Determination of Mercury in Milk by Cold Vapor Atomic Fluorescence

In the Laboratory

Determination of Mercury in Milk by Cold Vapor Atomic Fluorescence: A Green Analytical Chemistry Laboratory Experiment Sergio Armenta* and Miguel de la Guardia Department of Analytical Chemistry, University of Valencia, 50 Doctor Moliner Street, E-46100 Burjassot, Valencia, Spain *[email protected]

Green analytical chemistry attempts to change the mentality and practices of analytical chemists by emphasizing considerations about the toxicity and hazards of reagents, the reduction of reagents and solvents, and the minimization and decontamination of analytical wastes (1, 2). The introduction of green analytical chemistry principles to undergraduate students is desirable. It is appropriate to introduce didactical green experiments in analytical chemistry laboratories and to define green parameters in the method selection. In this experiment, analysis of mercury (Hg) concentration in commercially available milk is proposed to introduce undergraduate students the following concepts: (i) reagent toxicity and operator risks involved in method application, (ii) quantification of reagents consumed in method evaluation, (iii) quantification of waste generation and the aspects regarding their treatment, (iv) atomic fluorescence spectroscopy (AFS) as a fast and sensitive method for Hg determination at the trace levels, (v) characteristics of Hg determination, (vi) sample pretreatment for Hg trace determinations, and (vii) practice of online passivation and reduction of wastes. The Mercury Problem Mercury is a toxic element present in the environment at low concentration levels because of the natural degassing of the earth's crust. However, the most important sources of mercury are the industrial activities involving Hg extraction and the use of Hg compounds in different fields from mining to amalgam fillings in human teeth (3). All chemical forms of mercury can cross the placental barrier and also are secreted to milk; thus, it is important to control the presence of mercury in milk samples. Milk is important in human nutrition especially during the developing years, and thus, there is a responsibility to minimize the Hg concentration in human milk, infant formulas, and commercially available cow and goat milk. The concentration reported for Hg in human milk varied from less than 0.2 to 6.86 μg L-1 (4) and from 0.25 to 11.7 μg L-1 (5). The high quantities of Hg in mothers' milk result mostly from the presence of amalgams filings in mothers' teeth and fish consumption. The average Hg concentration found in the human milk at the Faroe Islands was 2.45 μg L-1 (6) where the typical diet includes whale meat and that in Sweden only 0.6 ng g-1 (7). In infant formula Hg levels varied from 0.4 to 2.5 μg L-1 (4), and in commercially available cow and goat milk, the Hg content varied from 0.1 to 0.48 μg L-1 (8). 488

Journal of Chemical Education

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Atomic Fluorescence Spectrometry Determination of the low concentrations of Hg in milk requires the use of highly sensitive analytical procedures such as atomic fluorescence spectrometry (AFS). Atomic fluorescence is the optical emission from gas-phase atoms that have been excited to higher energy levels by absorption of electromagnetic radiation. To avoid high background levels, the detector and the source are mounted at a 90° angle and a filter system can be used for wavelength selection. The AFS is a simpler process than the molecular fluorescence because of the absence of molecular transitions and the resonance between the excitation and emission wavelength, 254 nm for Hg. However, difficulties arise because AFS needs the atoms in the ground state and in the gaseous form. The available AFS instrumentation requires the online reduction of Hg ions and Hg compounds to Hg0 vapor. To generate Hg0 vapor, NaBH4 or SnCl2 could be used. However, because of the toxicity of those compounds, SnCl2 is strongly recommended over NaBH4 for cold vapor (CV) Hg generation. The experimental setup employed for CV-AFS determination of Hg is shown in Figure 1. Sample Pretreatment Mercury in milk can be present as Hg(II) or methylated species; thus, it is necessary to treat the samples to obtain an acidic solution of mercury ions. Sonication of milk with aqua regia (HCl:HNO3) for 10 min is enough to extract Hg from the milk. The addition of antifoam A, hydroxylamine hydrochloride solution, HCl, KBr, and KBrO3 before dilution provides an acid slurry of milk suitable to feed into the AFS system (8). Sonication Sonication is a room-temperature low-energy sample treatment that is faster than the wet- or dry-ashing classical treatment used to obtain solutions from food samples. Milk is sonicated in a closed tube to avoid the contact of acid vapor with the analysts. The selection of an HCl:HNO3 mixture permits the quantitative extraction of Hg in a short period of time without matrix destruction, thus, simplifying the matrix effects on the following analytical steps. Soft sample treatments save energy, time, and reagents. The main goal is to make the pretreatment methods greener, but additionally, it is useful to avoid matrix interferences in the measurement step, demonstrating that it is possible to green methods and to obtain accurate and precise results.

