In the Laboratory
The Quantitative Analysis of an Analgesic Tablet An NMR Experiment for the Instrumental Analysis Course Thomas A. Schmedake and Lawrence E. Welch* Department of Chemistry, Knox College, Galesburg, IL 61401
We always try to keep our Instrumental Analysis course as up to date as possible, incorporating any new instrumentation that the department is fortunate enough to obtain. So what can we do with our new FTNMR? There are plenty of characterization experiments following organic synthesis (1, 2) and procedures that focus on the theoretical behavior of various spectral features (3, 4). In the best tradition of analytical chemistry, we wanted to produce a good old-fashioned quantitative procedure for an unknown sample using the NMR. This can be done with relative ease using proton NMR (5). Carbon 13 NMR is not well known as a source of quantitative data, but it can be obtained with some effort. The search for the proper solution and instrumental parameters to obtain quantitative 13 C data provides an excellent framework for an undergraduate investigation, culminating with the determination of an unknown. One factor limiting quantitative 13C NMR applications is poor sensitivity, attributable largely to the low native abundance of carbon 13 (1.10% [6]). Modern FTNMR instrumentation has extended the useful concentration range, but trace analysis is not currently possible. The typical practice of acquiring carbon 13 signals while performing broadband proton decoupling also works against quantitative experiments, as this causes signal enhancement of carbons in proximity to hydrogen atoms via the nuclear Overhauser effect (NOE [7]). This enhancement is specific to the local environment of each carbon in the molecule, making it impossible to normalize integrated peak areas from the different carbons. The NOE can be removed by not decoupling protons during acquisition, but this creates other problems. When not decoupled, many of the carbon 13 resonances will be split into multiplets, creating spectral overlap and worsening the signal-to-noise ratio of the peaks. As a result, integration accuracy becomes much poorer. Even with the NOE gone, the long relaxation times of the carbon 13 nuclei relative to the typical delay time in the standard carbon 13 pulse sequence (default of 1 s) creates intensity variations for the resonances from the carbons within the same molecule. Gated Decoupling Methods To achieve a proportional response from each carbon in the molecule, nonstandard decoupling measures are necessary. Gated decoupling methods (8) utilize broadband proton decoupling, but only at specific times throughout the pulse sequence. The standard gated decoupling routine (note that the jargon is not completely standardized and that some sources have alternate *Corresponding author.
names for this experiment) turns off the proton decoupler during the 13 C acquisition period. The decoupler is turned on during the relaxation period between the completion of one pulse cycle and initiation of the next (Fig. 1a). Since the coupling/decoupling processes are rapid, the 13C signals will be split by nearby protons. The NOE process is relatively slow, so some enhancement in the 13C peak intensities will remain even though the decoupler is turned off just prior to acquisition. By increasing the length of the relaxation period, a greater degree of NOE enhancement is developed. For nuclei with relatively large T1 (spin lattice relaxation time) values, the standard gated decoupling experiment can yield confusing data due to the antagonistic effect on peak size from NOE enhancement and from spin lattice relaxation during the relaxation period. By inserting a relaxation period (termed the relaxation delay), with the decoupler off, between the acquisition period and the regular relaxation period (termed the NOE time), one can minimize T 1 effects on this experiment (9) (Fig. 1b). An alternative decoupling approach is the inverse gated decoupling program (8), which runs the decoupler during acquisition but not during the relaxation period (Fig. 1c). This produces singlet resonances from each of the carbons, and with sufficient relaxation time the NOE enhancement decays and the peak intensities approach a proportional response. Unfortunately, allowance for requisite relaxation time is rarely possible during the time allotted for an undergraduate laboratory experiment. The introduction of paramagnetic compounds into the sample can be used as an alternative or supplement to the inverse gated decoupling routine. The paramagnetic additive increases the relaxation rate due to a dipole–dipole interaction between the unpaired electron and the carbon nuclei (8). In theory, the concentration of paramagnetic additive can be increased until the NOE is completely eliminated, but the amount required tends to produce excessive peak broadening. The solubility of the paramagnetic compounds is a limitation as well. By combining the use of a paramagnetic additive with inverse gated decoupling, nearly quantitative response can be obtained without excessive peak broadening. Starting with the use of a standard sample, the different decoupling procedures can be demonstrated. By monitoring peak areas and multiplicities, the observed spectral features seen with the different routines can be correlated with the structure of the standard compound. Since the “ideal” peak areas are known, the student can evaluate which procedure provides the nearest approach to a quantitative response. At this point a true unknown sample is assigned. We have chosen to use an analgesic tablet containing aspirin and acetaminophen as major components, each to be determined by 13C NMR. To solve for the amount of each, a known amount of acetophenone is added to the sample to act as an internal stan-
