Chromium speciation in waste waters by HPLC-ICPMS. Performance comparison between MassNeb® and Micromist® nebulizers

1. Introduction

Chromium is naturally found in rocks, soil, plants and animals, but can also be introduced into the environment as a result of human activity. Chromium toxicity, bioavailability and transport properties depend on the specific form in which element is present in the environment rather than its total amount. Cr can exist in several oxidation states from 0 to VI. The two forms of chromium commonly found in the environment are Cr(III) and Cr(VI).

The intermediate states: Cr(II), Cr(IV), Cr(V) are unstable products in oxidation and reduction reactions of trivalent and hexavalent chromium, respectively. In trace amounts, trivalent chromium (Cr (III)) is considered an essential nutrient that promotes insulin, sugar, and lipid metabolism. In contrast, hexavalent chromium (Cr (VI)) is toxic and can lead to respiratory tract, stomach damage, and intestinal irritation, anemia, and is known to be a human carcinogen.

Due to significant differences in the biochemical properties of both Cr species there is an obvious need for the accurate quantification.

Along with the improvement of analytical methods, speciation analysis has attracted more attention based on the use of HPLC-ICP-MS. In this study, we evaluate the performance of MassNeb® inert, robustness and durability nebulizer combined with a One-Piece High Pressure specific connector for speciation analysis by HPLC-ICP-MS in comparison with the conventional connection kit for speciation analysis by HPLCICP-MS when employing the Micromist® nebulizer for this purpose.
Chromium speciation in waste waters

2. Experimental

Reagents and solutions

All reagents employed in this Application Note were of the highest available purity.
Ultrapure water (18.2 MΩ cm-1, Millipore Milli-Q™, Merck Millipore, Saint Quentin in Yvelines, France) was used throughout the study. Ethylene diamine tetraacetic acid (EDTA, 99.995%, Sigma-Aldrich, France) was used as complexing agent for Cr(III). HNO3 and H2O2 (Suprapur) used as an eluent was purchased from Merck (Fontenay sous-Bois, France). Standard stock solutions (1000 mg L-1) of Cr(III) and Cr(VI) (Inorganic Ventures, Christiansburg, USA) were used to prepare the calibration levels. All standard mixture were spiked with EDTA to give a final concentration of 10 mM to help with the preservation of Cr(III).


Anion exchange chromatography analysis was carried on an Agilent 1260 HPLC system comprising a quaternary pump, autosampler and vacuum degasser was coupled to an Agilent 7900 ICP-MS. Chromium species were separated on an Agilent speciation column for testing chromium by HPLC-ICP-MS (4.6 mm x 30 mm, Part No G3268-80001). Samples and standard solutions were injected via a 200 µL sample loop. Temperature of the column was kept at 30°C during chromatographic separation. Chromium compounds were separated by employing an isocratic elution 5 mM EDTA (2Na)* – 5 mM NaH2PO4 /15 mM Na2SO4 , pH = 7.0 adjusted with NaOH. The flow rate of mobile phase was kept constant at 1.2 mL min−1 during chromatographic separation. Total time for separating of chromium species was 4 min.

The Agilent 7900 ICP-MS (Agilent Technologies) provides sensitive and specific analysis of Cr isotopes.
In this study, MassNeb® nebulizer is proposed as an appropriate nebulizer in speciation analysis based on the use of HPLC-ICP-MS instrument configuration using the One-Piece High Pressure for Speciation Analysis by HPLC-ICP (Figure 1) (Part Number: CN2530050, Ingeniatrics Tecnologías S.L.). On the other hand, the Agilent LC connection kit for speciation analysis by HPLC-ICP-MS (Part No: G1833-65200) was employed using Micromist nebulizer. Operating conditions for chromium species quantification optimized are indicated in Table I.

Chromium speciation in waste waters

Figure 1. High pressure connector ON / MS speciation 0.125 mm ID for HPLC-ICP-MS speciation analysis.

Conventionally, the internal standard is mixed with the calibration standards and samples using a Y connection, when one inlet nebulizer in employed.
However, the novel MultiNeb® has been developed which allows a high mixing efficiency between two liquids, miscible or immiscible, since the mixing takes
place under turbulent conditions of high pressure at the tip of the nebulizer. For internal standard introduction a tygon tubing for ISTD 0.19 mm i.d. was employed (Part No. G1820-65220, Agilent Technologies). The instrumental configuration employed, and the optimized operational conditions are shown at Table I.

Chromium speciation in waste waters

Table I. Operational conditions for 7900 ICP-MS and 1260 HPLC optimized for chromium speciation by HPLC-ICP-MS (Agilent Technologies).

EDTA was also included in the mobile phase to stabilize the Cr(III)-EDTA complex during chromatographic separation. Furthermore, it was determined that a pH of 7 was optimal for species stabilization and chromatographic speciation.
The EDTA present in the eluent could lead to the formation of a significant level of 40Ar12C polyatomic ions, which would contribute to the background signal at m/z 52. However, Agilent ICP-MS systems include the ORS collision/ reaction cell (CRC) that uses helium (He) collision mode with kinetic energy discrimination (KED) to control common polyatomic ions, including carbon-based interferences. When the cell is pressurized with He, polyatomic ions such as ArC+ are filtered out effectively, while analyte ions such as Cr+ are transmitted through the cell. He mode therefore minimizes the background signals from ArC polyatomic ions, allowing Cr to be measured at low concentrations at mass 52.

Sample Preparation

For the preservation of Cr species in the waste waters samples, EDTA (with final solution concentration of 10 mM) were added to the samples on collection help with the preservation of Cr(VI).

