Laboratory Experiment pubs.acs.org/jchemeduc
A Solvent Microextraction Approach for Environmental Analysis: Colorimetric Assay for Phosphorus Determination in Natural Waters Francisco 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 for students in an upper-division undergraduate analytical laboratory for the UV−vis spectrophotometric determination of phosphorus in natural waters using a current microextraction technique. The method is based on the formation of a colored ion pair and its subsequent extraction in a microdrop of organic solvent. The proposed laboratory experiment allows the students to gain experience in developing a green sample preparation approach. Furthermore, the extraction of a colored product allows them to better understand different issues that govern solvent microextraction. Optimization of some experimental parameters and the chemical analysis of natural waters 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
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colored ion pair is formed between molybdophosphate and malachite green. Upon addition of a low-density organic solvent such as methyl isobutyl ketone (MIBK), the color ion pair can be extracted. This experiment was designed to introduce students into the philosophy of green analytical chemistry by using SME, an environmentally friendly alternative to SE. Students are involved in the development of a miniaturized methodology with the assessment of the main experimental parameters that affect the extraction process using univariate optimization, preparation of the phosphorus calibration curve, and determination of the corresponding figures of merit. Thus, they can evaluate the greenness of the method without compromising the analytical characteristics, that is, using low volumes of organic solvents with the minimum amounts of waste and speed. The motivation of the students increases when they deal with the analysis of environmental samples. Therefore, this experiment can lead to, or be preceded by, a discussion of environmental concepts and the biogeochemical cycling of phosphorus. In addition, students become familiar with other SME approaches to 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 upperlevel instrumental analysis course. Results and questions proposed in the lab documentation were discussed in a separate classroom session with particular emphasis on comparison with
olvent extraction (SE) is employed to perform educational exercises in undergraduate laboratories of chemistry and chemical engineering.1 In analytical procedures, SE allows the separation and preconcentration of target compounds as well as an efficient sample cleanup. In spite of this, it is not free from limitations; for example, large volumes of organic solvents are usually consumed, tedious procedures are involved, and large quantities of waste are generated. Therefore, when SE is considered from the standpoint of green analytical chemistry, its general applicability is questionable. In the last years, different efforts have been devoted to the achievement of more efficient and greener alternatives to SE. Solvent microextraction techniques (SME) such as single-drop microextraction (SDME)2 and dispersive liquid−liquid microextraction,3 among others, are nowadays considered powerful analytical tools, especially when a minimum organic solvent consumption along 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 best of our knowledge, there is only a single laboratory experiment addressing an SME technique, specifically headspace SDME.10 However, this approach requires significant staff and student training, making it troublesome to obtain results in a few working sessions. Thus, we propose a laboratory experiment based on the use of directly suspended droplet microextraction (DSDME), that is, an SME technique first introduced by Yangcheng et al. in 2006,11 involving tasks that can be easily accomplished by untrained personnel such as students. DSDME is used in this work for the determination of phosphorus in natural waters.12 When natural water is mixed with a solution containing molybdate and malachite green, a © 2014 American Chemical Society and Division of Chemical Education, Inc.
Published: January 9, 2014 586
dx.doi.org/10.1021/ed300855t | J. Chem. Educ. 2014, 91, 586−589
Journal of Chemical Education
Laboratory Experiment
classical SE procedures. The use of very small volumes of toxic reagents was highlighted by students.
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EXPERIMENTAL DETAILS
Chemical Reaction
The reaction between phosphorus and molybdate in acidic medium gives rise to the formation of 12-molybdophosphate (eq 1), which forms a colored ion pair with a cationic triphenylmethane dye such as malachite green (MG+) (eq 2) H3PO4 + 12MoO3 → H3PMo12O40
(1)
H3PMo12O40 + MG+ → (MG+)(H 2PMo12O40−) + H+ (2)
This colored ion pair can be extracted into an organic solvent (eq 3):
Figure 1. DSDME procedure (clockwise from top left): Addition of extractant phase to the stirred aqueous sample; microextraction process after 3 min; microextraction process after 5 min; deposition of the enriched drop for absorbance measurement by UV−vis microspectrophotometry.
(MG+)(H 2PMo12O40−)aq ⇌ (MG+)(H 2PMo12O40−)org (3)
Theoretical aspects of solvent microextraction are considered. Further information can be found in the Supporting Information.
