A Solvent Microextraction Approach for Environmental Analysis:Colorimetric Assay for Phosphorus Determination in Natural WatersFrancisco Pena-Pereira, Marta Costas, Carlos Bendicho, and Isela Lavilla*

Departamento de Química Analítica y Alimentaria, Universidad de Vigo; As Lagoas-Marcosende s/n, 36310 Vigo, Spain

*S Supporting Information

ABSTRACT: We describe a hands-on experience designed forstudents in an upper-division undergraduate analytical laboratory forthe UV−vis spectrophotometric determination of phosphorus innatural waters using a current microextraction technique. Themethod is based on the formation of a colored ion pair and its sub-sequent extraction in a microdrop of organic solvent. The proposedlaboratory experiment allows the students to gain experience indeveloping a green sample preparation approach. Furthermore, theextraction of a colored product allows them to better understanddifferent issues that govern solvent microextraction. Optimization ofsome experimental parameters and the chemical analysis of naturalwaters are also addressed in this laboratory experiment.

KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Environmental Chemistry, Laboratory Instruction,Hands-On Learning/Manipulatives, Microscale Lab, Quantitative Analysis, Separation Science, UV−vis Spectroscopy

Solvent extraction (SE) is employed to perform educationalexercises in undergraduate laboratories of chemistry and

chemical engineering.1 In analytical procedures, SE allows theseparation and preconcentration of target compounds as wellas an efficient sample cleanup. In spite of this, it is not free fromlimitations; for example, large volumes of organic solvents areusually consumed, tedious procedures are involved, and largequantities of waste are generated. Therefore, when SE isconsidered from the standpoint of green analytical chemistry,its general applicability is questionable. In the last years,different efforts have been devoted to the achievement ofmore efficient and greener alternatives to SE. Solvent micro-extraction techniques (SME) such as single-drop microextra-ction (SDME)2 and dispersive liquid−liquid microextraction,3

among others, are nowadays considered powerful analyticaltools, especially when a minimum organic solvent consumptionalong with a high enrichment factor are pursued.In recent years, few experiments using microextraction

techniques have appeared in this Journal,4−10 and to the bestof our knowledge, there is only a single laboratory experimentaddressing an SME technique, specifically headspace SDME.10

However, this approach requires significant staff and studenttraining, making it troublesome to obtain results in a fewworking sessions. Thus, we propose a laboratory experimentbased on the use of directly suspended droplet microextraction(DSDME), that is, an SME technique first introduced byYangcheng et al. in 2006,11 involving tasks that can be easilyaccomplished by untrained personnel such as students.DSDME is used in this work for the determination ofphosphorus in natural waters.12 When natural water is mixedwith a solution containing molybdate and malachite green, a

colored ion pair is formed between molybdophosphate andmalachite green. Upon addition of a low-density organic solventsuch as methyl isobutyl ketone (MIBK), the color ion pair canbe extracted.This experiment was designed to introduce students into the

philosophy of green analytical chemistry by using SME, anenvironmentally friendly alternative to SE. Students are in-volved in the development of a miniaturized methodology withthe assessment of the main experimental parameters that affectthe extraction process using univariate optimization, prepara-tion of the phosphorus calibration curve, and determination ofthe corresponding figures of merit. Thus, they can evaluate thegreenness of the method without compromising the analyticalcharacteristics, that is, using low volumes of organic solventswith the minimum amounts of waste and speed. The moti-vation of the students increases when they deal with the ana-lysis of environmental samples. Therefore, this experiment canlead to, or be preceded by, a discussion of environmental con-cepts and the biogeochemical cycling of phosphorus. Inaddition, students become familiar with other SME approachesto better understand the theoretical aspects of SME.This experiment was successfully carried out by students,

receiving a positive feedback from both students and instructor.It was performed in two laboratory sessions of 4 h in an upper-level instrumental analysis course. Results and questions pro-posed in the lab documentation were discussed in a separateclassroom session with particular emphasis on comparison with

Published: January 9, 2014

Laboratory Experiment

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© 2014 American Chemical Society andDivision of Chemical Education, Inc. 586 dx.doi.org/10.1021/ed300855t | J. Chem. Educ. 2014, 91, 586−589

classical SE procedures. The use of very small volumes of toxicreagents was highlighted by students.

