZ)#+ standard solution was then diluted with DDHzO to 5 X lo-*, lo+,5 X lo+, and lo* M. 5 X lo", lo", 5 X ZZ. Aerosol Samples. A 2- or 4-cm2aliquot of an aerosolsample collected on a filter over the North Pacific Ocean was leached in 0.2 mL of doubly distilled hydrochloric acid (DDHCl) and 2.0 mL of DDHzO for at least 3 h. After the sample was filtered through a 0.4-pm Nuclepore filter, 25% NH3 was added to the filtrate to adjust the pH to the range of 4-6; 0.1-0.2 mL of a M solution of FZ was then added to the sample. ZII. Chinese Loess. Chinese loess was the major source of the mineral aerosols over the North Pacific. To determine the relationship of Fe(1I)in mineral aerosolsand ita source,the Fe(II) in Chinese loess was also measured. A 1.0- or 2.0-mg sample was leached in 0.2 mL of HC1 and 2.0 mL of DDHzO for at least 3 h. The sample was then treated the same way as the aerosol sample. ZV. Rain and Seawater. The samples were prepared by using a reversed-phase C18 Sep-Pak cartridge for the solid phase extraction procedure. Rainwater samples were collected from Narragansett, a coastal area of Rhode Island. Coastal seawater samples were collected from Narragansett Bay, RI. As soon as a sample was collected, it was filtered with a 0.4-pm Nuclepore filter. The rainwater or seawater (100-500 mL) was passed through the pre-prepared Sep-Pak cartridge at a rate of 10 mL/ min. Each Sep-Pak cartridge had been cleaned with 10 mL of methanol and 10 mL of DDHzO and then loaded with 2 mL of 10-3M FZ. A 5-10-mL aliquot of DDH20 was used to rinse the Sep-Pak. The complex [Fe(FZ)s]Z+and FZ were eluted with 1 mL of methanol. A 4-mL portion of DDHzO was added to the eluent in order to make the ratio of methanol to water in the
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eluent the same as that in the mobile phases, and this eluent was used for injection in the HPLC system. V. Blank Samples. A blank Whatman 41 filter of the type used for aerosol sampling was used as a blank sample for aerosols, and DDHzO was used as a blank sample for rain and seawater. The procedures for blank sample preparation were the same as those described in sections I1 and IV. Sample Recovery. The recovery of Fe(I1)from the Sep-Pak was determined by addition of known amounts of Fe(I1)standards to sample matrixes of rainwater or deionized water. The complex ion, [Fe(FZ)#+, was extracted using the sample preparation procedure described in section IV. Detection Limit. To determine the detection limit, a 10-10 M standard [Fe(FZ)3I2+solution was prepared by the dilution of 1mL of 5 x IO4 M [Fe(FZ)3I2+standard solution to 500 mL M FZ was passed through a with DDH20. After 1 mL of Sep-Pak, 500 mL of 10-loM [Fe(FZ)3I2+was passed through the Sep-Pak prepared with FZ. The Sep-Pak was then rinsed with 5 mL of H2O. The [Fe(F2)3l2+adsorbed on the Sep-Pak was eluted with 1mL of methanol. The methanol in the eluent was evaporated with helium gas to -0.2 mL, and DDHzO was added to 1mL. A 50-pL portion of the sample was then injected into the HPLC system. Peak Identification. Identification of the peaks of interest was based on retention time, co-injection of standards with the samples, and determination of the ratios of peak area at various wavelengths (254,270,290,310,330,350,450,546, and 562 nm) to the peak area at 230 nm. Interferences. Interferences from Fe(III),Ni(II),Co(II),and M Fe(III), Ni(II), Co(II), Cu(1) were tested. Solutions of and Cu(1)were prepared from Fe, Ni, and Co standard solutions (NBS) and CuBr, respectively;0.1 mL of M FZ was added to 5 mL of 10-5M Fe(III), Ni(II), Co(II),and Cu(I),respectively. The pH of the Fe(II1) solution was -4.0, and the pH of the Ni(II), Co(I1) and Cu(1) solutions was 5.5. Since Cu(1) is lightand air-sensitive,the Cu(1)solution was wrapped with aluminum foil, and FZ was added into M Cu(1) solution immediately after CuBr was dissolved in DDH20. A capillary electrophoresis (CE) method for ~ations2~J3 was also used to determine Fe(II), and the results were compared with those obtained by HPLC. A Waters Quanta 4000 capillary electrophoresis system, equipped with a positive voltage power supply, was used in this study. Details were in the literature.22.a
RESULTS AND DISCUSSION The peaks of [Fe(FZ)3I2+and FZ were baseline resolved (Figure 1). By measuring the absorbance of [Fe(FZ)#+ at 254 nm, iron(I1)concentrationswere determined. [Fe(FZ)#+ was separated from ferrozine and was detected in 6 min. The peaks prior to 5 min are not identified. The retention times, peak areas, and peak shapes were reproducible on 10samples of a 10-7 M standard solution; the relative standard deviation of the retention times and the peak areas were 0.6% and 4.33'% , respectively. There was a good linear response (regression coefficient = 0.997) in the concentration range of 10-5-10-8 M.
