Interference from Ethanol-Stabilized Chloroform in the Infrared Spectrometric Determination of Certain Deuterated Compounds S. Edward Krikorian Department of Medicinal Chemistry, University of Maryland, School of Pharmacy, Baltimore, Md. 2 120 1
One consequence of the failure to remove the ethanol used as stabilizer in commercial reagent-grade chloroform employed as an infrared solvent is demonstrated. The ethanol can serve as an agent for de-deuterating certain labeled solutes having deuterium situated at a readily exchangeable site. Thus, N-d-+caprolactam is shown to undergo significant conversion to its N-protio form in commercial chioroform solution via an in situ D-H exchange reaction with the hydroxyl group of the atcohot. Removal of the stabilizer can be effected by treating the solvent with Type 4A molecular sieve and eliminates the Interference.
The value of deuteration as a tool for elucidating the origin or character of absorption bands in the infrared spectra of organic molecules is well known (1).However, it is important that, during the spectroscopic measurement, care be taken that the deuterium label in the molecule being investigated is not lost via in situ deuterium-hydrogen exchange processes, especially where the deuterium is situated a t a readily exchangeable site, such as on a heteroatom. This is probably best accomplished by analyzing the compound in its pure solid, liquid, or vapor state or, in the case of a solid, in an inert matrix such as a mineral oil mull or KBr pellet. Where solutions of the labeled compound must be analyzed, then an aprotic solvent such as CC14 or CS2 would be expected to be most suitable for ensuring the preservation of the deuterium label. Chloroform is a useful solvent for infrared spectroscopy and, because of the low lability of its hydrogen atom, also would be expected to pose little threat to compromising the label in deuterated compounds via potential D-H exchange processes. It has generally been recommended that the ethanol normally present as a stabilizer in commercial reagent or spectrophotometric grade chloroform be removed prior t o use of the latter as an infrared solvent (2, 3 ) , although the reasons for the recommendation have not always been made clear. This precaution is easy to disregard, usually with no serious consequences when routine infrared spectra of organic compounds are being determined for structural elucidation or identification. However, we wish to report in the present study a case in which the alcohol can be a serious source of interference by acting as an agent for de-deuterating certain labeled compounds when ethanol-stabilized chloroform is used as solvent therefore. Thus, it would appear that its removal is especially to be prescribed when such chloroform is used for this purpose.
EXPERIMENTAL Reagents and Chemicals. The solvents employed were reagent grade. Ethanol used as stabilizer (0.50% by volume, 0.086 M )was removed from reagent-grade chloroform by allowing the solvent to stand overnight over 8-12 mesh beads of Type 4A molecular sieve ( 4 ) under an atmosphere of nitrogen in a stoppered flask kept in the dark. The purified solvent was used directly upon decantation from the molecular sieve as needed and within a day of preparation. N-d-+Caprolactam was prepared by repeated hydrogen-deute190
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rium exchange reactions ( 5 ) of 10 mmol of +caprolactam (Aldrich, 99.9+%) with five successive fresh 5-ml portions of D20 (99.75 atom % D),the final product being dried over P205 in a vacuum desiccator after evaporation of the last portion of spent D20.All solutions of the caprolactams used for infrared spectral studies were prepared a t a concentration of 0.10 M in the appropriate solvents. Infrared Spectral Measurements. All infrared spectra were obtained using a Perkin-Elmer Model 257 spectrophotometer scanned at medium speed and sodium chloride cells of 0.1-mm pathlength. Spectra of solutions were recorded vs. the pure solvent as reference.
RESULTS AND DISCUSSION During the course of infrared studies on certain lactams ( 6 ) , we were struck by an apparent disagreement in the degree of N-deuteration achieved upon hydrogen-deuterium exchange of the lactams with D20, as determined by a comparison of infrared spectra of CC14 and CHC13 solutions of the products. This observation led us to conduct the following investigation. A sample of N-d-e-caprolactam was shown to be completely deuterated by the absence of N-H stretching bands (u") a t 3450-3100 cm-l and the presence of N-D stretching bands ( U N D ) a t 2550-2250 cm-l (5, 7) in the infrared spectrum of a CC14 solution of the compound (Figure 1A). However, when the same sample of the lactam was rerun in CHC13 containing 0.5%ethanol as stabilizer, bands for both U N H (3420 cm-l) and U N D (2540 cm-') were present (Figure 1B), indicating that the sample was now a mixture of deuterated and undeuterated lactams. Based on the relative intensities of the 3420 cm-l U N H band for a sample of pure undeuterated 6-caprolactam and that for the N-deuterio derivative determined a t the same concentration in ethanol-stabilized CHC13 solution, it is calculated that 60% of the deuterium label of the latter was lost via an in situ deuterium-hydrogen exchange process occurring in the solvent (within 30 min or less of preparing the solution). Two features of Figure 1B unequivocally implicate the ethanol specifically as the agent responsible for the partial conversion of N-deuterio to N-protio lactam, viz., the negative absorption peak a t 3630 cm-l and the absorption band a t 2680 cm-l (arrows). The former undoubtedly is a manifestation of the loss of an hydroxylic compound ( Y O H for unassociated ethanol) (8) from the solution contained in the sample cell. The second feature can then be clearly ascribed to UOD (calcd 2650 cm-', from i j o ~= 0.73 i j o ~obtained from the Hooke's law expressions for the 0-H and 0-D stretching vibrations) arising from the conversion of EtOH to EtOD due to H-D exchange with the N-d-lactam in the sample cell. If this is the case, the 60% loss of deuterium from the N-d-lactam observed above represents 70% of the theoretical loss possible if this exchange reaction were complete, based on the relative molar amounts of ethanol and N - d 6-caprolactam present (see Experimental section) in the chloroform solution analyzed. T o prove the suggested mechanism for the partial loss of deuterium from the labeled compound, the above procedure was repeated with ethanol-free chloroform to circumvent the exchange process. Although many chemical meth-
