Improvement on simultaneous determination of chromium species in aqueous solution by ion chromatography and chemiluminescence detection

Analytica Chimica Acta 354 (1997) 107±113

Improvement on simultaneous determination of chromium species in aqueous solution by ion chromatography and chemiluminescence detection Bente Gammelgaarda,*, Yi-ping Liaob, Ole Jùnsa a

Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Peking University, Beijing, People's Republic of China Received 4 April 1997; received in revised form 16 June 1997; accepted 20 June 1997

Abstract A sensitive method for the simultaneous determination of chromium(III) and chromium(VI) was developed using ion chromatography and chemiluminescence detection. Two Dionex ion-exchange guard columns in series, CG5 and AG7, were used to separate chromium(III) from chromium(VI). Chromium(VI) was reduced by potassium sulphite, whereupon both species were detected by use of the luminol±hydrogen peroxide chemiluminescence system. Parameters affecting retention times and resolution of the separator columns, such as eluent pH, eluent composition, reductant pH and concentration, and ¯ow rates were optimized. Furthermore, the stabilities of reductant and luminol solutions were studied. The linear range of the calibration curve for chromium(III) and chromium(VI) was 1±400 mg lÿ1. The detection limit was 0.12 mg lÿ1 for chromium(III) and 0.09 mg lÿ1 for chromium(VI), respectively. The precision at the 20 mg lÿ1 level was 1.4% for chromium(III) and 2.5% for chromium(VI), respectively. The accuracy of the chromium(III) determination was determined by analysis of the NIST standard reference material 1643c, Trace elements in water with the result 19.11.0 mg Cr(III) lÿ1 (certi®ed value 19.00.6 mg Cr(III) lÿ1). The method was applied to analyse the stability of chromium patches for contact dermatitis testing. # 1997 Elsevier Science B.V. Keywords: Chromium speciation; Ion chromatography; Flow injection analysis; Chemiluminescence

1. Introduction In environmental samples, chromium mainly occurs as chromium(III) or chromium(VI). The biochemical roles and effects of chromium are critically dependent on the oxidation state of the element. Chromium(VI) is toxic; the major sources of chromium(VI) are *Corresponding author. Tel.: +45 35370850; fax: +45 35375376. 0003-2670/97/$17.00 # 1997 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00421-2

anthropogenic and originate from the electroplating, steel, and textile industries from where chromium(VI) is transferred to the environment through air and water emissions. In contrast, chromium(III), which is widespread at trace levels in nature, is non-toxic and considered to be an essential nutrient [1]. Chromium is a contact sensitizer and is responsible for a high incidence of occupational dermatitis. Chromate, in contrast to chromium(III) compounds, is able

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to penetrate the skin [2]. Chromate is reduced to the trivalent form during diffusion through the skin, some protein-bound form of chromium(III) being considered to be the actual allergen [1]. Hence, there is a need for sensitive analytical methods able to distinguish between chromium(VI) and chromium(III). Speciation of chromium most often involves a separation step in form of ion chromatography prior to detection. The low inherent levels of chromium present in most samples of interest, i.e. concentrations at the low mg lÿ1 level, demands special attention due to the risk of contamination as chromium is a ubiquitous element and the risk of changing the ratio of chromium(VI) to chromium(III) in the samples during sample treatment and analysis [3]. Methods involving preconcentration by retainment of the chromium species on columns and subsequent elution have been reported using ¯ame atomic absorption spectrometry (FAAS) [4,5], direct current plasma atomic emission spectrometry (DCP±AES) [6,7] or inductively coupled plasma atomic emission spectrometry (ICP±AES) [8] for detection. Direct determination of the chromium species demands very sensitive detectors. A detector system being suf®ciently sensitive is inductively coupled plasma mass spectrometry (ICP±MS), which has been used in the determination of chromium species in aquatic samples [8±12]. However, such an instrument is not available in most laboratories. As an alternative, the sensitive chemiluminescence detection can be used. The ®rst attempt to use chemiluminescence detection based on the oxidation of luminol by hydrogen peroxide for the determination of chromium was made by Seitz et al. [13]. This system was later applied for the simultaneous determination of chromium(VI) and chromium(III) by Williams et al. [14]. They introduced a chromatographic separation system consisting of a guard anion exchange column in parallel with a guard cation exchange column using two eluents and four pumps. Chromium(VI) and chromium(III) were separated on the columns, whereupon chromium(VI) was reduced by sulphite before detection as chromium(III). This rather complicated separation system was simpli®ed using a single guard cation exchange column, a single eluent, and the same chemiluminescence detection system [15]. However, this method suffered from the disadvantage that chromium(VI) was not

