Determination of Unknown Concentrations of Sodium Acetate Using

Laboratory Experiment pubs.acs.org/jchemeduc

Determination of Unknown Concentrations of Sodium Acetate Using the Method of Standard Addition and Proton NMR: An Experiment for the Undergraduate Analytical Chemistry Laboratory Massy Rajabzadeh* Department of Chemistry, Western Connecticut State University, Danbury, Connecticut 06810, United States S Supporting Information *

ABSTRACT: In this experiment, students learn how to find the unknown concentration of sodium acetate using both the graphical treatment of standard addition and the standard addition equation. In the graphical treatment of standard addition, the peak area of the methyl peak in each of the sodium acetate standard solutions is found by integration using proton NMR. Using the calibration curve of the peak areas of the methyl protons of sodium acetate versus the concentration of the sodium acetate solutions, the concentration of the unknown is determined through extrapolation to the x axis. In a separate experiment, the unknown concentration of sodium acetate is found using the standard addition equation. In this case, the methyl peak areas in the unknown sodium acetate solution and the unknown sodium acetate spiked with a standard are obtained using proton NMR. The standard addition equation is then used to find the concentration of the unknown. This experiment has been successfully used in the undergraduate analytical lab and students have learned the method of standard addition to find unknown concentrations of sodium acetate in the millimolar range. It has also given them the opportunity to have hands-on experience using NMR spectroscopy as a quantitative tool. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Instrumental Methods, NMR Spectroscopy, Quantitative Analysis

T

resonance. However, the analysis can be performed on any compound if the analyte signal is observed without interference. With a few modifications, the method described here can be used to quantify biologically important substances such as acetate in biofluid samples. Acetate has been measured in plasma, urine, serum, and human amniotic fluid.10−14 High levels of acetate are an indication of a diseased state and it has been shown that concentrations of acetate are elevated in patients with severe liver disease and acidosis.10 One of the advantages of using NMR to quantify acetate levels in biological fluids is that sample handling and pretreatment of the sample is minimal. Moreover, by using this technique more than one metabolite in a complex mixture can be measured simultaneously in a single spectrum. In addition, the time required per sample is a few minutes for a simple one-dimensional proton spectrum and metabolite concentrations down to the low micromole per liter range can readily be detected by high-field spectrometers. The disadvantages of NMR are that it is most efficient in the millimole per liter concentration. The large signal of the water peak in the presence of the weak signals presents a problem in proton NMR. Nevertheless, high signal-to-noise ratios can be achieved by suppressing the solvent peak by continuous proton irradiation at the water frequency.15 NMR is limited to

he purpose of the experiment is for undergraduate students to practice the method of standard addition introduced in their analytical chemistry textbook1 and become familiar with using NMR. Both the standard addition equation and graphical treatment of standard addition are used to find the unknown concentration of sodium acetate. The advantage of using the graphical technique is that students can observe the linear relationship between solution concentration and the area under the peak in the NMR spectra. NMR is a tool that is widely available in chemistry departments and this experiment gives undergraduates an opportunity to have hands-on experience with an NMR instrument and use it as a quantitative device to determine the unknown concentrations of sodium acetate.2 NMR spectroscopy is commonly used in chemistry laboratory programs; however, its major application is in organic chemistry where it is used for solving structures of unknowns. Because the intensity of an NMR signal is proportional to the number of nuclei contained in the sample, quantitative analysis is possible. Many articles describing NMR for quantitative analysis in instrumental analysis laboratories have appeared in this Journal.3−9 Most of these articles use internal standards in their analyses3,6,7 whereas Hoffman et al. have used standard addition.4 The experiment described here is used for quantifying the concentration of sodium acetate solutions using standard addition. Sodium acetate has been chosen as the unknown as it demonstrates a single proton © 2012 American Chemical Society and Division of Chemical Education, Inc.