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Vol. 88 No. 4 April 2011 pubs.acs.org/jchemeduc r 2011 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100578g Published on Web 02/14/2011

In the Laboratory Table 1. Instrumental Conditions Employed for the Hg Determination in Milk by CV-AFS Parameter

Figure 1. Schematic diagram of a CV-AFS system employed for Hg determination in milk and for the online passivation of wastes.

Sample sonication does not decompose the matrix and in the case of milk only protein denaturization is observed. The Hg remains in the liquid phase and it is unnecessary to do a phase separation. After sonication, an acidic slurry is obtained from which the Hg0 can be generated and measured online in the AFS system. Antifoam A is added to avoid foam formation during the Hg0 generation by reaction with SnCl2. Hydroxylamine and KBr/KBrO3 are added to avoid the oxidation effect of HNO3 and the possible reduction of Hg before the addition of SnCl2. The addition of the reagents ensures the release of Hg during the measurement step and stability of the samples and standards for several hours. Cold Vapor Generation The experimental generation of Hg0 and its AFS determination involves vapor measurement at room temperature. Free mercury atoms in a carrier gas are excited by an ultraviolet light source at a wavelength of 254 nm, emitting fluorescence at the same wavelength to return to the ground state. Several analytical methods based on CV-AFS have been approved by the United States Environmental Protection Agency. For instance, US EPA Method 245.7 (9) is commonly used for Hg determination in water using CV-AFS. Online Decontamination of Wastes The liquid effluent obtained from the CV-AFS system is very acidic. The merging of this waste with a solution of Fe(III) and NaOH provides the online co-precipitation of the heavy metals remaining in solution and a neutralization of the waste acidity. The neutralization of the acidic waste to a pH around 7.5 with NaOH generates the precipitation of iron as Fe(OH)3 and the heavy metals present in the waste stream precipitate or coprecipitate with iron, providing a reduced volume of a solid waste in which toxic trace elements are surrounded by a Fe(OH)3 cover. This simple treatment reduces the quantity of toxic wastes to be treated from the liter scale of diluted solutions to a gram scale of passivated residues. Apparatus and Reagents A CV-AFS system equipped with a liquid-gas phase separation chamber and a drying unit is necessary for Hg

r 2011 American Chemical Society and Division of Chemical Education, Inc.

