Laboratory Experiment pubs.acs.org/jchemeduc
An Analysis of Ethanol in Commercial Liquors via Quantitative NMR Spectroscopy Rebecca A. Hill† and Christopher P. Nicholson*,‡ Department of Chemistry, University of West Florida, Pensacola, Florida 32514-5750, United States S Supporting Information *
ABSTRACT: NMR spectroscopy is traditionally introduced to students during Organic Chemistry as a tool used for structure elucidation and conformational analysis. NMR spectroscopy also has numerous applications beyond structure determination, including quantitative analysis of samples. For this study, an experiment was developed to use quantitative analysis functions of a 400 MHz NMR to quantify the ethanol content in different liquor samples during an Instrumental Analysis course. The concentrations of alcohol in vodka and other liquors were determined using the electronic reference to access in vivo concentrations (ERETIC) method embedded in the NMR processing software. ERETIC was used to analyze a proton NMR spectrum to define the concentration of ethanol in the reference sample, generate a calibration curve, and calculate the concentrations of ethanol in commercial samples. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Hands-On Learning/Manipulatives, Laboratory Instruction, Alcohols, NMR Spectroscopy, Quantitative Analysis
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chemical and cultural importance.11−14 In the ERETIC method, a calibrated reference signal based on a known sample is generated by the instrument. Comparison of the generated reference signal to a sample peak enables quantitative determination of the amount of ethanol in an sample. Methods of quantitative NMR analysis exist dating back to the 1960s,15,16 but ERETIC has become increasingly popular in the past two decades. In spite of its increasing industrial applications, only one prior example of an ERETIC application in a teaching lab has been published.17 The method itself is a feature embedded within some NMR processing software and as such can serve as a convenient tool for students to use in the context of Instrumental Analysis courses. The process relies on calibrating a reference signal with a known concentration of analyte. The integration is performed, and the program records the concentration (in mM) and the number of protons in the integration. For future analyses, the corresponding peak is integrated, and the ERETIC method calculates the concentration of analyte that would generate that peak. By the use of basic arithmetic, concentration units (mM) can be converted to percent alcohol (ethanol) by volume (% ABV) for comparison.
he traditional application of NMR spectroscopy in the undergraduate curriculum is focused primarily on the determination of structure in the Organic Chemistry lecture and laboratory curriculum.1−3 Although some experiments have been developed to incorporate features of NMR spectroscopy into Analytical Chemistry and Physical Chemistry,4−7 often as the curriculum progresses NMR spectroscopy falls out of use for all but research applications. We sought to expand the use of our department’s 400 MHz NMR spectrometer by developing an experiment to bring quantitative NMR spectroscopy to the Instrumental Analysis laboratory curriculum. Previous applications of quantitative NMR spectroscopy in the undergraduate curriculum have largely focused on peak intensity and calibration of intensity using the standard addition method.5 Experiments typically involve the addition of specific analytes in known quantities followed by the determination of peak intensities. Graphical analysis and back calculation then affords initial quantity and concentration analysis. Analyte addition represents an internal standard methodology that is also commonly taught in GC and HPLC analysis.8,9 NMR spectroscopy represents an opportunity to employ an external reference via the use of a synthetic reference pulse. Pulse analysis can be accomplished by a variety of methods. For the purposes of this experiment it is accessed using the electronic reference to access in vivo concentrations (ERETIC) method.10 This experiment was developed to study ethanol as an analyte in commercial liquor samples by simple dilution with D2O. Indeed, the study of ethanol as an analyte in Analytical Chemistry and Instrumental Chemistry is popular for both its © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: February 10, 2017 Revised: August 21, 2017
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DOI: 10.1021/acs.jchemed.7b00117 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 1. Student ERETIC calibration curve of ethanol solutions in units of percent alcohol by volume (% ABV) and millimoles per liter (mM). The horizontal axis is the ERETIC-measured ethanol concentration, and the vertical axes are the calculated concentrations based on serial dilution of known ethanol solutions in D2O.
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EXPERIMENTAL PROCEDURE
peak is best resolved from impurities. If both peaks are wellresolved either can be used for analysis. Reference spectra for laboratory-grade ethanol and the liquor samples analyzed in this experiment are provided in the Supporting Information.