Vol. 73 No. 11 November 1996 • Journal of Chemical Education
1045
In the Laboratory
a)
Acquisition
on
C13
Relaxation Time
off
H1 on
Acquisition
b)
C13
on
Relaxation Delay
NOE Time
off
H1
on
decoupled 13C of this solution, and trial 10 was an inverse gated decouple with a 5.0-s relaxation time. The unknown samples were caplets of Extra Strength Excedrin™, each reported to contain 250 mg of aspirin, 250 mg of acetaminophen, and 65 mg of caffeine. Not listed on the bottle, but also present, are a number of minor ingredients (10). Each caplet was added to 2.0 mL of 99.8%-d Unisol (a mixture of DMSO-d6, CDCl3, and CD 2Cl2) from Norell (Landisville, NJ). As the internal standard, 0.17 mL of acetophenone (Aldrich) was added, and 0.0304 g of 2,2,6,6-tetramethyl-1piperidinyloxy (TEMPO, Aldrich) was mixed in to serve as a paramagnetic relaxation agent. Insoluble components were removed by filtration through a syringe-tip Nylon-66 filter (Model 2047, 0.45 µm pores, Alltech, Deerfield, IL) before addition to the NMR tube. The sample was run using the inverse gated decouple routine with a 3.0-s relaxation time. Results and Discussion
Acquisition
c)
C13
on off
Relaxation Time
H1 on
Figure 1. Instrumental protocol for the various gated decoupling routines. (a) Standard gated decoupling. (b) Gated decoupling for nuclei with large T1 values. (c) Inverse gated decoupling.
dard. Peak areas are integrated for carbons from each of the compounds, and mole ratios relating the analytes to the internal standard are found by equating them to the peak area ratios for each of the compounds. Experimental Procedure The NMR spectrometer used was an Eclipse 270 from JEOL (Peabody, MA) with Delta software. Most of the instrumental parameters were maintained at the default settings defined by the acquisition software. One hundred scans were acquired for all trials except the analgesic unknown, which required 500 scans. The sweep parameters were set to give a resolution of 1.099 Hz for all of the experiments. The samples were placed in Model 528-PP8 5-mm NMR tubes from Wilmad (Buena, NJ). Acenapthene (see Fig. 2) from Aldrich (Milwaukee, WI) was used as the standard compound for the initial trials, with 99.8%-d CDCl3 (Aldrich) as the solvent. The acenapthene concentration was ca. 1.8 M, chosen rather high to necessitate fewer scans during acquisition. Trial 1 collected a standard 13 C spectrum with broadband proton decoupling. Trial 2 repeated the collection with the decoupler turned off. Trial 3 was a repeat of Trial 2, with the relaxation time increased from 1.0 to 6.0 s. Trial 4 collected the 13C data with gated decoupling, using a relaxation delay of 6.0 s and NOE time of 5.0 s. Trials 5 through 8 utilized inverse gated decoupling, with relaxation times of 1.0, 2.0, 4.0, and 6.00 s, respectively. For the final two standard trials, the acenapthene standard solution was exchanged for a second sample identical except for the addition of 0.1 M of the paramagnetic Cr(acac)3 from Aldrich. Trial 9 was a broadband proton-
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Acenapthene is a good choice for a standard compound, as a wealth of literature regarding its NMR spectra is available (11, 12). It is also a difficult candidate in which to induce proportional response, as the T1 values range widely from 150 s for C-11 of Figure 2 to 3.1 s for C-1 and C-2 (10), and the carbons differ in their degree of protonation. The chemical shift is tabulated for each carbon in Table 1, along with its normalized peak area for each trial. The normalization was done by assigning the carbon signal at 139.6 ppm (usually the smallest signal) from each trial an area value of 1.00. The acquisition time required for each trial is also tabulated; note that this does not account for field shim time, experimental setup, or postcollection processing. Observing the peak areas from the standard 13C experiment, it is surprising to note how distant they are from a proportional response. Two coupled 13C trials with differing relaxation times were run to allow a fair comparison of both coupled vs. uncoupled spectra and coupled vs. gated decouple, which required a longer relaxation time to minimize T 1 effects. Doing this also allowed examination of the effect of increasing relaxation time in a system where no NOE is present. Evaluation of broadband decoupled vs. coupled shows a very obvious difference in multiplicity and signal-to-noise ratio. Comparing the coupled trials with differing relaxation times, one can observe the expected decline in relative peak area as a function of T 1 value, as well as the larger absolute peak size as the relaxation time is increased. Comparing the coupled vs. the gated decouple clearly illustrates the NOE enhancement acquired by the protonated carbons during the gated experiment. The trend in peak areas with increased relaxation time for the in2
1
9
10 11
8
3
4
7
12 6
Figure 2. Acenapthene.