ods (9) as well as adsorbing agents, such as alumina (IO, 1 1 ) and silica gel (2), are useful for removal of ethanol from chloroform, we found that simple overnight standing of commercial chloroform over Type 4A molecular sieve was extremely effective for this purpose. This was shown by comparing infrared spectra of the commercial, ethanol-stabilized CHC13 measured vs. itself and vs. the sieve-treated CHC13 as reference. Whereas in the former case a well compensated straight line was obtained, in the latter case the spectrum of a dilute solution of ethanol was recorded (e.g., bands a t 3630, 2980, 2900, 1050, and 880 cm-'), indicating successful removal of ethanol from the CHC13 in the reference cell by the molecular sieve treatment. Indeed, when the infrared spectrum of the sample of Nd- +caprolactam was remeasured in the ethanol-free chloroform (vs. the same chloroform as reference) no evidence for deuterium-hydrogen exchange was discerned (absence of U N H absorption a t 3420 cm-l), and the spectrum was that for the pure N-d-lactam (Figure 1C). (The U N D region differs from that observed in CC14 solution, Figure l A , because of the difference in the degree of hydrogen-bonded self-association of the lactam in the two solvents.) This result (cf. Figures 1B and 1C) clearly confirms the interference from ethanol in the obtaining of spectra of the N-dlactam in commercial chloroform solution. We have noted this interference further with other N-d-lactams and also with N-deuterio open-chain primary and secondary amides as well. Presumably such D-H exchange would also be a problem with any compound having readily exchangeable deuterium atoms, such as 0-d-carboxylic acids and alcohols, although an investigation of the full extent of the problem has not been undertaken. In any case, as a precaution the admonition to remove ethanol from commercial chloroform prior to using it as a solvent for deuterated compounds should be heeded.
CONCLUSIONS When commercial reagent or spectrophotometric grade chloroform is used as a solvent in infrared spectrophotometry, failure to remove the ethanol used as stabilizer therein can cause loss of the isotopic label of certain deuterated compounds via deuterium-hydrogen exchange involving the alcohol. Since the procedure for removing the ethanol can be as simple as allowing the solvent to stand over molecular sieve for a time, this operation could well be made a matter of routine practice generally whenever chloroform is used as a solvent for infrared spectrometric work. However,
3500
4000
1100
3000 *IYIYU*IIR
1000
8lm-1,
Figure 1. Partial infrared spectra of 0.10 Msolutions of N-d-c-caprolactam
(0.I - m m pathlength cell).
(A) in CCI4, ( B ) in commercial reagent-grade CHC13 containing 0.5% ethanol as stabilizer, ( C ) in ethanol-free CHC13
the purified chloroform should be protected from bright illumination and used immediately, since HC1 (as well as phosgene) is generated upon exposure of the unstabilized solvent to light and air (9). This precaution is especially important in the spectrometric determination of basic compounds, whose infrared spectra may be affected by protonation by dissolved HC1.
LITERATURE CITED (1) F. Halverson, Rev. Mod. Phys.. 19, 87 (1947). (2) A. R. H. Cole in Technique of Organic Chemistry", Vol. Xi, Part 1, A. Weissberger, Ed., Interscience Publishers, New York, N.Y., 1963, p 137. (3) R . T. Conley, "Infrared Spectroscopy", Aiiyn and Bacon, Boston, Mass.; 1966, p 64. (4) L. F. Fieser and M. Fieser, "Reagents for Organic Synthesis", John Wiley and Sons, New York, N.Y.. 1967, p 703. (5) A. Koshimo, J. Appl. Po/ym. Sci., 9, 55 (1965). (6) S. E. Krikorian, T. A. Andrea, and M. Mahpour. submitted for publication in Spectrochim. Acta. (7) A . V. logansen and M. Sh. Rozenberg, Zh. Prikl. Spektrosk.. 9, 1027 (1968). (8) U. Liddel, Ann. N.Y. Acad. Sci., 69, 70 (1957). (9) J. A. Riddick and W. B. Bunger. in "Techniques of Chemistry", Vol. 11, 3rd ed., A. Weissberger, Ed., Wiley-lnterscience, New York. N.Y., 1970, p 771. (10) G. Wohlleben, Angew. Chem., 66, 752 (1956). (1 I)K.B. Whetsel and J. H. Lady, J. Phys. Chem., 68, 1010 (1964). "
RECEIVEDfor review August 7, 1975. Accepted October 1, 1975.
Infrared Analysis of Weathered Petroleum Using Vacuum Techniques Chris W. Brown* and Patricia F. Lynch Department of Chemistry, University of Rhode Island, Kingston, R.I. 0288 7
Treatment of unweathered oils with warm salt water and subsequent evacuation of the dried sample, was found to simulate the natural weathering of oils which had been in the marine environment for 2-7 days. This treatment provides a quick, accurate method for correctly matching the fingerprint of a spill sample to the source. Furthermore, It
gives Insight into the Initial changes of an oll during weathering.
The identification of the source of weathered oil presents a problem to investigators in the environmental-analytical field. It is a problem because the chemical components of ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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