retained, but appeared in the void volume. Beere and Jones [16] used the same chemiluminescence system but improved the separation by applying an analytical anion exchange column containing a small proportion of cation exchange groups. This system complied with the wish to retain both ions. Escobar et al. [17] investigated the chemiluminescence system based on ¯ow injection without columns for the determination of chromium(III) alone, while Zhang et al. [18] used a chemiluminescence detection system involving hexacyanoferrate(II) for the determination of chromium(VI) in water. The aim of this study based on our previous work [15] was to improve the separation of chromium(VI) from the void volume and improve the stability of the reagents, especially the luminol solution and the reduction reagent. Furthermore, the large number of parameters of the system was optimized to improve sensitivity. 2. Experimental 2.1. Reagents Eluent: 0.055 M potassium sulphate and 0.095 M potassium nitrate adjusted to pH 3.0 with concentrated nitric acid. Reducing reagent: 0.010 M potassium sulphite adjusted to pH 4.0 with concentrated nitric acid. Chemiluminescence reagent: 0.010 M luminol stock solution diluted to 0.0015 M with 0.2 M carbonate buffer to 1 l, added 2 ml of 30% hydrogen peroxide and adjusted to pH 12.5 with 10 M sodium hydroxide. The luminol stock solution was prepared by dissolving luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) in 1 M potassium hydroxide. These reagents were all made 110ÿ3 M with respect to EDTA. All chemicals were analytical grade and from Merck (Darmstadt, Germany), except luminol (Sigma, St Louis, MO ). All solutions were prepared with ultra-pure water from a Milli-Q System (Millipore, Milford, MA). Concentrated nitric acid was puri®ed by sub-boiling. Standard solutions: Chromium(III) standard solutions were diluted from a 1.000 g Cr lÿ1 Titrisol

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standard solution (Merck, Darmstadt, Germany) and chromium(VI) standard solutions were diluted from a 1.000 g Cr lÿ1 Chrom-Standard (Riedel-deHaeÈn, Seelze, Germany). Final dilutions of the standards and samples were made by the eluent. 2.2. Apparatus A Jasco Model 880-PU high-performance liquid chromatography pump (Japan Spectroscopic, Tokyo, Japan) forced the eluent through the columns with a ¯ow rate of 1.6 ml minÿ1. The columns were 5 cm Dionex Ionpac AG7 and CG5 guard columns (Dionex Corporation, Sunnyvale, CA). A FIA-08 (Bifok, Sweden) peristaltic pump fed the system with reducing agent (2.2 ml minÿ1) and luminol reagent (1.6 ml minÿ1). The injection volume was 50 ml introduced by a Valco 10 port injection valve with titanium stator (Valco Instruments, Houston, TX), PEEK tube (Upchurch Scienti®c, Oak Harbor, WA) was used for connections. The autosampler was build in the laboratory workshop. The chemiluminescence detector was constructed by placing two photomultiplier tubes (RCA IP 28, RCA, Harrison, NJ) on each side of a rod winded with a PTFE tube with a capacity of 300 ml as previously described [15]. The detector was connected to a DP 700 data processor (Carlo Erba, Milan, Italy). 2.3. Procedure A ¯ow diagram of the analytical arrangement is shown in Fig. 1. Chromium(VI) and chromium(III) are separated on the columns placed in series. Chro-

Fig. 1. Flow diagram of the analytical system.

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mium(VI) is reduced in the stream of reducing agent applied after the separation. The luminol±hydrogen peroxide reagent is added just before the entrance to the detector. The retention time of the species was about 2.8 min for chromium(VI) and about 4.6 min for chromium(III), respectively, resulting in two well resolved peaks. All the measurements were made in triplicate. 3. Results and discussion 3.1. Columns and elution reagents The choice of columns and the composition of the elution reagent are important parameters in the separation of chromium(III) and chromium(VI). In a previous study [15], the use of a single cation guard column resulted in elution of the chromium(VI) peak very close to the solvent front. In some instances, this could result in disturbance of the chromium(VI) signal. In an attempt to delay the chromium(VI) peak, several anion exchangers were examined in combination with the Dionex CG5 cation exchanger used in the previous study. The Dionex AG5 anion exchanger was used successfully in a large number of experiments. In this system, the chromium(VI) peak was delayed for about 2 min and the chromium(III) peak for about 5 min in a 0.09 M potassium sulphate eluent. However, when this column was worn out and exchanged by a new one, it appeared that the column material had been changed. The new column behaved as a cation exchanger, actually the retention time for chromium(III) was longer on this column when compared with the CG5 column. When applying different columns to the system it appeared that some of the anion exchangers possessed cation exchange capability as well. This is shown in Table 1. To separate the species on the combination of AG11 and CG5 or (AG5) the eluent concentration had to be lowered to 0.075 M potassium sulphate, which resulted in peak broadening. On this system the chromium(VI) peak was delayed for about 1.5 min. The AG4A column in combination with CG5 or AG5 could separate the species, however the delay of chromium(VI) was only about 0.6 min in 0.09 M potassium sulphate and could be extended to

