'H NMR Analysis of Mixtures Using Internal Standards A Quantitative Experiment for the Instrumental Analysis Laboratory Jim Peterson The University of Alabama,Tuscaloosa, AL 35487
Quantitative NMR spectroscopy still appears to be largely ignored in standard analytical chemistry courses and textbooks. In fact, there is a t least one recently published instrumental text which contains no discussion of NMR a t all ( I ) , and there is certainly a paucity of detailed reports of quantitative procedures suitable for use in the analytical teaching laboratory This is a n unsatisfactory state of affairs given the wide applicability of 'H NMR and 13C NMR to qualitative product analysis i n synthetic chemistry (2)and also, the numerous situations in which NMR methods can be usefully and sometimes uniquely applied to quantitative determinations (3-6). However, the procedures involved in these measurements are not trivial and the instrumentation necessary not available in undergraduate laboratories. consequekly, the 'H NMR experiment described here was developed as a teaching exercise, illustrating the general principles of quantitative NMR methodology, for use in the instrumental analysis course a t The Universitv of Alabama. Accentable data can routinely be collected in about a n hour on a 60 MHz continuous wave spectrometer. The Experiment ACS certified reagents are used throughout (Fisher Scientific) and 5 mm thrift NMR tubes (Wilmad). Spectra are recorded on Varian EM 360A spectrometers. Students taking the instrumental course a t The University of Alabama are seniors who have been previously introduced to 'H NMR spectroscopy in organic chemistry courses and who have all taken a t least one semester of physical chemistry At the start of the laboratory session, the students are instructed to make up their standard solutions following the normal volumetn~procrdureiiemploying 1-mL and 10ml. maduated oioets fitted with bulbs and 20-ml. volumetric flasks. Warnings are issued concerning the hazardous nature of chlorinated hydrocarbons. Attention is drawn to a suitable container reserved for storage of this type of waste prior to its disposal in a n EPA-licensed incinerator a s prescribed (7). The following written instructions are provided.
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'H NMR Analysis of Trichloroethane Mixtures The composition of mixtures can be quantitatively analyzed by NMR spectroscopy if distinguishable signals from each component can be integrated separately. The use of a n internal standard renders the integration a n absolute measurement. In this particular experiment the composition of a mixture of l,l,l-trichloroethane and 1,1,2trichloroethane is determined by 'H NMR spectroscopy. The chemical shifts of interest, in ppm on the 6 scale, are trichloroethene
: 6.5
I ,2-dichloroethane
: 3.7
1,I ,Btrichloroethane
: 3.9 (doublet);5.8 (triplet)
Reagents Use 1% (vlv) TMS in carbon tetrachloride a s the solvent to prepare five standard solutions with composition:
Prepare a second set of five standards using 1,1,2trichloroethane instead of l,l,l-trichloroethane. Procedure Introduce approximately 0.5 mL of a standard solution into a n NMR tube and record the 'H NMR spectrum between 8.0 and 0.0 on the S scale. Integrate the signals. Continue in the same manner with the other solutions to prepare calibration curves for l,l,l-trichloroethane and 1,1,2-trichloroethane, using both internal standards (i.e., generate four curves in all). Obtain from your instructor a n unknown consisting of a mixture of l,l,l-trichloroethane and 1,1,2-trichloroethane in carbon tetrachloride~TMS.Add l,2-dichloroethane to 5% (vlv) and trichloroethene to 15% (vlv) a s internal standards. Proceed to record the 'H NMR of this solution over
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Calibration curves for trichloroethane isomers. All concentrations are expressed as percent (vlv).Internal standards were 15%trichloroethene (0)and 5% 1,2-dichloroethane(A). Lett: Calibration curves for 1,l.l-trichloroethane.The ordinate axis is the ratio: integrated intensity of l,l,t-trichloroethanepeak divided by integrated intensity of internal standard peak. Right: Calibration curves for l,i ,Ptrichloroethane.The ordinate axis is the ratio: integrated intensity of t ,I 2trichloroethane triplet divided by integrated intensity of internal standard peak. the same range a s the standards and integrate the signals obtained. Determine the wmposition of the unknown mixture by comparing the measured integrated intensity ratios (unknownlstandard) of the signals with those of the calibration curves. Use the data sets generated with each internal standard indeoendentlv to obtain two results. Report these in terms of the composition of the mixture as it was suoolied to vou (i.e.. before the addition of the intrrnal standarks) quoting the 95% confidence limits. Explain which of the two results you think is the more reliable and why. How can the experiment be improved? Student Results I n the figure are shown some typical calibration curves for l,l,l-trichloroethane and 1,1,2-trichloroethane using Determination of the Composition of Trichloroethane Mixtures
Internal Standard:trichloroethene 1,I ,I-trichioroethane
1,1,2-trichloroethane (% vol.)