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HAZARDS Sulfuric acid is extremely corrosive and highly toxic, being harmful or fatal if swallowed. Skin contact can cause severe burns. Diluted sulfuric acid solutions must be prepared by carefully adding acid to water, not the reverse. Hydrochloric acid is irritant and corrosive and can be very hazardous in case of ingestion or skin or eye contact. Hot hydrochloric acid can cause both thermal and acid burns. Malachite green is harmful in case of ingestion and slightly hazardous in case of inhalation or skin or eye contact. Severe overexposure to malachite green may be fatal. MIBK is flammable and can lead to potential acute health effects in case of skin or eye contact, ingestion, or inhalation. Methanol is hazardous in case of skin or eye contact, ingestion, or inhalation Safety glasses, lab coat, and gloves are mandatory for personal protection.
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 conventional UV−vis spectrometers in undergraduate teaching laboratories. Thus, commercially available micro or ultramicro cuvettes compatible with conventional spectrophotometers could be used for phosphorus quantification, even though some dilution of the enriched drop would be unavoidable after the extraction process. In this work, an Uvikon XS UV−vis spectrophotometer (Secomam, Domont, France) equipped with 50 μL cells was employed as an alternative. Other microvolume UV−vis spectrometric systems also applicable in combination with 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 the range 60−1600 rpm was used to carry out extractions.
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RESULTS Several variables can affect the performance of the DSDME method.11 In this laboratory experiment, students evaluated the impact of three relevant parameters, namely, organic solvent volume, stirring rate, and extraction time, by carrying out univariate optimizations. The results of the optimizations are shown in Figure 2. According to the obtained results, students showed that the optimal conditions were as follows: 100 μL of organic solvent, 1200 rpm of stirring rate and 7 min of microextraction time. Nevertheless, a reduced extraction time (5 min) was selected in this work in order to increase the sample throughput, bearing in mind that complete equilibrium need not to be attained for accurate and precise analysis, as pointed out in the literature.14 A calibration curve for phosphorus determination was built under optimal conditions by subjecting ten blanks and five standard solutions of phosphorus (ranging from 0.1 to 1.5 μM) to the described procedure. Corrected absorbance values were calculated by subtracting the blank absorbance from the absorbance values of standards and samples. The calibration curve was obtained by plotting the corrected absorbance versus the concentration of phosphorus. The equation for the calibration curve obtained by students was Abs =0.4605[P] + 0.0031,
DSDME Procedure
A 5 mL natural water sample was placed in a 7 mL glass vial containing a magnetic stir bar (10 mm × 3 mm). Then, 500 μL of 4.5 M H2SO4 and 500 μL of the mixed reagent solution were added. The stirring rate was set at 1200 rpm, and 100 μL of 4-methyl-2-pentanone (MIBK) was carefully added to the stirred sample. During the extraction process, the sample vial was capped. After 5 min, the cap was removed and 2 μL of the colored drop were collected with a Hamilton syringe and deposited onto the lower pedestal of the microvolume UV−vis spectrophotometer for analysis. All glassware was previously cleaned with hot hydrochloric acid (0.5 M) and then rinsed several times with water. The instructor prepared the mixed reagent solution and the sulfuric acid solution before class. The mixed reagent solution consists of an aqueous solution containing ammonium heptamolybdate tetrahydrate, malachite green oxalate, tartaric acid, and sulfuric acid. The preparation of the mixed reagent solution is described in the lab documentation. The DSDME procedure is shown in Figure 1. 587
dx.doi.org/10.1021/ed300855t | J. Chem. Educ. 2014, 91, 586−589
Journal of Chemical Education
Laboratory Experiment
Figure 2. Effect of experimental parameters on the DSDME for phosphorus determination: (A) organic solvent volume with conditions 1200 rpm, 5 min; (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.
Table 1. Concentration of DRP in Water Samples
where Abs is the corrected absorbance and [P] is the concentration of phosphorus (μM). The students obtained the analytical figures of merit of the method. Under optimal conditions, the students obtained the limits of detection (LOD) and quantification (LOQ) of 9.4 nM and 31.4 nM of phosphorus, respectively. The precision, obtained from five consecutive extractions and expressed as relative standard deviation (RSD), was 4.5%. Alternatively, the students obtained the analytical figures of merit of the method using the conventional UV−vis spectrophotometer equipped with 50 μL cells by dilution of the microdrop with methanol. Under these conditions, the equation for the calibration curve was Abs =0.1695[P] + 0.0003. The LOD and LOQ were 15.5 and 51.7 nM, respectively, and the precision of the method was 4.2%. The calibration curves obtained by the students are shown in Figure 3. Further information on the calculations to obtain the analytical characteristics can be found in the Supporting Information.
sample
DRP/nM
river water well water rain water
62 ± 4a