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Table 1 Retention times for Cr(VI) and Cr(III) on single columns. Eluent: 0.055 K2SO4‡0.095 M KNO3 Column

Retention (times/min) Cr(VI)

Cr(III)

CG5 AG5 AG11 AG4A AG7

0.74 0.80 1.09 0.81 1.82

1.94 2.84 0.52 1.24 1.27

1.2 min in 0.045 M eluent. As the retention times could not be predicted on basis of the ionic strength alone, experiments were performed by adding potassium nitrate in different combinations with potassium sulphate. Addition of potassium nitrate to the eluent was found to enhance the resolution, and a mixed eluent containing 0.055 M potassium sulphate and 0.095 M potassium nitrate was found to give a satisfactory resolution. The ®nal choice of anion exchanger was AG7 in combination with CG5 as this system delayed the chromium(VI) peak for 2.8 min and the chromium(III) peak for 4.6 min. 3.2. Luminol and reduction reagents The noise and the signal to noise ratio were primarily dependent on the composition and ¯ow rate of the luminol±hydrogen peroxide reagent. Also the stability of the reagents was important for practical use, as previous studies showed that the luminol and reduction reagents had to be prepared daily due to their instability. The effects of luminol and hydrogen peroxide concentrations together with the pH of the solutions were studied. The concentration of luminol giving the best sensitivity was found to be 0.015 M. The best luminol reagent composition was a 0.2 M carbonate buffer, pH 12.5 with a content of 0.06% hydrogen peroxide. This solution was stable for a week. At more alkaline pH, the solution was stable and resulted in less noise, but the sensitivity was decreased. With a higher hydrogen peroxide concentration, more noise was observed, probably as a consequence of hydrogen peroxide decomposition. At more acidic pH and lower hydrogen peroxide concentration, the sensitivity decreased.

Table 2 Optimized parameters for chromium speciation Reagent Eluent 0.055 M K2SO4‡0.095 M KNO3 Luminol reagent 0.0015 M Luminol‡0.06% H2O2 Reductant 0.010 M K2SO3

pH

Flow rate

3.0

1.6 ml minÿ1

12.5

1.6 ml minÿ1

4.0

2.2 ml minÿ1

The concentrations of luminol and hydrogen peroxide and the pH of the solution used in this work are higher compared to the concentrations and pH in the solution used by Beere and Jones [16], but apparently these authors had no problems with stability as their solutions were constantly purged with nitrogen. The remaining parameters involved in the system, e.g. ¯ow rates of reagents and pH and concentration of the reducing reagent, were ®nally optimized using the program COPS (Chemometrical Optimization by Simplex, Elsevier, Amsterdam) [19]. As lowering of ¯ow rates resulted in peak broadening, the response parameter used as criterium for optimization was the peak height of the second peak, the chromium(III) peak. The optimized parameters are summarized in Table 2. The reducing reagent was stable for one week, when the potassium sulphite concentration was 0.010 M and pH was 4. 3.3. Validation The calibration curves based on peak area measurements for chromium(VI) and chromium(III) are shown in Fig. 2. A slightly higher sensitivity for chromium(VI) when compared to chromium(III) is observed. Some days the two curves were merging, but the system was found to be sensitive for age of columns. Beere and Jones [16] reported chromium(III) to have the highest sensitivity. In theory, the sensitivity for the species should be equal as the chemiluminescence detection is based on the catalytic effect of chromium(III) in both cases. However, the chromium(III) species formed in situ from reduction could have another catalytic ef®ciency. When optimizing the eluent composition, one of the aims was to obtain equal sensitivity for the two species. Injection

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Fig. 3. Chromatogram of chromium(VI) and chromium(III) in the range 1±5 mg lÿ1. (Correlation coefficients > 0:999 based on peak height measurements). Chromium(VI) appears at 2.8 min, chromium(III) appears at 4.6 min.

