Arsenic Speciation By Atomic Fluorescence Spectroscopy Biology Essay

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The occurrence of inorganic arsenic in food has received worldwide concern during recent years due to its toxicity and adverse health effects. It is known to be a human carcinogen and can cause skin and bladder cancer. Inorganic arsenic (Arsenite and Arsenate) are not only commonly found in the ground water, they are also found to be the predominant species in some food such as rice. Reliably monitoring of inorganic and organic arsenic level in our environment is the one of the most important task in order to assess its impact on human health.

Several extraction methods have been developed for the extraction of arsenic species from food and biological samples. In this report we were discuss different type of extraction method. But nitric acid based extraction method was found to be most suitable method for digestion of biological and non-biological samples. We can use different column for the speciation of inorganic arsenic, DMA, MMA and arsenobetaine. But anion-exchange liquid chromatography is one of the very popular technique for arsenic speciation. pH play an important role in speciation. Experimentally it is proof pH 6-7 give 95% of recovery and by using this mobile phase it is easy to separate As (iii) from As (v).

Glossary of Abbreviations


Atomic fluorescent spectrometry


High performance liquid chromatography


Hydride generation


 Monomethlyarsonic acid


Dimethylarsinic acid





As (III)


As (V)



As we know that arsenic is a very toxic element to humans, animals and plants {1}. Arsenic poisoning is a major public health problem because it is carcinogenic to humans. There are many countries such as Bangladesh, India, Japan and China etc. where arsenic is widely present in soils and the well water may contain as much as 300-4000 mg L_1 of arsenic {2}. Arsenic toxicology is a complex phenomenon because arsenic also considered as being an essential element. Generally two type of toxicity occurs in human being acute and sub-acute. The major cause of acute toxicity is ingestion of contaminated food or water and it required prompt medication. These are the common syndromes of acute such as dryness of the mouth and throat, dysphasia, colicky abnormal pain, projectile vomiting. Sub-acute arsenic toxicity act on the respiratory system, cardiovascular, nervous system and gastro-intestinal system. It may cause loss of appetite, nervous weakness, tingling of the hands and feet, jaundice and diarrhoea {3}

Basically arsenic is present in a different number of valency states and the level of toxicity depends on the chemical form of the arsenic like arsenic As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Inorganic arsenic species are more toxic than the organic ones {4}. Arsenic (V) is significantly less toxic than Arsenics (III), (Petrick et al., 2000; Styblo et al., 1999, 2000) {5}. In recent years there has been increasing researches in the speciation of these elements in environmental and biological samples and many methods have been developed, for the determination of the trace element. Atomic fluorescence is one of the best techniques for determination of arsenic because it is sensitive, selective, good linear dynamic range and less spectral interference. Especially in speciation of arsenic, we can use hydride generation coupled with AFS because arsenic can easily form volatile and covalent hydrides {6}. Generally boosted hollow cathode lamp (electrode discharge) is used as an excitation source. It excites the arsenic atomic vapour and argon hydrogen diffusion flames work as an atom reservoir {7}.

Atomic fluorescence spectrometry

Atomic fluorescence spectrometry (AFS) can be described simply as the absorption of energy of a particular wavelength by the free ground state analyte atoms. These atom are excited to higher energy levels and after some time it returns to the ground state (through radiation deactivation), they emit energy of a particular wavelength. It can measure with the help of atomic fluorescence spectrometry. Basically AFS has four main type of fluorescence: Resonance, Direct Line, Stepwise and Thermally Assisted Anti-stokes Fluorescence. Particularly in resonance Fluorescence, the wavelength of excitation is same as the wavelength of emission. This is a most intense and commonly occurring type of atomic fluorescence. In Direct Line and Stepwise Atomic Fluorescence, the excitation wavelength is shorter than that of emission and at last Thermally Anti-stokes Fluorescence, the wavelength of excitation is longer than that of emission. The intensity of the fluorescence radiation depends on different factors such as intensity of the excitation source, the quantitative efficiency of the process, the concentration of the atoms means atomiser and extent of any self-absorption in the atomiser {8}.