For the determination of the total Cr content in all samples studied, 20 mL was microwave digested in 2.0 mL concentrated HNO3 and 0.5 mL of 30 % w/w H2O2 in XP1500 vessels in CEM Mars5 microwave system. The samples were diluted to the final volume of 25 mL with ultrapure water. Each sample was prepared in triplicate. The resulting solution was filtered through Iso-Disc poly(vinylidene difluoride) filters (25 mm diameter, 0.45 µm pore-size). The total content of chromium was measured by ICP-MS using as internal standard scandium (Sc) to 1 µg L-1.

A blank containing the reagents used for the total chromium determination was run simultaneously with the sample preparation.

On the other hand, for speciation analysis, the sample extraction and clean-up procedures constitute a crucial step when analyzing biota samples, due to possible analyte losses, changes of the species or incomplete extraction of the chromium compounds, which may lead to poor or erroneous results. In this study, recollected samples were incubated 60 min at 60 ºC (for species complexation and extraction) by heating using a heating block system type DigiPREP MS (SCP Science, Courtaboeuf, France). Then, resulting solutions were diluted five times with the mobile phase and filtered through Iso-Disc poly(vinylidene difluoride) filters (25 mm diameter, 0.45 µm pore-size) prior to analysis by HPLC-ICP-MS.

3. Results and discussion

Chromatographic optimization

Chromium species were speciated under operational and experimental conditions optimized, previously described in less of 4 min. The resulting chromatogram is shown in Figure 2.

Figure 2. Chromatographic profile obtained for 1 µg Kg-1 (as Cr) of Cr(III) and Cr(VI) species. A) Chromatogram obtained using MassNeb® in combination with the OnePiece High Pressure for Speciation Analysis for HPLCICP (Part Number: CN2530050, Ingeniatrics Tecnologías S.L.); B) On the other hand, the Agilent LC connection kit for speciation analysis by HPLC-ICP-MS (Part No: G1833-65200) was employed using Micromist nebulizer.

In Figure 2, we can observe chromium species were speciated under operational and experimental conditions optimized, as previously described, in less of 4 min. Firstly, the results obtained show higher sensitivity using MassNeb® and the high-pressure connector designed by Ingeniatrics Tecnologias S.L. for this purpose, this could be attributed to the fact that MassNeb® nebulizer uses Flow Blurring nebulization technology instead of the traditional Venturi effect, as Micromist® nebulizer. This allows the generation of a very fine droplet aerosol with a narrow size distribution (most droplets are smaller than 10 μm), more efficiently desolvated and excited in plasma. Related to this, a greater proportion of the sample will reach the plasma, minimizing the proportion of larger droplet size aerosol that is drained to waste.
Table II. Experimental mean values for total chromium and Cr(III) and Cr (VI) species concentrations in the waste waters samples analyzed, as well as the SD and recoveries obtained for three replicates using HPLC-ICP-MS.
On the other hand, it has been demonstrated that the novel high-pressure connector for speciation analysis by HPLC-ICP-MS designed by Ingeniatrics Tecnologias S.L. present some advantages, such as minimize dead volume and peak broadening (higher chromatographic resolution and sensitivity), as a consequence of increased I.D. and perturbations in the pressure elution causing by interface connection kit normally employed for HPLC-ICP-MS coupling when Micromist® is used as nebulizer (Figure 2). In addition, this connector is simple to use, to allow quick, cheap, reliable and easy connection of your HPLC to your ICP instrument. Additionally, the method detection limits (MDLs) were determined by analyzing five replicate injections of the calibration blank and multiplying the standard deviation obtained by three. The results, shown in Table II, support the previously mentioned higher sensitivity.

Chromium speciation in waste waters samples

In order to validate the method performance in real waste waters samples a spike recovery test was performed using the mixed Cr species standard solution at 10 µg kg-1. The recoveries obtained were shown in Table II. The samples were donated by a control analysis laboratory located in Huelva (Southwest of Spain).

Table II summarizes the experimental registered values for total chromium and each species, as well as the standard deviation (SD) and recoveries results obtained for 3 replicates. Generally, the results reported when MassNeb® nebulizer was employed shown better precision and reproducibility in comparison with the results obtained with Micromist® nebulizer.

Accurate and sensitive determination of chromium species in waste waters was successfully demonstrated using anion exchange chromatography after conversion of Cr(III), which is cationic, to its anionic form by complexing with EDTA. The analysis is rapid, taking only about 4 minutes to complete, and is capable of measuring both species at concentrations less than 100 ng kg-1 (ppt).

4. Conclusions

In this study, it has been demonstrated that the novel MassNeb® nebulizer offers higher precision, sensitivity, signal stability and reproducibility in total chromium determination using ICP-MS and Cr(III) and Cr(VI) species quantification employing HPLCICP-MS in combination with the One-Piece High Pressure for Speciation Analysis by HPLC-ICP (Figure 1) (Part Number: CN253005, Ingeniatrics Tecnologías S.L.), a specific connector for OneNeb® or MassNeb® nebulizers (one liquid inlet) with a 50 mm PEEK capillary to connect the exit of the chromatographic column directly to nebulizer.

In this regard, the high-pressure connector designed by Ingeniatrics Tecnologias S.L. presents some advantages, such as resistance to blockage, fast washout, minimized dead volume and peak broadening (higher resolution and sensitivity).

Additionally, it is designed to be user-friendly, facilitating quick and easy connection of your HPLC system to your ICP instrument. This connector provides a convenient, cost-effective, reliable, and efficient solution for seamless integration of HPLC and ICP instrumentation.

Let's Make Something Great Together