If Sep-Pak CU solid phase extraction cartridges were used, the detection limit was 10-lOM. When the SepPak extraction procedures were not used, the detection limit at 254 nm was decreased to loe8M, and the mass detection limit was 0.028 ng. Even though the wavelength 562 nm has been reported asthe best,21baseline noise was greater at 562 nm. In addition, the peak areas at 230,310,290,330, and 254 nm are greater than that at 562 nm (Table I). Therefore, detection limits were lower at the wavelengths 230,310, and 254 nm than at 562 nm. Since some models of UV/visible detectors can only be used at one wavelength (usually at 254 nm), the wavelength of 254 nm was chosen in this work. (22) Weston, A,; Brown, P. R.; Jandik, P.; Jones, W. R.; Heckenberg, A. L.J. Chromatogr. 1992,593, 289-296. (23) Weston, A.; Brown, P. R.; Heckenberg, A. L.; Jandik, P.; Jones, W. R. J . Chromatogr. 1992,602, 249-256.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
erable error in the Fe(I1) concentrations. For example, when a 5 X 10-6 M Fe(I1) standard reacted with 2 X M FZ solution was measured at 562 nm, the excess FZ concentration was 5 X 10-6 M. The ratio of the peak area of [Fe(FZ)#+ to the peak area of FZ was 10. As reported in the measurement of Fe(I1) in seawaterin which a C18 Sep-Pak preconcentration step was used,21 the concentration of FZ was -10-3 M and the measured Fe(I1) concentration was only -4 nM. In this case the absorbance from the excess FZ was much greater than that of Fe(I1). Therefore, the calculation of Fe(I1) obtained by subtracting the FZ “blank” from the total absorbance may cause considerableerror. Fortunately, with our HPLC method, [Fe(FZ)J2+ was completely separated from FZ and other constituents in the samples,and thus there was no interference from the FZ peak. In blank samples for Chinese loess, aerosol, rainwater, and seawater, [Fe(FZ)J 2+ was not detectable. To determine if any Fe(I1) was released from Sep-Pak cartridges during the sample preparation, lo00 mL of double distilled HzO was used as a blank to pass through the same type of Sep-Pak cartridge, and there was no detectable Fe(I1) ferrozine complex peak due to Fe released from the Sep-Pak cartridge. It was reported that Cu(I), Co(II), and Ni(I1) are the only metals other than Fe which may form colored complexeswith FZ.17,24When 0.1 mL of 10-3 M FZ was added to 5 mL of 106 M Ni(II), Co(II), and Cu(1) solutions, there are no additional peaks to respond the possible Ni(II), Co(II), and Cu(1) ferrozine complexes either in the range of Fe(I1) peak or in other ranges of the chromatograms even up to 2 h after the addition of FZ. It was also found that when 0.1 mL of M FZ was added to 5 mL of M Fe(II1) solutions, there is no Fe(I1) peak to respond the possible reduction of Fe(II1) by FZ to Fe(I1) in the range of the Fe(I1) peak even up to 2 h after the addition of FZ. Only 0.1 % of 18 p M Fe(II1) was reduced by FZ to Fe(I1) 2 h after addition of FZ. Since all the samples were analyzed within 30 min of addition of FZ, there would be no error caused by reduction of Fe(II1) to Fe(I1). Similarly, because there is no interference from the presence of Ni(II), Co(II), and Cu(I), these ions which may form colored complexes with FZ would not cause any errors in the Fe(I1) concentrations in any of the samples. Peak identification of [Fe(FZ)#+ was carried out by retention time, by addition of known amount of standard, and by comparison of peak area ratios at various wavelenghs.26 The ratios of peak areas at various wavelengths to the peak area at 230 nm for a standard solution and for an aerosol sample solution (sample ID: EN 162) are shown in Table I. The [Fe(FZ)3I2+peak was identified when the ratios for the standard solution and for the sample were similar or identical. These results indicate that there was little or no absorption due to other substances, such as possible organic compounds, in the aerosol samples. It would be unlikely that the organic compoundswould have the same absorbance ratios at so many different wavelengths. The recovery of 10-klO-10 M [Fe(FZ)312+by preconcentration with the Sep-Pak CIScartridge was in the range of 92-99% (Table 11). To determine if any Fe(I1) was produced due to the reduction of Fe(II1) in aerosol samplesby other factors during the storage, a standard Fe(II1) solution was added to several blank Whatman41 fiiters (-1 pmol/cm2filter,-5-400 times higher than that in the measured aerosol samples). These filters were measured for Fe(I1) by the same procedures used for measuring Fe(I1) in the aerosol samples 7,14,35, and 91 days after addition of Fe(II1). In all of these cases there was
-
I 0
I
5
10
Time
15
a,
25
(minutes)
Flgue 1. Achrometogamof 20pLof5 X 10-6M[Fe(FZh]*+standard solution. Chromatographiccondbns are describedIn theExperimental Sectlon. UV/vlsdetector, 254 nm; senslthrlty, 0.01 AUFS (Absorbance Unlt Full Scale): response tlme, 500 ms.
