Digitization and Processing of Nuclear Magnetic Resonance Spectra Roger E. Rondeau and Vincent L. Donlan Air Force Materials Laboratory, Wright-Patterson Air Force Rase, Ohin 45433 THEADVENT of high speed computers has greatly simplified the problem of extracting chemical shifts and coupling constants from multispin paramagnetic resonance (PMR) spectra. The spectral parameters of complicated spectra are now usually obtained through the use of iterative computer techniques ( I , 2). Spectral analyses are far from being automatic, however; the main task is still the assignment of individual experimental frequencies to the many possible transitions between levels in an energy level diagram (3). In making these assignments, the experimental lines must be accurately measured-not only to ensure the correct determination of the energy levels but also t o check the agreement between a calculated spectrum and a n experimental one. It is preferable, therefore, that as large a number of experimental frequencies be measured with as high an accuracy as possible. Experimentally, this means that peaks from several scans must be measured and averaged. Also, spectra must be scanned in both directions (low field to high and high field t o low) to correct for peak asymmetries. Thus, the careful manual measurement of experimental frequencies from several spectra is an essential but extremely tedious and time-consuming process. By using an analog-to-digital converter to digitize the PMR spectra and by formulating a computer program to edit and manipulate the digitized data, we’ve not only eliminated the time and tedium of hand measurements but also significantly increased the accuracy of the measurements. Once the ability to digitize and record spectra is available, the speed and precision of a digital computer can be invoked to treat the raw data in a variety of ways. One method which we have used to process the digitized data is described in this paper. Benzisoxazole, whose spectrum is a complex ABCD system, was used to demonstrate the technique. EXPERIMENTAL
Apparatus. The P M R spectra were recorded with a Varian HA-601L spectrometer operating in the frequency sweep mode. A sealed sample tube was used which consisted of neat, degassed benzisoxazole and 3 tetramethylsilane for use as an internal lock signal and reference peak. Digitization of the spectrometer output was performed by a n analog-todigital (AID) converter system designed and fabricated by Technology Incorporated 7400 Colonel Glenn Highway, Dayton, Ohio. The A/D system converts spectrometer voltages to binary coded decimal (BCD) values and stores these values on magnetic tape along with channel identification, signal polarity, and decimal point location. The control logic of the T I converter sequences and governs the operation of the three basic units in the system: a Vidar Model 520 integrating digital voltmeter, a Vidar model 604 analog scanner (Vidar Corporation, 77 Ortega Ave., Mountain View, Calif.), and a Kennedy Model DS 370 stepping tape recorder (Kennedy Company, 275 Halstead St., Pasadena, Calif.). Data are recorded in seven tracks o n a l/n-inch tape at a character (1) J. D. Swalen and C. A. Reill). J . Chem. Phys., 37, 21 (1962). (2) S. Castellano and A. A. Bothner-By, ibid., 41, 3863 (1964). (3) S. M. Castellano and A . A. Bothner-By, ibid., 47, 5443 (1967).
density of 556 per inch and a t a rate compatible with standard IBM computers. All computations were performed on a n IBM 7090/7094. A Calcomp plotter operating at a resolution of 5 mils was used for plotting the taped data. Procedure. With the data acquisition system wired to monitor the Varian spectrometer recorder pen voltage, a typical run proceeds as follows. The minimum sweep width required t o include all of the resonances of interest is selected. A convenient offset frequency is then dialed at the spectrometer console. This new frequency setting in frequency units (Hz)from the internal TMS lock signal, is calibrated with a frequency counter and serves as the reference point for all of the spectral peaks. A sweep time T and sweep direction are then decided upon. If the sweep direction is from left to right, the field increases from H, to H , 4- W ; on a right to left sweep, W to H , during the sweep. the field decreases from H, The sweep is started, and the pen begins to record the spectrum on the chart paper. The magnetic tape drive is then turned on. The tape transport is activated by a stepping motor which advances the tape at a constant, preselected stepping rateP(0.188, 0.375. 0.75, 1.5, 3, 6. or 12 sec-I). At each advance of the tape, the data acquisition system commands the digital voltmeter to measure the pen voltage. The voltmeter obtains the average pen voltage during some integrating time interval (which can be selected to be I,B, or ‘/e00 sec). The binary coded voltage digits, together with a sign bit and the decimal point position, are sensed at the digital voltmeter output terminals, packed into a 36 bit word, and transmitted to the write head of the tape recorder. Before the end of the sweep is reached, the tape recorder is stopped and an end of file mark is written on the tape. The same procedure is followed on each subsequent sweep. Computer Program. A computer program called EDREM (Editing Digitized, Repetitive Experimental Magnetic Resonance Data) has been. written to measure and average the positions of the absorption peaks. In order to distinguish between real peaks and noise peaks, and between real peaks and ringing peaks, several precautions and tests were incorporated into the computer program. First, the raw data are smoothed by means of a parabolic point by point smoothing routine. Second, every peak below a certain “discrimination” voltage is ignored. A convenient value for this threshold voltage is easily determined from visual inspection of the chart recorded spectra. Third, any peak that occurs within a certain minimum distance of the previous valley is ignored. This minimum distance is determined by the resolution R (in Hz) of the spectrometer. In terms of data points, the computer program ignores peaks occurring within (RTP. W data points of a previous valley. For our instrument, we use a value R = 0.3 Hz. Fourth, the peak to valley voltage difference on either side of a maximum must be greater than some “noise” voltage, which is chosen by visually inspecting the pen records. Finally, only peaks which occur at approximately the same position in each sweep are retained. The criterion applied is that a peak be within 2 R of the average position of one of the peaks retained from the preceding sweeps. It is this last criterion that is most effective in assuring that the spurious ringing peaks are discarded. Output from EDREM consists of tables of spectral peaks fcr each data file (NMR spectrum). These maxima are tabulated in terms of the data point at which the maximum