(% VOI.)
Sample a b c d
15 20 20 25
Sample a b e f
Supplied
Supplied
Found
Supplied
13(+2) 18(+3) 261~3) . . 25(+2)
25 30 15 15
Numbers in parentheses are 95% confidence limits.
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Found
i5(d) 25 26(*3) 20(*1) 30 30 k3) 19(d) 30 27k4) 15 13(+3) 24(+2) Internal Standard: 1,Pdichloroethane
Supplied 15 20 25 25
Found
Journal of Chemical Education
Found 23(+2) 27(+5) 16(+1) . . 17(+3)
both internal standards, where the solid lines are the best fits to the data points. Most students are able to obtain calibration data of this apparent quality; i.e., reasonably linear plots that extrapolate close to the origin. However, on closer inspection, it becomes clear that these data are not entirely satisfactory. For instance, the ratio of the slopes of the two calibration curves for each isomer with the same internal standard should theoretically be 1.33, but, in the left and right sides of the figure, this ratio is 1.56 and 2.14, respectively. The discrepancy cannot be explained on the basis of differential losses of volatile inteeration standards from solutions. since the two comwunds In questlon have smular b o h g points (83 "C and 8 i O C fur 1.2-dshlorocthane and trlchloroethcnc. resoectivelv) Additionally, neither l,l,l-trichloroethane (d.p. 74% "C), nor 1,1,2-trichloroethane (b.p. 110-115 'C) are especially volatile liquids; therefore, losses due to evaporation are not significant in this experiment. Approximately two thirds of the students performing the analysis obtain calibration data exhibiting this kind of discrepancy and not surprisingly, their final results are in error. Poor volumetric technique in preparation of standards and failure to ensure that the spectrometer remains properly tuned during the entire procedure seem to be the main problems here. A referee has pointed out t h a t preparing standards by weight using a n analytical balance would avoid the volumetric difficulty and lead to improved precision. This is quite correct and indeed, if state-of-the-art instrumentation were being employed, would be the method of choice. However, the reproducibility of integrations performed on the spectrometers that we use for teaching purposes varies by f3% (or more) and wnsequently, the increased precision to be obtained in preparing standards by weight is nnwarranted. I n addition, we feel students benefit from the opportunity afforded by this experiment to practice their volumetric technique. Stock solutions of unknowns for analysis are made up volumetrically in 100-mL quantities by a n instructor prior to the laboratory session. Generally, a different stock solution is prepared for every three students expected to be in the class. This is then divided into four individual unknowns, each with a unique identification label. Three of these are given to students for analysis and the fourth retained in case it subsequently becomes
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necessarv to confirm the com~osition.C o m ~ a r i nthe ~ resnlts of students with identical unknowns irovides a convenient means of checking for possible errors in their preparation. Giving students unknown samples in 25-mL quantities enables them to add their internal standards by pipeting the appropriate amounts of trichloroethene and 1.2-dichloroethane directly into a 20-mL volumetric flask and then diluting up to the mark with the unknown as supdied. Most students need to be directed by an instructor to proceed in this fashion, but some of the better ones solve the problem for themselves. In the table are given some examples of actual analyses performed by various students with reference to both internal standards. These data are representative of the one third (or less) of our students who produce satisfactory results. I t should be pointed out that we observe no significant difference between the two internal standards in terms of the aualitv of the results obtained by students. This is a littlesurpksing since there is some interference between the spectrum of 1,2-dichloroethane and the analyte 1,1,2-trichloroethane. The interference is deliberately included in our procedure. Students are asked by the instructor to comment on this and make recommendations to overcome the problem. The use of shift reagents, or other internal standards, are frequently suggested approaches. While these are certainly valid suggestions, we are primarily seeking to use this question as an opportunity to impress upon the student the value