Table I. Peak Area Ratios (R= A, nm/Amnm) for a Standard Solution and a Sample Solution at Various Wavelengths wavelength (nm) 230 254 270 290 310 330 350 450 564 562
standard solution0
sample solutionb
ratio area, nm (A, d A m nm) 9227400 4641900 0.50 4581300 0.50 6741 300 0.73 6993200 0.76 5074900 0.55 3323400 0.36 1016800 0.11 2218300 0.24 2881300 0.31
ratio area (A, d A z m nm) 7 080 300 3 581 100 0.51 3 574 400 0.50 5 343 100 0.75 5 462 900 0.77 3 940 100 0.56 2 592 500 0.37 862 340 0.12 1 809 200 0.26 2 341 500 0.33
4 Standard solution: 50 p L of 5 X 10” M [Fe(FZ)#+. Sample: 50 pL of aerosol sample solution from an aerosol sample collected at Enewetak, an island in the North Pacific (sample ID EN162).
The important advantage of our HPLC method is that the [Fe(FZ)3I2+ complex can be separated from the excess ferrozine and other constituents in the solution. As mentioned above, in previous methods the absorbance from the excess FZ, as measured in a blank of FZ, had to be subtracted from the totalabsorbanceof the sample. In a system with very low Fe(I1) concentrations, this calculation could cause consid-
(24)Bet-Pera, F.;Jaselakie, B. Analyst 1981,106, 1234-1237. (25)Krstulovic, A.M.;Rosie, D. M.; Brom, P. R. Anal. Chem. 1976, 48,1383-1386.
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Table 11. Recovery Efficiency of Fe(I1) Measured by Preconcentration with C18 Sep-Pak Cartridges added concn (nM)
measured concn (nM)
10 5 1 0.1
10.6 6.0 2.12 0.099
recovery concn (nM) 9.4a
recovery (%)
94 96 92 99
4.8" 0.92' 0.0996
a The recovery was determined by addition of known amounts of newly prepared fresh Fe(I1) standards to a rainwater sample. The recovered Fe(I1) concentration is the measured Fe(I1) concentration minus the Fe(I1) concentration in the rainwater sample (1.2f 0.4 nM; n = 4). * T h e recovery was determined with 0.1 nM Fe(I1) standard in the iron-free deionized water. The recovered Fe(I1) concentration is the measured Fe(I1) concentration.
T i m (minutes) Figure 3. A chromatogramof rainwater collected from Narragansett, RI, (sampling date 12/8/1990). Sample preparation and chromatographic condltions are described in the Experimental Sectlon. Fe(I1) in rainwater was preconcentrated50 times with C18 Sep-Pak. UV/& detector, 254 nm; sensitivity, 0.02 AUFS; response time, 500 ms.
0
5
ID
15
a0
25
Time ( m i n u t e s ) Figure 2. A chromatogramof an aerosol Whatman 41 fitter sample (EN 161). Sample preparation and chromatographic conditions are described in the Experimental Sectlon. UV/vis detector, 254 nm; senslthrity, 0.05 AUFS; response tlme, 500 ms.
no detectable Fe(I1) (i.e., Fe(I1)