■ EXPERIMENTAL DETAILS

Chemical Reaction

The reaction between phosphorus and molybdate in acidicmedium gives rise to the formation of 12-molybdophosphate(eq 1), which forms a colored ion pair with a cationic tri-phenylmethane dye such as malachite green (MG+) (eq 2)

+ →H PO 12MoO H PMo O3 4 3 3 12 40 (1)

+ → ++ + − +H PMo O MG (MG )(H PMo O ) H3 12 40 2 12 40(2)

This colored ion pair can be extracted into an organic solvent(eq 3):

⇌+ − + −(MG )(H PMo O ) (MG )(H PMo O )2 12 40 aq 2 12 40 org

(3)

Theoretical aspects of solvent microextraction are consid-ered. Further information can be found in the SupportingInformation.

Apparatus

A commercial microvolume UV−vis spectrophotometer(Nanodrop ND-1000 spectrophotometer, Thermo Scientific,Wilmington, DE, U. S. A.) was employed in this work;however, the procedure can be modified to use conventionalUV−vis spectrometers in undergraduate teaching laboratories.Thus, commercially available micro or ultramicro cuvettescompatible with conventional spectrophotometers could beused for phosphorus quantification, even though some dilutionof the enriched drop would be unavoidable after the extrac-tion process. In this work, an Uvikon XS UV−vis spec-trophotometer (Secomam, Domont, France) equipped with50 μL cells was employed as an alternative. Other microvolumeUV−vis spectrometric systems also applicable in combinationwith the DSDME procedure can be found in the literature.13

Measurements were made at 627 nm. A magnetic stirrer(P-Selecta, Barcelona, Spain) with selectable stirring rates in therange 60−1600 rpm was used to carry out extractions.

DSDME Procedure

A 5 mL natural water sample was placed in a 7 mL glass vialcontaining a magnetic stir bar (10 mm × 3 mm). Then, 500 μLof 4.5 M H2SO4 and 500 μL of the mixed reagent solution wereadded. The stirring rate was set at 1200 rpm, and 100 μL of4-methyl-2-pentanone (MIBK) was carefully added to thestirred sample. During the extraction process, the sample vialwas capped. After 5 min, the cap was removed and 2 μL of thecolored drop were collected with a Hamilton syringe anddeposited onto the lower pedestal of the microvolume UV−visspectrophotometer for analysis.All glassware was previously cleaned with hot hydrochloric

acid (0.5 M) and then rinsed several times with water. Theinstructor prepared the mixed reagent solution and the sulfuricacid solution before class. The mixed reagent solution consistsof an aqueous solution containing ammonium heptamolybdatetetrahydrate, malachite green oxalate, tartaric acid, and sulfuricacid. The preparation of the mixed reagent solution is describedin the lab documentation. The DSDME procedure is shown inFigure 1.

■ HAZARDSSulfuric acid is extremely corrosive and highly toxic, beingharmful or fatal if swallowed. Skin contact can cause severeburns. Diluted sulfuric acid solutions must be prepared bycarefully adding acid to water, not the reverse. Hydrochloricacid is irritant and corrosive and can be very hazardous in caseof ingestion or skin or eye contact. Hot hydrochloric acid cancause both thermal and acid burns. Malachite green is harmfulin case of ingestion and slightly hazardous in case of inhalationor skin or eye contact. Severe overexposure to malachite greenmay be fatal. MIBK is flammable and can lead to potential acutehealth effects in case of skin or eye contact, ingestion, orinhalation. Methanol is hazardous in case of skin or eye contact,ingestion, or inhalation Safety glasses, lab coat, and gloves aremandatory for personal protection.