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Quantity

Wavelength

254 nm

Measurement mode

Peak height

Argon flow rate

330 mL min-1

Air flow rate

2.5 L min-1

Sample flow rate

4.0 mL min-1

SnCl2 flow rate

4.0 mL min-1

Reaction coil length

100 cm

determination. (We used a PSA Millennium Merlin 10025 from PS Analytical, Orpington, U.K.) An ultrasound water bath with at least 50 W power and 50 Hz frequency is necessary for sample treatment. The 2.5% (m/v) SnCl2 3 2H2O solution is prepared by dissolving the salt in 50 mL concd HCl, heating for 10 min, and diluting with water to 100 mL. The 10% (m/v) hydroxylamine hydrochloride solution is prepared by dissolving the salt in distilled water. The 0.1 M KBr and 0.02 M KBrO3 solutions are prepared (daily) by dissolving the corresponding salts in distilled water. A solution of 1000 mg L-1 Hg(II) is used as stock standard solution. A 100 μg L-1 Hg(II) dilution is prepared weekly and is used to prepare the corresponding calibration solutions from 0.02 to 1 μg L-1 Hg(II) in 3 M HCl, 8 mM KBr, 1.6 mM KBrO3, and 1% (m/v) hydroxylamine hydrochloride medium. The carrier blank solution is prepared by adding the appropriate quantity of HCl to obtain 3 M acid medium and the same quantities of KBr, KBrO3, and hydroxylamine hydrochloride as employed for standard solutions. The 40 mg L-1 Fe(III) solution is prepared by dissolving FeCl3 3 6H2O in acidified distilled water. The 6 M NaOH is prepared by dissolving NaOH pellets in distilled water. Procedure A small volume, 2 mL, of sample is weighed accurately and 2 mL of aqua regia is added to the vessel. After shaking in an ultrasound water bath for 10 min, 0.5 mL of antifoam A, 2.5 mL of 10% (m/v) hydroxylamine solution, and 6.25 mL of concd HCl are added to the mixture. Finally, 2 mL of 0.1 M KBr and 2 mL of 0.02 M KBrO3 are added and the mixture is diluted to 25 mL. The obtained slurry can be directly introduced in the AFS system (8). Hg concentration in milk samples is calculated by interpolation of the sample signals in a calibration line obtained by the measurement of Hg solutions from 0.02 to 1 μg L-1 Hg(II) in the same experimental and instrumental conditions as the samples. The instrumental conditions are summarized in Table 1. Hazards Concentrated hydrochloric and nitric acids are corrosive and harmful if inhaled or absorbed through the skin. Concentrated nitric is also a strong oxidant. Sodium hydroxide pellets are caustic and irritate the skin upon contact. Hg is highly toxic. Long-term exposure to the metal may be fatal. Inhalation may

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Vol. 88 No. 4 April 2011

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In the Laboratory Table 2. Concentration of Hg in Commercial Milks

Table 4. Green Parameters of the CV-AFS Method for the Determination of Hg in Milk

Mercury Concentration/(ng g-1)

Sample

Green Parameters

CV-AFS Hg Determination

Full-cream cow milk

0.33 ( 0.03

Sample consumptiona

Partially skimmed cow milk

0.22 ( 0.01

Reagent Consumption

Skimmed cow milk

0.10 ( 0.02

Hydroxylamine hydrochloridea

2.5 g

Full-cream cow milk with Ca

0.21 ( 0.03

Aqua regiaa

200 mL

Partially skimmed cow milk with propolis

0.59 ( 0.03

Antifoam Aa

50 mL

Vegetable-base full-cream milk (mainly from soy)

0.17 ( 0.02

HCla

∼700 mL

Powdered cow milk Semi-skimmed goat milk

a

KBr

2.4 g

0.25 ( 0.02

KBrO3a

0.7 g

0.48 ( 0.04

SnCl2 3 2H2Oa

18 g

Table 3. Figures of Merit of the CV-AFS Method for the Determination of Hg in Milk Analytical Figures of Merit

CV-AFS Hg Determination

LODa

-1

0.00375 ng g

c

RSD

1.5% 0-1.5 μg L-1

Recovery

93-101%

Sampling time

120 s

a

LOD is the limit of detection established as 3 times the standard deviation of a blank measurement. b LOD regarding the original sample. c RSD is the relative standard deviation of nine independent measurements of a 0.05 μg L-1 Hg solution.

lead to liver, kidney, and central nervous system damage. There is a danger of cumulative effects and it is harmful by ingestion and by skin contact. SnCl2 and FeCl3 3 6H2O may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Hydroxylamine hydrochloride is corrosive, causes burns to any area of contact, and is harmful if swallowed or inhaled. KBrO3 is a strong oxidizer and is harmful if inhaled or absorbed through skin. The material safety data sheets (MSDS) of the aforementioned reagents are available free of charge at Sciencelab.com, Inc. (10).