Published: August 17, 2012 1454

dx.doi.org/10.1021/ed200577v | J. Chem. Educ. 2012, 89, 1454−1457

Journal of Chemical Education

Laboratory Experiment

observing only low molecular weight compounds13 because larger molecules such as lipoproteins have fast relaxation rates in NMR experiments and give rise to broad resonances and are more difficult to detect. Furthermore, NMR has little value for metabolites that do not have detectable protons or those present at low concentration. The magnitude of the longitudinal relaxation time, T1, of the nuclei is important when using NMR for quantitative analysis. T1 is a measure of the time required for the net magnetization vector to return to its equilibrium value after the spin system has been disturbed from its full length along the z axis by an applied radiofrequency field.16,17 To avoid nonquantitative situations, a waiting period of 5 times the longest T1 value among the nuclei being measured should be applied. If the delay between the pulses is too short, insufficient relaxation will cause inaccurate spectrum integrals. At 400 MHz, the T1 relaxation time of the methyl group in sodium acetate is around 4 s. Therefore, a delay of 40 s between each pulse for the sodium acetate samples ensures that the magnetization vector has returned to its equilibrium value. Higher field strengths may require longer delays to ensure complete longitudinal relaxation. T1 information can come from literature values, approximation, or preferably, measurement. The T1 relaxation time for the protons of sodium acetate can be measured using the inversion recovery method.18 A 180° pulse is used to invert the magnetization vectors of all nuclei in the sample. After a recovery time, τ, a 90° pulse converts the z magnetization to xy magnetization. The recovery time that produces the spectrum in which the signal intensity of the observed nucleus is zero is used to find the value of T1 using eq 1:16 τ = T1 ln 2

Graphical Procedure of Standard Addition

The students prepare 50 mL of 100 mM sodium acetate stock solution. Students then prepare four sample solutions using 10 mL of the unknown sodium acetate solution, 2.5 mL of deuterium oxide (D2O), and the appropriate volumes of 100 mM stock solution necessary to prepare standard addition sample concentrations of 10, 20, 30, and 40 mM sodium acetate (detailed instructions and a table are available in the Supporting Information). Students prepare the solutions using normal volumetric procedures and employ micropipets, graduated pipets with bulbs, and volumetric flasks. Around 0.80 mL of each sample solution is placed in a labeled NMR tube. These four samples are then used to determine the unknown concentration of sodium acetate by the graphical procedure of standard addition. Standard Addition Equation Procedure

To determine the unknown concentration of sodium acetate using the standard addition equation, 0.80 mL of the unknown sodium acetate solution is placed in a labeled NMR tube and 0.80 mL of a spiked unknown solution is placed in another NMR tube. This spiked solution contains a mixture of 5 mL of the unknown solution and 5 mL of 50 mM sodium acetate solution made from the 100 mM sodium acetate stock solution. The details are available in the Supporting Information.

■

DATA ACQUISITION Each student in a group acquires the proton spectra for three samples under the supervision of the instructor. After acquiring the spectrum, students are guided through the analysis of the unprocessed raw spectral data of their first sample. This involves Fourier transformation of the free induction decay (FID), introduction to the zoom operations for data inspection, and phasing the spectrum. The methyl peak of the sodium acetate appears at around 1.83 ppm in the proton NMR spectrum. Students integrate the methyl peak and set a value of 1 for the peak area of the 10 mM sample solution. Then, the students acquire the rest of the samples and the integrated methyl peak areas for the other three sample solutions (20, 30, and 40 mM) are calibrated with respect to the 10 mM solution. The sodium acetate concentrations and their respective methyl peak areas are recorded. For calculating the unknown sodium acetate concentration using the standard addition equation, students initially acquire the spectrum for the unknown sample and set the peak area of the methyl peak to 1. Then, the spiked unknown is acquired and the peak area of the methyl peak is integrated and calibrated with respect to the methyl peak of the unknown.