Sample introduction method may also enhance the sensitivity. We can use nebulisation technique for the introduction of sample but there is a drawback with this technique. It allows only 1-2% of the actual analyte into the detector. An alternative technique which is frequently used is vapour generation. In this technique analyte is directly converted into a vapour and it allows 100% of the analyte into the detector. This is helpful to increase the sensitivity. That technique is very popular because it has many advantages like it separates the analyte from the matrix thus reducing both spectral and matrix interferences. But it also has few disadvantages such as quenching and scattering effects. Quenching occurs when the excited atom collide with diatomic gas molecules, it may cause reduction in the fluorescence signal. Scattering can occur when particular material enter into the detector and interact with the excitation source and produce noise. To overcome these potential problems we can use argon gas instead of air or nitrogen, its help to reduce the quenching effect, and secondly, we can use vapour generation and gas liquid separation means. Which allows only gas and retain particulate material is introduced into the detector; it helps to avoid the scattering effects. This technique is very popular for the speciation of the arsenic and its give better results {15}.

High performance liquid chromatography (HPLC-AFS) for Arsenic speciation

In order to study the different species of arsenic, high - resolution speciation technique have to be used. For arsenic this means separation in HPLC mode combined with atomic fluorescent spectrometry. Generally, anion exchange column and C-18 column have been used for the separation of arsenic species such as As III, As V, DMA, MMA, and AsB.

3.1 Principle

Sample containing arsenic species is injected into the loop of HPLC valve then injected into mobile phase carrier stream by HPLC pump. After that it goes to the column where the species are separated. Acidify the solution prior to mixing with sodium boro hydrides. Those arsenic species present in sample react with the NaBH4 and form volatile covalent hydrides and continuously flow help to carry sample to gas-liquid separator which separate the volatile hydride from the liquid. Then the hydride are atomised in the hydrogen flame and different species of arsenic give different fluorescent signal after excitation from boosted discharge hollow cathode lamp {9}.

There are two type of arsenic categories present in our environment, one is reducible and other is non-reducible. Reducible form is generally present in water sample such as As(III), As(V), DMA or MMA. On the other hand sample such as urine usually contain non - reducible forms of arsenic for example arsenobetaine. Non-reducible forms of samples create more difficulty in determination process {10}. Millennium Excalibur by P.S.Analytical Kent (U.K) manufacture that instrument which is capable to detect arsenic in both form determination of reducible form is a simple process only need to use few reductant. But those sample containing non reducible arsenic species (Arsenobetaine and Arsenosugars) required photo oxidation treatment in this process first sample treat with oxidising agent and it break down the non reducing arsenic species then it will react with sodium boro hydride and then follow the above principle {11}.

3.2 Instrumentation and Apparatus

Generally, there is not too much difference between AAS and AFS instrument except that the light source and detectors are placed at right angles and a high intensity light source is required. Atomic fluorescence spectrometry instrument has been categorized into two parts, dispersive and non-dispersive. It depends on the type of wavelength. Usually low resolution monochromator is used in dispersive instrument but for continuum radiation source high resolution monochromator is required. On the other hand for non - dispersive instrument no need to use any monochromator, it makes the instrument simple in design and reduces the cost. Non-dispersive instrument can prone to interference because of the background emission from the atomizer. The basic requirements of atomiser are efficient and rapid production of free atoms with minimal background noise, low quenching properties and long residence time for the analyte in the optical path {12}.

This instrument consists of Hydride generation unit and Atomic fluorescence detector and HPLC system. Arsenic species are generally present in soluble forms. That's why we need to use liquid separation technique. It is also compatible with other element-specific detectors. pH play an important role in the chromatography separation process. According to a modern review, Anion exchange column totally depend on the pH of mobile phase. So we need to maintain the pH for the speciation of arsenic. Experimentally it is proof pH 6-7 give 95% of recovery and by using this mobile phase it is easy to separate As (iii) from As (v) {13}. Generally high performance liquid chromatography system consist of HPLC-Pump with 5ml/min stainless steel pump head, 6-port sample injector with 20-250 µl sample loop and a reverse phase HPLC-column {14}.

3.3 Reagents and standard

Generally, sodium tetrahydroborate and potassium chloride are used as a reductant. Need to prepare freshly each day do not keep in closed container because of pressure build up due to hydrogen evolution.

HCl is used as a reagent blank because it is best for hydride generation. Basically reagent blank represent the added constituents to the sample.