Fig. 2. Calibration curves of chromium(VI) and chromium(III) in the range 5±400 mg lÿ1. (Correlation coefficients > 0:9995 based on peak area measurements). (*): chromium(VI), (~): chromium(III).

of the species separately without use of the columns resulted in equal sensitivity. The chromium standards were diluted by eluent. If the chromium(III) standards were diluted with water, a higher sensitivity was observed for this species. Diluted standards were prepared daily. The ratio of chromium(VI) to chromium(III) in the standards did not change for at least a day. The linear range could be extended to 400 mg lÿ1 which was the upper limit of the input signal of the data processor. In the concentration range below 5 mg lÿ1, measurement of peak heights gave the best precision. According to the IUPAC de®nition, the limit of detection was determined as the mean of the blank plus three times the standard deviation of the blank. As blank gave no signals at all, the detection limit was calculated as the concentration giving three times the standard deviation on basis of analysis of a standard solution containing 2 mg lÿ1 of chromium(VI) and chromium(III) (nˆ10). A recorder trace at this concentration level is shown in Fig. 3. The limit of detection was 0.09 mg lÿ1 for chromium(VI) and 0.12 mg lÿ1 for chromium(III), respectively based on peak height measurements. The limit of determination, de®ned as the concentration giving ten times the standard deviation of the blank (measured on the basis of 2 mg lÿ1 solution), was 0.3 mg lÿ1 for chromium(VI) and 0.4 mg lÿ1 for chromium(III), respectively based on peak height measurements.

The precision (coef®cient of variation) at the 20 mg lÿ1 level was 2.5% and 1.4% for chromium(VI) and chromium(III), respectively, based on peak area measurements. The precision was improved compared to previous studies due to sample introduction by an autosampler. As there is still no reference material for chromium speciation available, the NIST standard reference material 1643c, Trace elements in water was used for the determination of accuracy. Six analyses of the material using standard addition resulted in a mean of 19.1 mg chromium(III) lÿ1 with a standard deviation of 1.0 mg lÿ1, which is in agreement with the certi®ed value of 19.00.6 mg lÿ1. There was no detectable chromium(VI) in the reference material, which has also been observed by others [8]. 3.4. Interferences The following ions were reinvestigated for interferences: Cu2‡, Mn2‡, Ni2‡, Co2‡, Al3‡ and Fe3‡. Concentrations of 10 and 20 mg lÿ1 of the interferents were added to solutions containing 20 mg lÿ1 of chromium(VI) and chromium(III). Examples of the resulting chromatograms are shown in Fig. 4. The separation system together with the addition of EDTA, which masked other metal ions, protected against most interferences. When adding 20 mg lÿ1 nickel(II) or manganese(II), no difference in signals were observed. Addition of cobalt(II) and copper(II) resulted in peaks in the solvent front but did not disturb the chromium signals. Addition of 10 mg lÿ1 iron(III) gave rise to a small shoulder on the chromium(VI) peak. Adding aluminium(III), resulted in a minor shift of the chromium(III) signal towards the front, but the peak area of the signal was almost unchanged.

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Fig. 5. Chromatogram of a dissolved patch test sample analysed by means of 2 standard additions of 20 and 40 mg lÿ1 chromium, respectively Three peaks appear ± front peak, chromium(VI) and chromium(III). (Correlation coefficient for chromium(VI) 0.9993).

Fig. 4. Result of addition of different potential interfering ions to a standard containing 20 mg lÿ1 chromium(VI) and chromium(III). 1a. No nickel, 1b. 10 mg lÿ1 nickel, 1c. 20 mg lÿ1 nickel. 2a. No copper, 2b. 10 mg lÿ1 copper, 2c. 20 mg lÿ1 copper, 3a. No aluminium, 3b. 10 mg lÿ1 aluminium, 3c. 20 mg lÿ1 aluminium; the dashed line is the blank signal of 20 mg lÿ1 aluminium.

When pH of samples was below 3, a signal in the front was observed. However, this signal was well separated from the chromium(VI) signal and caused no disturbance. 3.5. Applications The system has been used to examine the conversion of chromium(VI) to chromium(III) in biological

media as e.g. arti®cial gastric juice. It appears that the reduced chromate only partly appears as chromium(III) in the chromatograms. This ®nding is similar to ®ndings of Beere and Jones [16], who followed the decrease in chromate concentration in a simulated fresh water sample. Apparently, some of the chromium is converted to a species which is not retained on the column. The analytical system was used to examine the stability of patch test preparations used for examination for chromium contact allergy in clinical practice. The patches are prepared from a gel containing a dichromate solution. As only chromium(VI) is able to penetrate the skin, it is of importance that the chromate salt is not converted to chromium(III) during storage. The patches were dissolved in 1 M hydrochloric acid in an ultrasonic bath, followed by dilution with eluent and analysed by means of two standard additions. Only few of the patches showed any chromium(III) content, an example is shown in Fig. 5. The precision was 3.9% (nˆ6) on analysis of a patch containing 5.4 mg Cr(VI) cmÿ2. Hence, the method could be used for quality control of the patches.

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