Journal of Chemical Education • Vol. 73 No. 11 November 1996
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In the Laboratory
Table 1. Normalized Peak Areas for Acenapthene Trial Number
Experiment
Relaxation Acquisition Time (s) Time (s)
Carbons 1,2 Carbons 3,8 Carbons 4,7 Carbons 5,6 Carbons 9,10 Carbon 11 30.6 ppm 119.4 ppm 128.1 ppm 122.5 ppm 146.3 ppm 139.6 ppm
Carbon 12 131.9 ppm
1
Standard broadband decouple
1
202
11.34
9.90
10.19
10.05
3.17
1.00
1.44
2
Coupled
1
202
5.24
4.87
4.53
4.86
2.46
1.00
1.29
3
Coupled
6
722
3.07
2.73
2.87
3.18
2.30
1.00
1.27
1248
5.31
4.70
5.08
5.24
2.74
1.00
1.34
Relax = 6 NOE = 5
4
Gated decouple
5
Inverse gated decouple
1
202
10.26
9.77
9.62
9.70
3.13
1.00
1.34
6
Inverse gated decouple
2
306
7.94
8.69
8.58
8.73
3.23
1.00
1.38
7
Inverse gated decouple
4
514
4.77
6.39
6.08
6.35
3.00
1.00
1.42
8
Inverse gated decouple
9 10
Inverse gated decouple with Cr(acac)3 Inverse gated decouple with Cr(acac)3
6
722
3.62
5.39
5.33
5.40
2.96
1.00
1.46
1
202
2.64
2.21
2.29
2.37
2.08
1.00
0.97
5
618
2.20
2.13
2.20
2.08
2.22
1.00
0.97
verse gated decouple experiments likewise is easily visible. Even longer relaxation times would be a nice addition, but time constraints prohibit this. For the sake of comparison, we do like to provide the students with the data from an inverse gated decouple with the software maximum 429 s delay time run outside of class (it required more than 12 hours). Literature sources suggest that a relaxation period of 3T 1,max (8) or greater (12, 13) is necessary for quantitative trials. By selecting a compound (such as acenapthene) with exceedingly long T1 values, it is clear that gated decoupling does not always produce quantitative data, whereas the opposite conclusion might be reached if an alternative standard with more rapid 13C equilibration were chosen. The Cr(acac) 3 worked well as a paramagnetic supplement at 0.1 M levels in chloroform, but its solubility was poor in some other solvents tested. No peak broadening or significant alteration of the chemical shifts was observed with this concentration of the Cr(acac)3. Only a single gated decouple trial was done to save time. Changing the relaxation time does not cause significant changes unless the times chosen were small (less than 1 s). The Extra Strength Excedrin™ worked well as an unknown, as would many others: different analgesics, linseed oil, and aromatic hydrocarbon mixtures (12). Two major concerns were crucial in selecting the unknown sample and experimental conditions. First, the analyte components should be fairly concentrated, to avoid long acquisition times. For reasonable quantitative results, the peak must be considerably larger than what is necessary for a qualitative trial. Carbon signals with smaller S/N suffer greater experimental error due to peak area uncertainty. For example, the determination of caffeine in the analgesic samples was possible, but the accuracy was poor. A second concern is that the analyte components must be completely soluble, which is made more difficult by the high concentrations in use. The internal standard must also be completely soluble, although one has the luxury of choosing a standard with similar polarity to the analyte(s). For solubility purposes, TEMPO was used as a paramagnetic additive instead of Cr(acac)3. The concentration used was slightly higher than used with the Cr(acac)3 (0.15 M vs. 0.1 M). Due to the slightly higher concentration and the differing paramagnetic nature of the TEMPO, the chemical shifts were observed to increase when compared to the same solution without TEMPO: the average shift was 0.365 ppm. The TEMPO