(1)

The experiment has been incorporated into the undergraduate quantitative analysis laboratory and students work in pairs. Although students do not use a real matrix, the use of standard addition procedure for cases where matrix effects in complex samples, such as blood, cell suspensions, and other biological samples, cause insensitivity and severe problems is discussed during lecture. The experiment is also appropriate for upper-level undergraduate instrumental laboratory and can be used with minor alteration of this procedure. Upper-level undergraduates could use sample preparation involving real samples that would demonstrate matrix effects. For example, students could quantify the amount of acetate in bovine serum albumin or urine. An in-depth description of sample preparation and data acquisition is presented in the Beckonert et al. protocol.15 Students are made aware that the graphical treatment of standard addition is used for destructive techniques such as atomic absorption spectroscopy, whereas the standard addition equation is used for nondestructive techniques such as NMR spectroscopy. The graphical treatment and the standard addition equation are also demonstrated with examples. Indepth explanation of the NMR instrument is not discussed; however, a basic overview is presented.19−21

■

HAZARDS Precautions to prevent skin contact, inhalation, and ingestion should be taken during the preparation of the samples. Sodium acetate is an irritant and is slightly hazardous in case of skin or eye contact. Deuterium oxide is hazardous in case of ingestion. Students should wear protective eyewear and can use protective gloves to prepare solutions.

■

■

SAMPLE PREPARATION During the 3-h lab period, two students prepare six samples and acquire the NMR spectra to investigate the two standard addition procedures and find the concentration of the unknown. The unknown is given to each group at the beginning of the lab.

CALCULATIONS

Graphical Procedure of Standard Addition

To determine the concentration of the unknown sodium acetate by the graphical treatment of standard addition, students are instructed to plot the calibration curve of the 1455

dx.doi.org/10.1021/ed200577v | J. Chem. Educ. 2012, 89, 1454−1457

Journal of Chemical Education

Laboratory Experiment

Although the standards could be prepared by mass using an analytical balance (by adding solid sodium acetate to the NMR tubes) to circumvent the huge relative errors caused by mediocre volumetric skill, the goal of the experiment is for students to practice their volumetric technique. To improve the statistical evaluation of the two methods in future analytical laboratories, the same concentration of acetate will be given to four different groups in the lab. After the groups have finished the lab, the data will be pooled and students will be asked to perform error analysis, compute 95% confidence intervals and execute statistical comparison of results.

signal (methyl peak area) obtained from the proton NMR spectra versus the concentration of the respective standard addition solutions (10, 20, 30, and 40 mM). These data are fit to a line: the calibration curve. The calibration curve equation is used to find the intercept on the x axis, Csa (i.e., setting y or the NMR peak area to zero). Csa can be used to find the concentration of the unknown, C0, using C0 = −Csa

Vflask Vunknown

(2)

where Vflask is the final volume of the solutions and Vunknown is the volume of unknown added to the solutions. The derivation of eq 2 is nicely presented by Zellmer.22 Standard Addition Equation Procedure

■

To find the unknown concentration using the standard addition equation, students are instructed to use eq 3,

The student handout and instructor notes. This material is available via the Internet at http://pubs.acs.org.

[X ]i I = X [S]f + [X ]f IS + X

ASSOCIATED CONTENT

S Supporting Information *

■

(3)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

where

⎛V ⎞ [X ]f = [X ]i ⎜ X ⎟ ⎝V ⎠

(4)

The authors declare no competing financial interest.

⎛V ⎞ [S]f = [S]i ⎜ S ⎟ ⎝V ⎠

(5)

V = VX + VS

(6)

ACKNOWLEDGMENTS The author thanks the students who participated in this experiment.

Notes

■ ■

and [X]i is the unknown concentration, IX is the peak area of the unknown, IS+X is the peak area of the spiked sample, the subscript “f” denotes the final diluted volume, VX is the initial volume of the unknown, VS is volume of solution with initial concentration of [S]i that is spiked in the unknown.1

■

RESULTS AND DISCUSSION The results using both methods are tabulated in Table 1 and the relative errors have been calculated. The relative errors Table 1. Student Results for the Determination of Sodium Acetate Concentration/mM Unknown Group Concentration/ Number mM 1 2 3 4 5 6 7 8