Standard Solution Preparation
Students are instructed during prelab lecture on the operation of the instrument and the ERETIC software routine within the NMR operation software. After the prelab, the students are asked to reserve a block of time during the following week to perform the experiment at the instrument. The work is not restricted to the traditional laboratory block of time because of the availability of only a single instrument and the project-based nature of the experiment. The first goal is to develop a calibration curve relating known ethanol concentrations and integration values. To do this, a series of standards are prepared using laboratory-grade ethanol and D2O. Because very small volumes are needed for the NMR analyses, these samples can be prepared using micropipettes. A series of serial dilutions are prepared beginning with a concentrated sample containing 280 μL of 200-proof ethanol diluted to 5.00 mL in a volumetric flask. The choice of concentrated reference sample preparation is based on the necessity of ERETIC to have an input concentration in millimoles per liter with an upper threshold of 999 mM. More dilute reference samples can also be used. Serial dilutions are carried out starting from the concentrated sample using clean, disposable vials. A micropipette is used to pipet 750 μL aliquots of the concentrated sample and pure D2O. This dilution is repeated three more times for a total of four serial dilutions, resulting in a range of ethanol concentrations appropriate for the analysis of liquor samples. The remaining 750 μL of each sample is sufficient for NMR analysis. The initial concentrated sample and each subsequent dilution are placed in labeled, clean, and dry NMR tubes for data acquisition.
Data Acquisition
Prior to obtaining the 1H NMR spectrum of the concentrated reference sample, students should calculate the concentration (in mM) of ethanol in the sample. Upon completion of the first 1 H NMR spectrum, this value will be required to define the reference signal. Because a precise line shape is essential for optimal comparison of peaks to the reference signal, the 90° pulse width calculation should be carried out prior to analysis of the first sample. A procedure for calibrating the pulse width is included in the student handout. The number of scans should be consistent across all samples, and the students are advised to carry out at least 16 scans per sample. The first sample is designated as the reference sample. Each subsequent dilution is analyzed, and the concentration is calculated using the ERETIC reference pulse. Finally, the commercial liquor dilution is analyzed by 1H NMR spectroscopy. Analysis of the commercial sample is performed multiple times for averaging and standard deviation determination. It is recommended that students conduct four independent analyses of the commercial sample. The sample does not need to be removed from the instrument between analyses, but four sets of data should be collected for comparison.
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HAZARDS Hazards associated with this experiment include safe operation around the NMR spectrometer. Manufacturers restriction recommendations should be followed where safety is concerned. Students with implants, pacemakers, or other medical issues impacted by strong magnetic fields should not perform this experiment.
Unknown Sample Preparation
Students are provided with a variety of small commercial liquor samples, with the highest being ∼50% ABV. Each liquor sample is diluted to 10% of its original concentration with D2O by pipetting 500 μL of the liquor sample into a 5.00 mL volumetric flask and diluting to the mark with D2O. Prior to analysis, a simple 1H NMR spectrum of the unknown should be obtained to determine whether the ethanol triplet or quartet
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DATA ANALYSIS Initially the students are asked to use the reference sample and subsequent serial dilutions to validate the linear response of the ERETIC methodology. By means of serial dilutions of the B
DOI: 10.1021/acs.jchemed.7b00117 J. Chem. Educ. XXXX, XXX, XXX−XXX
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variation in 90° pulse width and the resulting error in the peak area. Additional sources of error may arise from inaccurate pipetting of solutions, particularly when diluting liquor samples with higher viscosity. The sensitivity of the ERETIC method means that minor errors in sample preparation are manifested as large errors in the calculated sample concentration.
initial reference at the upper range of the ERETIC concentration calculations, a plot of theoretical versus actual concentration of ethanol in the sample can be generated. Students are asked to use the ERETIC output for the actual, or measured, concentrations in mM, but the theoretical values can be determined in a variety of units, including mM and % ABV. Presentation of calibration data using ERETIC from two separate student groups can be seen in Figure 1. Students are asked to apply their own thought processes to the preparation of the calibration graph. The results of their calibration are to be used to calculate the ethanol concentration from their commercial sample, but the necessary calculations are dependent on the units chosen for the calibration curve. Statistics for each of the representative calibration curves are given in Table 1. An integration of the triplet and quartet regions of ethanol
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STUDENT REPORTS As an upper-division experiment, students are asked to write up the experiment in a journal article template. A template based on ACS templates has been modified to incorporate the University of West Florida heading information, and students are asked to construct a journal article to express the significance of the experiment and the results of their analysis. Literature searches for background and theory are required, though the direction individual students choose for the scientific importance of the study is left to their discretion. Students tend to write either about the theory of NMR spectroscopy, various methods of commercial analysis, or background on the history and sociology of ethanol. Students then provide a Materials and Methods or Experimental Procedure section followed by calibration data and sample analysis. Finally, students are asked to draw conclusions related back to their Introduction and also to assess the accuracy of the manufacturer’s label and analyze any sources of error in their experimental procedure.