The laboratory experiment objective is to obtain mercury data in commercially available milk but also offers an opportunity to evaluate the main figures of merit of the methodology (accuracy, precision, limit of detection, etc.) and the green parameters of the methodology (volume of wastes and consume of reagents). The student data obtained in the laboratory are shown in Table 2. Very low concentrations of Hg, from 0.10 to 0.59 ng g-1, are found in the commercially available samples. The main analytical figures of merit of the methodology are shown in Table 3. The precision, as the relative standard deviation of five independent analysis of a same sample, is 1.5% and the limit of detection is 0.3 ng L-1, being 3.75 pg g-1 for the original sample. These data clearly indicate that the methodology is suitable for Hg determination in milk samples. Additional green parameters should be considered for the method's evaluation (Table 4). Those parameters concern the sample and reagent consumption, waste generation, and sample


480 mL

Waste generation with passivation

17 g

Ar consumedb

1730 mL

Throughput

32 h-1

throughput, which affect the green nature, cost, and analysis speed of the method. It is important to note that the strong reduction in reagent consumption and the passivation of wastes avoid extra costs in the method use, thus, providing economical opportunities for laboratories. Conclusions The proposed experiment can be employed to introduce green analytical chemistry principles to undergraduate students by Hg determination in milk by AFS and the online passivation of analytical wastes. The method was designed to feature two key requisites of a green analytical method: use of less toxic reagents and waste management. The inclusion of environmentally friendly experiments in the undergraduate curricula will contribute to the development of environmentally conscious new generation chemists. Literature Cited

Results and Discussion

490

b

Sample and reagent consumed corresponding to the analysis of 100 samples. b Waste generated and Ar consumed established for a 1 h working period.

0.3 ng L-1

Linear range

Waste generation without passivationb

a

b

LOD

1000 mL

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1. Armenta, S.; Garrigues, S.; de la Guardia, M. TrAC, Trends Anal. Chem. 2008, 27, 497–511. 2. Keith, L. H.; Gron, L. U.; Young, J. L. Chem. Rev. 2007, 107, 2695– 2708. 3. Leopold, B. R. Manufacturing Processes Involving Mercury. In Use and Release of Mercury in the United States. National Risk Management Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH. http:// www.epa.gov/nrmrl/pubs/600r02104/600r02104prel.pdf, 2002, Chapter 3. 4. Drasch, G.; Aigner, S.; Roider, G.; Staiger, F.; Lipowsky, G. J. Trace Elem. Med. Biol. 1998, 12, 23–27. 5. Drexler, H.; Schaller, K. H. Environ. Res. 1998, 77, 124–129. 6. Grandjean, P.; Weihe, P.; Needham, L. L.; Burse, V. W.; Patterson, D. G.; Sampson, E. J.; Jorgense, P. J.; Vahter, M. Environ. Res. 1995, 71, 29–38. 7. Oskarsson, A.; Schutz, A.; Skerfving, S.; Hallen, I. P.; Lagerkvist, B. J. Arch. Environ. Health 1996, 51, 234–241.


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In the Laboratory

8. Cava-Montesinos, P.; Rodenas-Torralba, E.; Morales-Rubio, A.; Cervera, M. L.; de la Guardia, M. Anal. Chim. Acta 2004, 506, 145–153. 9. U.S. Environmental Protection Agency, Office of Water, Office of Science and Technology, Engineering and Analysis Division (4303), EPA-821-R-05-001, Method 245.7, “Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry”, Washington, DC 20460, 2005.


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10. Material Safety Data Sheet Listing. http://www.sciencelab.com/ msdsList.php.

Supporting Information Available Instructions for the students; notes for the instructor. This material is available via the Internet at http://pubs.acs.org.


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Determination of Mercury in Milk by Cold Vapor Atomic Fluorescence

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