22 14 32 26 24 28 18 34

Graphical Treatment of Standard Addition (Relative Error) 31 (41%) 16 (14%) 33 (3%) 27 (4%) 13 (−46%) 24 (−14%) 15 (−17%) Incomplete Data

Standard Addition Equation (Relative Error) 21 15 35 28 28 36 17 34

REFERENCES

(1) Harris, D. C. Quantitative Chemical Analysis, 7th ed.; W.H. Freeman and Company: New York, 2007. (2) McGregor, M. A.; Euler, W. B. Undergraduate NMR Laboratory Experiments. In NMR Concepts; Traficante, D. D. Ed.; NMR Concepts: Warwick, RI, 1995. (3) Clarke, D. W. J. Chem. Educ. 1997, 74, 1464−1465. (4) Hoffmann, M. M.; Caccamis, J. T.; Heitz, M. P.; Schlecht, K. D. J. Chem. Educ. 2008, 85, 1421−1423. (5) Markow, P. G.; Cramer, J. A. J. Chem. Educ. 1983, 60, 1078− 1079. (6) Phillips, J. S.; Leary, J. J. J. Chem. Educ. 1986, 63, 545−546. (7) Peterson, J. J. Chem. Educ. 1992, 69, 843−845. (8) Schmedake, T. A.; Welch, L. E. J. Chem. Educ. 1996, 73, 1045− 1048. (9) LeFevre, J. W.; Silveira, A. J. Chem. Educ. 2000, 77, 83−85. (10) Tollinger, C. D.; Vreman, H. J.; Weiner, M. W. Clin. Chem. 1979, 25, 1787−1790. (11) Nicholson, J. K.; O'Flynn, M. P.; Sadler, P. J.; Macleod, A. F.; Juul, S. M.; Sonksen, P. H. Biochem. J. 1984, 217, 365−375. (12) Vreman, H. J.; Dowling, J. A.; Raubach, R. A.; Weiner, M. W. Anal. Chem. 1978, 50, 1138−1141. (13) Nelson, T. R.; Gillies, R. J.; Powell, D. A.; Schrader, M. C.; Manchester, D. K.; Pretorius, D. H. Prenatal Diagn. 1987, 7, 363−372. (14) Moolenaar, S. H.; Engelke, U. F. H.; Wevers, R. A. Ann.Clin.Biochem. 2003, 40, 16−24. (15) Beckonert, O.; Keun, H. C.; Ebbels, T. M. D.; Bundy, J.; Holmes, E.; Lindon, J. C.; Nicholson, J. K. Nat. Protoc. 2007, 2, 2692− 2703. (16) Wink, D. J. J. Chem. Educ. 1989, 66, 810−813. (17) King, R. W.; Williams, K. R. J. Chem. Educ. 1989, 66, A213− A219. (18) King, R. W.; Williams, K. R. J. Chem. Educ. 1990, 67, A93−A99. (19) Veeraraghavan, S. J. Chem. Educ. 2008, 85, 537−540. (20) Schwartz, L. J. J. Chem. Educ. 1988, 65, 959−963. (21) Hornak, J. P. The Basics of NMR [Online];1997−2011. http:// www.cis.rit.edu/htbooks/nmr/ (accessed Aug 2012).

(−4%) (7%) (9%) (8%) (17%) (29%) (−6%) (0%)

using the standard addition equation method range from −6% to 29%, whereas the relative errors calculated from the graphical treatment of standard addition range from −46% to 41%. The larger relative errors calculated for the latter standard addition method can be attributed to the poor volumetric skill in preparation of the standards: only one spiked standard is used with the former method whereas the latter method depends on four standard samples. 1456

dx.doi.org/10.1021/ed200577v | J. Chem. Educ. 2012, 89, 1454−1457

Journal of Chemical Education

Laboratory Experiment

(22) Zellmer, D. L. Standard Addition [Online] http://zimmer. csufresno.edu/∼davidz/Chem106/StdAddn/StdAddn.html (accessed Aug 2012).

1457

dx.doi.org/10.1021/ed200577v | J. Chem. Educ. 2012, 89, 1454−1457