of working at other frequencies and with other NMR nuclei. Should this aspect of the exercise not be required by some instructors, then it is relativelv easy to find an internal standard that does not interfere fromamong those suggested by Wallace (8). Additional Remarks I t is to be stressed that the motives underlying the development of this experiment were purely of a didactic uature. In fact, t h e composition of t h e mixtures of trichloroethane isomers employed is probably most conveniently quantified chromatographically, since they have quite different boiling points. If a "more realistic" experiment is required, i.e., a determination which cannot be performed routinely in some other manner, then analysis of the composition of mixtures of various trichloro-, or tetrachloro-, substituted benzenes is a good option. In either case, the use of organic chlorides in this laboratory exercise ~rovidesa convenient link to an important class of environmental contaminants (9)inclu-ding 2,3,7,8tetrachlorodibenzo-p-dioxin(TCDD), which continues to attract controversy(lO). If more than one laboratory period is available, a possible modification of the experiment is not to reveal the chemical nature of the analytes present. The analysis is then divided into two parts, the first involving qualitative
identification of the components of the mixture using previously suggested methods (11)and the second, the quantification of the composition in the manner described here. A very significant advantage of NMR determinations is that they do not require the availability of pure analyte for calibration purposis. The generation of a cdibration curve is, therefore, not absolutely necessary for this type of quantitative measurement, but there are some d i s t k t advantages to the particular procedure employed. Perhaps most importantly, it provides a convenient means of estimating the indeterminate error involved in a much more interestine fashion than s i m.~" l vr e ~ e a t i n the e same measurement several times. However, it is also useful for students to observe the linear relations hi^ between concentration and area under the peak (or multiplet) because unless demonstrated to the contrary, some of the weaker students seem to think that there should be a linear relationship between presumably by analogy concentration and peak height, . with optical spectroBcopic measurements more familiar t o them. In addition, this experiment presents the student with an e x a m ~ l eof the methodolow of determinations using internal standards in a very general form. The use of two internal standards is intended to reinforce the importance of selecting standards with resonance peaks close to those of the analytes. The instrumental analysis laboratory a t The University of Alabama is closely integrated with the theoretical part of the course. Consequently, the laboratory instructions given to students do not explain in detail how to analyze the data, since this will already have been covered. If the a .. ~ ~ r. o ~ r iDrocedure ate has been foreotten (or more likelv never properly Icarned~then the students are expected to look this uv for themselves in standard texts 112 . A l l ofour students are able to accomplish this task (sometimes following further consultation with an instructor) and a t the end of the exercise have a good appreciation of both quantitative NMR measurements and the method of internal standards.
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Literature Cited
Educ. 1 W 67.802. , S.nt.Imir. 1991,32,987-990 and references therein. 3. wsng. e; ~ o a h e rH. 4. Lundbere. -. P:Harmsen.. E.:, Ho. C.: Voeel. H. J A n d Biochem. 1990.191. . . 193-222 and references therein. 5. Allerhand. A,; Maple. S. R.Awl. Chom. 198'7 59,44m-4521). 6. Chen, C. -L: Robert, D. Method. inEnrymoiogy;Abelson andSimon. Eda. Academic Re%, 1988,161. pp 137-174. 7. Aidrich Cofolog11990-19911 ppF16.Fl7. The most recent version ofthiseatslog lor that ofsome other manufadurer) is nearly always the most readily available. up fo date source of sub information. 8. Wallace. T. J Chem.Educ. 1984 61. 1074. 9. Solomo& T.W G. Organic Chemistv, 4th ed.;Wiley: NewYork. 1988: pp 72&733. . J. C & E N 1991.69 132) 7-14. 10. Henson. D 11. Phlllips,J. $.;Lea- J.J. J . Chom. EduclSsB, 63,545-546. 12. Willard, H. H.; Memitt, L. L. Jr:Dean, J. A,; Settle, F. A. Jr. ImtrumntolMethods ofAnolpis, 7th ed.;Wadsworth Publishing Company, 1988: pp 454458.
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