Quantitative stock solution of arsenic is commercially available and it is also suitable for preparation of standard. Usually stock solution is use for the calibration purpose.

We can use different type of mobile phase according to the column. Basically anion exchange column required K2HPO4, KH2PO4 or NaH2Po4, Na2HPo4 and for C-18 column we can use ammonium phosphate and tetrabuto ammonium hydride.



5.1 Sample digestion

Digestion methods can be categorised into three type's on-line digestion, off-line digestion and wet digestion by using perchloric acid.

5.1.1 On-line digestion

An online microwave digestion method is used for determination of biological and food samples. This technique is based on the same principal as off-line digestion. Now a day's microwave digestion method has become very popular because of more reproducible, enhance accuracy and less time consuming than other methods. We can reduce the risk of contamination by using this technique {22}.

On-line UV-photo-oxidation is also comes in a same category only the mode of application is different. The purpose of using this technique is to determine the low level orgenoarsenic species of MMA, AsB, DMA and AsC. This is a simpler method as compare to microwave digestion. Generally, low power ultraviolet lamp is used as a source of reaction energy. Potassium persulfate and sodium hydroxide is used as an oxidant {23}.

5.1.2 Off-line digestion

For determine of total arsenic off-line digestion method is one of the most reliable and easiest method. If arsenic is present in trace level in food this method is useful. In this method sample should be digested with HNO3 (heated at 120 degree centigrade for 2 hours) or HCl (heated at 65 degree centigrade for18 hours).

5.1.3 Wet digestion

Wet digestion methods with nitric-sulfuric-perchloric acid mixture (Francesconi et al., 1985; Le et al., 1992) and a nitric-perchloric-chloric acids mixture (Norim and Christakopoulas, 1982) have been reported to decompose arsenic compound to arsenate. Usually strong base is required to convert arsenobetaine to trimethylarsenic oxide. This method has now been used for the analysis of many thousands of samples for nitrogen, potassium, Arsenic, sodium and calcium in analytical laboratories. This method also reported poor precision and few errors. But it can be minimised by minor modification. The major drawback of this method is perchloric acid; strong oxidising agent but anhydrous and monohydrated perchloric acids are highly corrosive and readily forms explosive mixtures. Because of these hazards, when we use perchloric acid need to take more precautions like always handled under special fume hoods.

5.2 Sample pre-treatment

Concentration of total arsenic is not sufficient for environmental considerations. As we know that arsenic has different form which is present in our environment. The detection is normally based on the concentration of As (III). So we need to convert arsenic (V) to arsenic (III) by using pre-reductant. Potassium iodide is a very popular reductant of As (V) to As (III), Which can be used with ascorbic acid, in order to prevent the oxidation of iodide to triiodide by air. Potassium iodide can reduce arsenic only in a strong acidic media.

5.3 Hydride generation

Hydride generation atomic fluorescence spectrometry (HG-AFS) is a very popular sample derivatization method used for detection of inorganic arsenic. The main principle of hydride generation is to separate the analyte from the matrix and gas - liquid separator improve the efficiency and sensitivity of this technique. Hydride generation is helpful to investigate different type of analyte in the specific sample. The combination of hydride generation and fluorescent spectrometry is first invented by Tsujii and Kuga {7}.This method is also capable for differential determination of As (III) and As (V). Sodium borohydrate is acting as a reductant for As (V) as well as a hydride source. Generally generation of arsenic from Arsenic (III) is faster and it gives a great sensitivity than generation from AS (V). Some time transition metals might interfere with the determination of arsenic. l-cysteine is one of the best reagent which has to be very useful for preventing iron interferences {17}.