■ RESULTSSeveral variables can affect the performance of the DSDMEmethod.11 In this laboratory experiment, students evaluated theimpact of three relevant parameters, namely, organic solventvolume, stirring rate, and extraction time, by carrying outunivariate optimizations. The results of the optimizations areshown in Figure 2. According to the obtained results, studentsshowed that the optimal conditions were as follows: 100 μL oforganic solvent, 1200 rpm of stirring rate and 7 min ofmicroextraction time. Nevertheless, a reduced extraction time(5 min) was selected in this work in order to increase thesample throughput, bearing in mind that complete equilibriumneed not to be attained for accurate and precise analysis, aspointed out in the literature.14

A calibration curve for phosphorus determination was builtunder optimal conditions by subjecting ten blanks and fivestandard solutions of phosphorus (ranging from 0.1 to 1.5 μM)to the described procedure. Corrected absorbance values werecalculated by subtracting the blank absorbance from theabsorbance values of standards and samples. The calibrationcurve was obtained by plotting the corrected absorbance versusthe concentration of phosphorus. The equation for the calibra-tion curve obtained by students was Abs =0.4605[P] + 0.0031,

Figure 1. DSDME procedure (clockwise from top left): Addition ofextractant phase to the stirred aqueous sample; microextractionprocess after 3 min; microextraction process after 5 min; deposition ofthe enriched drop for absorbance measurement by UV−vis micro-spectrophotometry.

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where Abs is the corrected absorbance and [P] is the con-centration of phosphorus (μM). The students obtained theanalytical figures of merit of the method. Under optimalconditions, the students obtained the limits of detection (LOD)and quantification (LOQ) of 9.4 nM and 31.4 nM of phos-phorus, respectively. The precision, obtained from five con-secutive extractions and expressed as relative standard deviation(RSD), was 4.5%. Alternatively, the students obtained theanalytical figures of merit of the method using the conventionalUV−vis spectrophotometer equipped with 50 μL cells bydilution of the microdrop with methanol. Under these con-ditions, the equation for the calibration curve was Abs=0.1695[P] + 0.0003. The LOD and LOQ were 15.5 and51.7 nM, respectively, and the precision of the method was4.2%. The calibration curves obtained by the students areshown in Figure 3. Further information on the calculations toobtain the analytical characteristics can be found in theSupporting Information.

Finally, students applied the analytical method to the deter-mination of dissolved reactive phosphorus (DRP) in water sam-ples provided by the instructor. DRP, defined as the fraction ofphosphorus that responds to chemical reactions without pre-vious hydrolysis or oxidation after filtration through 0.45 μmmembrane filters,15 was determined in three different watersamples, namely, river, well, and rainwater. Each sample wasanalyzed in triplicate. A summary of the students’ results isshown in Table 1.

■ CONCLUSIONSThis experiment successfully attracted upper-level analyticalchemistry students to green analytical chemistry topics throughnew extraction approaches. The students realized in practicethe benefits that miniaturized methods provide in the ana-lytical lab as compared to conventional sample preparation ap-proaches such as solvent extraction.In addition, the present experiment allows the students to

improve their skills on derivatization and miniaturized samplepreparation techniques in analytical chemistry. The extractionof a colored ion pair allows them to understand theoreticalaspects of liquid phase microextraction techniques more easilyby studying the effect of relevant parameters on extraction.

■ ASSOCIATED CONTENT*S Supporting Information

Instructions for students include background information(theoretical aspects of the microextraction technique, chemistryinvolved in the colorimetric method for phosphorus determi-nation, and biogeochemical cycle of phosphorus), as well as tipsfor the development of the laboratory experiment and pre-paration of the laboratory report. Instructor notes include therequired apparatus and glassware, reagents, and preparation ofsolutions and samples. This material is available via the Internetat http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*I. Lavilla. E-mail: isela@uvigo.es.Notes

The authors declare no competing financial interest.

