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Arsenic Speciation Analysis using High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry

BY: Dr. Suresh Kaushik | Category: Toxicology | Submitted: 2015-12-09 09:47:03
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Article Summary: "Speciation is the analytical activity of identifying and/or measuring in a sample the quantity of one or more individual chemical species. Arsenic has different toxicological properties dependent upon both its oxidation state for inorganic compounds as well as the different toxicity levels exhibited for organic arsenic compound.."

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Arsenic has different toxicological properties dependent upon both its oxidation state for inorganic compounds as well as the different toxicity levels exhibited for organic arsenic compounds. The field of arsenic speciation analysis has grown rapidly in recent years, especially with the utilization of high-performance liquid chromatography (HPLC) coupled to Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), a highly sensitive and robust detector system. Complete characterization of arsenic compounds is necessary to understand intake, accumulation, transport, storage, detoxification and activation of this element in the natural environment and living systems. The trivalent and pentavalent forms are the most common oxidation states. From both the biological and the toxicological points of view, arsenic compounds can be classified into three major groups such as inorganic arsenic compounds, organic arsenic compounds and arsenic gas. The most common trivalent inorganic arsenic compounds are arsenic trioxide, sodium arsenite and arsenic trichloride. Pentavalent inorganic compounds include arsenic peroxide, arsenic acid and arsenates such as lead arsenate and calcium arsenate. Common organic arsenic compounds are arsanilic acid, methylarsonic acid, dimethylarsinic acid (cacodylic acid) and arsenobetaine. Arsenic trioxide is only slightly soluble in water, in sodium hydroxide it forms arsenite and with concentrated hydrochloric acid it forms arsenic trichloride. Sodium arsenite and sodium arsenate are highly soluble in water. Interchange of valence state may occur in aqueous solutions depending on the pH and on the presence of other substances which can be reduced or oxidized. Arsenic is mainly transported in the environment by water. In oxygenated water arsenic usually occurs as arsenate but under reducing conditions e.g. in deep well-water, arsenates predominates. In water the methylation of inorganic arsenic to methyl and dimethyl arsenic acids is associated with biological activity. In oxygenated soil inorganic arsenic is present in the pentavalent form. Under reducing conditions, it is in the trivalent form.

Speciation Analysis

This is the analytical activity of identifying and/or measuring in a sample the quantity of one or more individual chemical species. Speciation is a growing field of analysis that is indispensable for accurately understanding the true nature of trace elements in the environment, industrial processes and biochemical pathways. Toxicity issues are the main driving force behind legislation and speciation analysis. Over the last two decade elemental speciation has become an established field of analysis as the scientific community recognizes that total element concentrations cannot provide the information necessary to draw valid conditions in a number of domains. The need to define and measure chemical species of an element lies in the fact that physiological factors such as toxicity, bioavailability, mobility and reactivity are dependent on the specific form of an element. Chemical species can be free ions, inorganic complexes, organometallic compounds, biomolecules and structures of high molecular mass.

Speciation analysis is performed in three distinct stages, namely sample preparation, separation of the chemical species and detection. Due to the wide range of sample matrices and chemical species a number of different methodologies for sample preparation and species separation have been developed. Speciation analysis demands a soft extraction technique in comparison to digestion methods used for total elemental analysis. Aqueous, enzymatic or solvent extraction is employed with the aid of agitation and heat. Ultrasonication, microwave extraction or accelerated solvent extraction are some popular techniques.

HPLC is the technique of choice in modern speciation analyses due to their resolution and the ease with which they are coupled to ICP-MS, allowing for on-line separation and detection. ICP-MS is often favoured as element-specific chromatographic detector due to its multi-element and multi-isotopic detection capability. For this, compatible HPLC mobile phase flow rates allow for a coupling connection from the outlet from the HPLC column directly to the ICP-MS nebulizer and chemical species are separate in accordance with their affinity to a mobile and stationary phase component. ICP-MS instruments with quadrupole analyser are the most popular for speciation analysis due to their robust nature, small footprint, low cost and their adaptability to changing configurations. ICP-MS with collision cell technology is typically operated using kinetic energy discrimination (KED) approach using an inert collision gas such as helium but reactive gases such as hydrogen or ammonia may also be used to attenuate specific polyatomic interferences. Commercially available speciation software and kits provide fully automated analysis and flexible chromatographic peaks integration features for a user-friendly analytical solution.


Speciation analysis has been perceived a highly sophisticated analytical tool due to the complexity of the samples but advances in sample preparation, separation science and analytical instrumentation have now greatly improved the robustness, accuracy and sample throughput capabilities for speciation analyses. The high specificity and sensitivity of the HPLC-ICP-MS technique provides scientists in the environmental and occupational exposures with a comprehensive information sources to monitor and maintain process control and to ensure compliance with key legislation.


1. European Virtual Institute of Speciation Analysis,

2. Ishinishi, N et al. (1986) Arsenic in: Friberg L., Nordbag GF and Vouk BV, ed. Hand book of the toxicology of metals, Vol. II, Amsterdam - New York-Oxford, Elsevier.

3. McSheehy, S and Nash, M; (2006) Elemental Speciation Analysis using ICP-MS, LabPlus International.

4. United States Environmental Protection Agency,

About Author / Additional Info:
A Researcher with Ph.D. in Molecular Biology and Biotechnology

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