5.4 Arsenic speciation:

We can use HPLC separation technique for the determination of different kind of arsenic species. Several analytical systems based on HPLC-HG-AFS have been reported such as PSA 10.055 Millennium Excalibur (P.S.Analytical Kent , U.K.). This instrument consists of three different units like HPLC, Hydride generation unit and atomic fluorescent spectrometry. This system is capable for the speciation of arsenite, arsenate, dimethylarsinic acid (DMA), and monomethylarsonic acid (MMAA) {18}.Speciation methods based on the use of liquid chromatography coupled to atomic spectroscopy. The standard solutions of a mixture of arsenic species are injected directly into the HPLC column through the injection port. Separation is take place into the separation column after that solution of hydrochloric acid and then sodium borohydrate are introduced into the column. Hydrides are generate in a mixing coil and separated with the help of gas - liquid separator. The gaseous products are flushed from the gas/liquid separator by a controlled stream of the purge gas carries the gaseous products into the measurement system. The hydrogen-argon diffusion flame is maintained by externally introduced hydrogen. The gaseous products from the gas/liquid separator pass through a perma pure dryer system to the detector. Detector gives a signal. But always the first step is determining the total concentration of arsenic then arsenic (v) is calculated by the difference of the total inorganic arsenic and As (III) {19}.


The potential interfering effects is due to some foreign species. Those generally present in environmental sample. Hydride generation and detection are also affected by the matrix. Some elements like Na, Ca, Fe, Cu; Se was investigated on chromatogram, detection and hydride generation process. These elements are generally present in water and food samples. During detection "Na" and "Ca" may produce spectral interference because of their relatively low ionisation potential. Some time Fe, Cu and Se decrease the production of hydrides {19}. There are little chemical interference occurs during the process associated with vapour / hydride generation atomic fluorescent spectrometry. Some time few species present in sample, which suppress the vapour generation step but it can be control by modifying the instrumental and chemical conditions. As we know that the transition metal is generally reduced to its metallic state by NaBH4. This metallic species get dispersed into the solution and decompose the hydride and produce interference. It can be minimize by increasing the concentration of HCl. It is possible to keep the transition metal ion in solution, so reduce the interferent effect {20}.

Certified Reference Materials

We can determine the certainty or uncertainty of our result with the help of certified reference material and certified value. The method compares the difference between the certified and measured values with its uncertainty. Validation of measurement procedure is a one of the most frequent applications of certified reference materials. According to European Commission - Joint Research Centre Institute for Reference Materials and Measurements (IRMM) Belgium, after the measurement of a CRM the absolute difference between the mean measured value and the certified value can be calculated as: {21}

Δm = l Cm - CCRM l

Δm = absolute difference between mean

Measured value and certified value

cm = mean measured value

cCRM = certified value

As-speciation analys requires suitable reference materials to be available to verify accuracy and to meet quality-assurance needs. For quality control, the adoption of certified reference materials (CRM) is the best choice {13}. A number of CRM for total arsenic are available, although only a few are available for speciation. Several easily available CRM for arsenic, relating to foods, are as follows:

NIES CRM 9 Sargasso (certified value: 115 ± 9 mg kg−1),

BCR CRM 422 cod muscle (21.1 ± 0.5 mg kg−1),

NIST SRM 1566 oyster tissue (14.0 mg kg−1),

BCR CRM 279 Ulva lactuca (sea lettuce) (3.09 ± 0.20 mg kg−1),

NIST SRM 1548 total diet (0.20 ± 0.020 mg kg−1),

NIST SRM 1570a spinach leaves (0.068 mg kg−1),

BCR CRM 185 bovine liver (0.024 ± 0.003 mg kg−1),

NIST SRM 8433 corn bran (0.002 mg kg−1).

CRM are produced by several institutions such as National Research Council of Canada, Community Bureau of Reference EC, Brussels, Belgium (Standards, Measurements and Testing Programme), International Atomic Energy Agency, Vienna, Austria, National Institute of Environmental Studies, Tsukuba, Japan, and National Institute of Standards and Technology, Gaithersburg, Maryland, USA {10}.


Arsenic contamination in environment is a worldwide problem because it is very toxic and carcinogenic to humans. That's why; it has become a challenge for the world scientists. In recent years there has been increasing researches in the speciation of these elements in environmental and biological samples and many methods have been developed for the speciation of arsenic, but atomic fluorescence spectrometry has become one of the most important analytical tools for the analysis of trace elements like arsenic. This is because of its excellent sensitivity, selectivity, and high accuracy. AFS is also capable to detect both types of species: organic and inorganic. It can be easily coupled with different instrument like HPLC, Hydride generator apparatus and get better result. Certified reference value play an important role in speciation process, with the help of CRM and CRV we can determine the percentage of accuracy and uncertainty of our results.