also caused a slight broadening of the peaks, although not by a statistically significant amount. We let the students decide which of the peaks for aspirin, acetaminophen, and the internal standard to use for integration. The acetophenone had a carbonyl carbon with a chemical shift larger than any of the compounds from the analgesic, so it was simple to pick out and free from interference. While developing the experiment, we used aspirin and acetaminophen peaks from the region of greatest chemical shift (150–200 ppm), finding the spectrum less cluttered here. The student choice was split pretty evenly between using these same peaks and using the peaks in the 20–30 ppm range for their quantitative analysis. Student results for the first trial of the experiment are shown in Table 2. It can be seen that the majority of students were relatively close with their findings, and those who were not invariably had low values. The low values were attributed to not getting all of the material into solution, which was generally the result of not grinding up the analgesic tablet well enough. The experiment took each student about 3.5 hours to complete. Some streamlining can be accomplished by either dropping one or two of the trials or by processing the spectral data off-line. It is unlikely that the students will be called upon to use quantitative 13C NMR in their future endeavors, but the lessons learned are of value. To prepare a lucid laboratory report on this experiment, students are forced to have some grasp of the way modern pulsed FT-NMR operates and an understanding of decoupling procedures and relaxation processes. From a theoretical point of view, students are challenged to consider whether an experiment will produce an analytical signal that is proportional to the concentration of the analyte. Acknowledgments Partial support for this work was provided by the National Science Foundation’s Instrumentation and Laboratory Improvement Program through grant DUE9352752. Additional support for T.A.S. was provided by Knox College in the form of a Presidential Internship. Literature Cited 1. Rablen, P. R.; Deuber, M. A.; Lim, A. C.; Dickson, R. M.; Wintner, C. E. J. Chem. Educ. 1991, 68, 796–797. 2. Cook, A. G. J. Chem. Educ. 1993, 70, 865–866. 3. Hughes, J. G.; Lawson, P. J. J. Chem. Educ. 1987, 64, 973–974. 4. Hoff, J. L.; Furtsch, T. A.; Mills, J. L. J. Chem. Educ. 1979, 56, 125–126.
Vol. 73 No. 11 November 1996 • Journal of Chemical Education
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In the Laboratory
Table 2. Student Quantitative Results Student
Aspirin Weight (mg) Acetaminophen Weight (mg)
1
246
227
2
196
175
3
105
105
4
163
150
5
255
242
6
248
245
7
259
235
8
217
179
9
252
242 225
10
212
Mean
215
203
Label Value
250
250
5. Sawyer, D. T.; Heineman, W. R.; Beebe, J. M. Chemistry Experiments for Instrumental Methods; Wiley: New York, 1984. 6. Weast, R. C., Ed. CRC Handbook of Chemistry and Physics, 70th ed.; CRC: Boca Raton, FL, 1989. 7. Gunther, H. NMR Spectroscopy; Wiley: New York, 1980. 8. Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy, 3rd ed.; VCH: New York, 1987. 9. Neuhaus, D.; Williamson, M. The Nuclear Overhauser Effect in Stereochemical and Conformational Analysis; VCH: New York, 1992. 10. Huff, B. B., Ed. Physician’s Desk Reference, 43rd ed.; Medical Economics: Oradell, NJ, 1989. 11. Becker, E. D. High Resolution NMR, 2nd ed.; Academic: New York, 1980. 12. Field, L. D.; Steinhell, S., Eds. Analytical NMR; Wiley: New York, 1989. 13. Atta-ur-Rahman. Nuclear Magnetic Resonance; Springer: New York, 1986.
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