Table 1. Student Calibration Curve Statistics parameter
% ABV units
mM units
Slope Intercept Standard deviation of the slope
5.907 x 10−5 mM−1 0.000% ABV 6.3 × 10−7
0.9908 5.990 mM 8.3 × 10−3
done on a pure D2O sample yields no measurable peaks. This zero value is incorporated in the calibration curve and the related statistical analysis. With the calibration curves generated, students take average of the four runs of their single dilution of the commercial liquor sample to determine the millimolar concentration of ethanol and use the calibration data to back-calculate the % ABV value. Depending on instrument access, replicate analyses can also be conducted at varying dilutions of the commercial sample. Table 2 provides a summary of the four liquor samples analyzed by
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DISCUSSION One benefit of this experiment is that it broadens student appreciation of the applications of NMR spectroscopy. While there are many research examples of quantitative NMR spectroscopy in the broad scientific literature, applications that reach down to the undergraduate course level are much less common. Furthermore, there are numerous potential samples for analysis. The first set of samples included simple liquors, but similar analysis can be performed on any liquor, wine, or beer sample. A similar study regarding the analysis of a home-brewing class is ongoing in conjunction with the Kugelman Honors Program at UWF. Additionally, there are numerous opportunities within the experiment for advanced student projects to study the impact of automatic versus manual free induction decay processing (phasing, etc.), acquisition pulse sequence, and baseline treatment on the quality of quantitative data. The format is also amenable to areas outside of food chemistry. The analysis of ethanol in fuels is another possible extension of this process, as is analysis of ethanol in synthetic blood plasmas for a forensic chemistry analysis of intoxication. Each of these analyses affords students an opportunity to apply a concentration analysis using NMR spectroscopy to a commercial project and to use their data to assess manufacturers’ reporting and labeling of products or medical reporting. These experiments more than any other engage students’ curiosity and connect their coursework to real-world applications.
Table 2. Representative Student Analyses of Commercial Liquors % ABV Commercial Liquor
Manufacturer
Student-Calculateda
% Error
Skyy vodka Margaritaville tequila Tanqueray gin 99 Whipped
40.0 40.0 47.3 49.5
46.0 41.12 46.39 48.93
15.0b 2.80 1.92 1.15
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Student-calculated values are exactly as reported by student analysis. The significant figures associated with the % ABV were as determined by student calculations. bThe percent error associated with the Skyy vodka sample was likely due to pulse width calibration on a different peak than that used for analysis (see Data Analysis for discussion).
students in the Fall 2015 section of the Instrumental Analysis Laboratory. The class, with a population of 19 students, was divided into five groups, with each group choosing a liquor sample for analysis. Although the data were collected in smallgroup sessions, each student was required to conduct his or her own calibration and analysis and write an independent report. In most cases, students were able to determine the concentration within 3% of the manufacturer’s reported concentration. One major source of error that arises from calibration is based on the peak chosen for line shape and 90° pulse width calibration. For example, calibrating on the basis of the ethanol quartet signal but analyzing on the basis of the ethanol triplet signal causes errors in the concentration due to a slight
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00117. Student handout with detailed instructions for 90° pulse width calibration (PDF, DOCX) C
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(14) Mendes, L. S.; Oliveira, F. C. C.; Suarez, P. A. Z.; Rubim, J. C. Determination of ethanol in fuel ethanol and beverages by Fourier transform (FT)-near infrared and FT-Raman spectroscopies. Anal. Chim. Acta 2003, 493, 219−231. (15) Smith, W. B. Quantitative Analysis Using NMR. J. Chem. Educ. 1964, 41 (2), 97−99. (16) Bharti, S. K.; Roy, R. Quantitative 1H NMR spectroscopy. TrAC, Trends Anal. Chem. 2012, 35, 5−26. (17) Simpson, A. J.; Mitchell, P. J.; Masoom, H.; Liaghati Mobarhan, Y.; Adamo, A.; Dicks, A. P. An Oil Spill in a Tube: An Accessible Approach or Teaching Environmental NMR Spectroscopy. J. Chem. Educ. 2015, 92, 693−697.
NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Christopher P. Nicholson: 0000-0003-2659-5404 Present Addresses † R.A.H.: Department of Chemistry, Mississippi State University, Mississippi State, MS 39762. ‡ C.P.N.: Department of Chemistry and Biochemistry, RoseHulman Institute of Technology, Terre Haute, IN 47803.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Carrie Simpson and the Fall 2015 University of West Florida Instrumental Analysis Laboratory course (CHM4130L) for piloting this experiment. We also thank Pamela Vaughan for helpful discussion in paralleling the FTIR quantitative experiment.
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REFERENCES
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