■ REFERENCES(1) Rydberg, J.; Cox, M.; Musikas, C.; Choppin, G. R. SolventExtraction-Principles and Practice, 2nd ed.; Marcel Dekker, Inc, NewYork, 2004.(2) Jeannot, M. A.; Cantwell, F. F. Mass transfer characteristics ofsolvent extraction into a single drop at the tip of a syringe needle. Anal.Chem. 1997, 69, 235.

Figure 2. Effect of experimental parameters on the DSDME for phosphorus determination: (A) organic solvent volume with conditions 1200 rpm, 5min; (B) stirring rate of the sample with conditions 100 μL of MIBK, 5 min; (C) microextraction time with conditions 100 μL of MIBK, 1200 rpm.

Figure 3. Calibration curves for phosphorus determination obtainedusing the microvolume UV−vis spectrophotometer (■) and using theconventional UV−vis spectrophotometer equipped with 50 μL cells(□).

Table 1. Concentration of DRP in Water Samples

sample DRP/nM

river water 62 ± 4a

well water <LOQb

rain water <LOQaAverage concentration ± standard deviation (n = 3). bLOQ = 31.4nM phosphorus.

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(3) Rezaee, M.; Assadi, Y.; Milani Hosseini, M. R.; Aghaee, E.;Ahmadi, F.; Berijani, S. Determination of organic compounds in waterusing dispersive liquid-liquid microextraction. J. Chromatogr. A 2006,1116, 1.(4) Galipo, R. C.; Canhoto, A. J.; Walla, M. D.; Morgan, S. L.Analysis of volatile fragrance and flavor compounds by headspace solidphase microextraction and GC−MS: An undergraduate instrumentalanalysis experiment. J. Chem. Educ. 1999, 76, 245.(5) Hardee, J. R.; Long, J.; Otts, J. Quantitative measurement ofbromoform in swimming pool water using SPME with GC−MS: Anundergraduate instrumental analysis experiment. J. Chem. Educ. 2002,79, 633.(6) Knupp, G.; Kusch, P.; Neugebauer, M. Identification of flavorcomponents in perfumes by headspace solid-phase microextractionand gas chromatography-mass spectrometry. J. Chem. Educ. 2002, 79,98.(7) Mowery, K. A.; Blanchard, D. E.; Smith, S.; Betts, T. A.Investigation of imposter perfumes using GC-MS. J. Chem. Educ. 2004,81, 87.(8) Liang, P.; Adhyaru, B.; Pearson, W. L.; Williams, K. R. Thebinding constant of estradiol to bovine serum albumin. An upper-levelexperiment utilizing tritium-labeled estradiol and liquid scintillationcounting. J. Chem. Educ. 2006, 83, 294.(9) King, A. G. Research advances: Ancient hair dye based onmodern nanotechnology. J. Chem. Educ. 2007, 84, 570.(10) Riccio, D.; Wood, D. C.; Miller, J. M. Using single dropmicroextraction for headspace analysis with gas chromatography. J.Chem. Educ. 2008, 85, 965.(11) Yangcheng, L.; Quan, L.; Guangsheng, L.; Youyuan, D. Directlysuspended droplet microextraction. Anal. Chim. Acta 2006, 566, 259.(12) Pena-Pereira, F.; Cabaleiro, N.; de la Calle, I.; Costas, M.; Gil,S.; Lavilla, I.; Bendicho, C. Directly suspended droplet microextractionin combination with microvolume UV−vis spectrophotometry fordetermination of phosphate. Talanta 2011, 85, 1100.(13) Pena-Pereira, F.; Costas-Mora, I.; Romero, V.; Lavilla, I.;Bendicho, C. Advances in miniaturized UV−vis spectrometric systems.TrAC, Trends Anal. Chem. 2011, 30, 1637.(14) Psillakis, E.; Kalogerakis, N. Developments in single-dropmicroextraction. TrAC, Trends Anal. Chem. 2002, 21, 53.(15) Spivakov, B. Y.; Maryutina, T. A.; Muntau, H. Phosphorusspeciation in water and sediments. Pure Appl. Chem. 1999, 71, 2161.

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