+
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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ANALOG
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!“
I, t
DIGITIZED
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I
Figure 1. Spectrum of benzisoxazole Top: 60 MHz spectrum of neat, degassed benzisoxazole recorded from left to right. Offset frequency was 100 Hz with a sweep width of 100 Hz. Peaks correlated by program EDREM are denoted by vertical lines. The “noise” and discrimination voltages used were 5 mV. Data were recorded at a rate of 3 points per second, with a total sweep time of 985 sec Bottom: Calcomp playback of the digitized spectrum recorded simultaneously with the top trace. Intensity is in volts and field in total data points 1700
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was recorded, the voltage at the maximum, and its distance from a spectral reference peak. After correlating the peaks from the several files, the program averages the correlated peaks and tabulates them in terms of H z from TMS. TO ensure that the pen voltage is faithfully monitored and that no time scale distortion occurred in the data conversion, the program has the option of plotting the raw data on a Calcomp plotter. Each analog spectrum, therefore, can be visually compared with its digitized version. The program will also punch the correlated frequencies o n cards and in the format required by LAOCN3 (2). A Fortran IV listing of program EDREM is available upon request. RESULTS AND DISCUSSION
A typical N M R spectrum of benzisoxazole is shown in Figure 1 (top). The spectrum is reproduced from the pen record of a single left to right sweep-;.e., a low field to high field sweep. The bottom part of Figure 1 shows a Calcomp playback of the same sweep, as recorded simultaneously on our data acquisition system. T o obtain the peak frequencies of benzisoxazole, we recorded ten such sweeps, five in each direction, as input to EDREM. The parameters for these sweeps were: W = 100 Hz, P = 3 sec-I, T = 985 sec, integration time per data point = ‘ / e sec. The discrimination and “noise” voltages used were 5 mV. The correlated and averaged peak positions found by EDREM from these ten sweeps are shown as vertical lines in the top part of the figure. There is sufficient difference in the apparent peak positions between one sweep and the next, especially between sweeps in opposite directions, to require averaging over several sweeps.
One reason for these measured differences is the ringing phenomenon evident on the right hand side of the peaks shown in the figure. The ringing prevents the pen from following each resonance faithfully and thereby gives a false indication of the peak positions. The average position of a peak, however, in the limit of a large number of sweeps taken in alternate directions, approaches the true position. In the case of the ten spectra, five in each sweep direction, recorded in the benzisoxazole analysis, the positions of 38 peaks (see Figure 1) were correlated and averaged by EDREM, which punched the computed frequencies onto cards in the format required by LAOCN3 ( 2 ) . In the absence of a data acquisition system, the usual procedure had been t o generate several spectra (typically three in each direction), manually measure the position of each peak in each spectrum, and compute the average positions. With the ability to automatically digitize and record the pen voltage, it is now feasible to carry out the peak identification and position averaging rapidly and without error on a digital computer. Eliminating the manual labor also allows for an arbitrarily large number of sweeps to be included in the averaging, up to the limit imposed by instrument drift. The only hand computation remaining is that required to average the occasional peaks missed by EDREM. The utility of o u r technique is limited only by the spectrometer ability to resolve a particular spectrum-whether it be that of a solution or a neat liquid. RECEIVED for review April 20, 1971. Accepted June 16, 1971.
3’,5’-Cyclic Adenosine Monophosphate Phosphodiesterase Assay Using High Speed Liquid Chromatography Sam N. Pennington Dirision of’kledicaf Sciences and Department of Chemistry, East Carolina Unicersity , Greenaifle, N . C. 27834
THEIMPACT of cyclic adenosine monophosphate (Cyclic-AMP) on biochemical research has been extremely large and research publications dealing with this compound now number in excess of 4000 ( I ) . One of the particular areas of interest in Cyclic-AMP is that of the enzyme 3’,5’-Cyclic-AMP phosphodiesterase (PDE). This enzyme catalyzes the following reaction:
More thari 300 publications have appeared between 1957 and 1969 dealing with this particular enzyme ( 1 ) . A number of assay met nods exist for PDE including potentiometric ( 2 ) , isotopic (3), and spectrophotometric (3, 4). Spectrochemical methods gerierally involve coupled enzymatic reactions to yield inorganic phosphate which is determined by the method of Fiske-SubbaRow ( 5 ) and have good sensitivity. The potentiometric method lacks the sensitivity necessary to d o tissue level assay and isotopic methods have the problem of expense and hazards associated with radioactivity. We desired a rapid method capable of doing tissue level (liver) assays and preferred to measure the reaction products directly for the obvious analytical reasons. (1) “Cyclic AMP 1957-1969,” N. S. Semenuk and H. Zimmerberg,
adenosine-5’monophosphate ’ (AMP)
HO OH
Ed., E. R. Squibb and Sons, Inc., Research and Development, Science Information Department, New Brunswick, N. J. 08903, @ 1970. (2) W. Y. Chwng, Ami. Biochem., 28, 182 (1969). (3) P. S. Schonhofer, I. F. Skidmore, G . Krishna, and H. R. Bourne, 2. .4nal. Chem., 252 182 (1970). (4) F. Eckstein and Hans-Peter Bar, Biochim. Biophys. Acta, 191 316 (1969). (5) C. H. Fisk.e and Y. SubbaRow. J. B i d . Chem., 66 375 (1925).
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