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  • Virtual Spectrometer Tutorial for Microsoft Windows 3.1, Windows-95, Windows NT and Macintosh Acorn NMR Inc. 46560 Fremont Blvd., #418 Fremont, CA 94538-6491 Telephone: (510) 683-8595 FAX: (510) 683-6784 Email: info@acornnmr.com http://www.acornnmr.com 1
  • Virtual NMR Spectrometer Tutorial This tutorial covers a series of exercises intended to illustrate the effects of the basic NMR data acquisition parameters. It is assumed the NUTS program has been successfully installed and initiated. See the NUTS User’s Guide for details. Note, in the current version, the shims are perfect (pure lorentzian lineshape) and relaxation effects have not been included. Also, the only choice for experiment is Single-Pulse. Future versions will include shimming, relaxation, and decoupling experiments. On-line Help is available, which gives a description of each parameter. For each of the exercises below, a description of how to perform the basic operations is provided. However, often there is more than one way of accomplishing a task and all choices are not given. Refer to the NUTS manual or on-line Help for details on how to use the NUTS program. These exercises are intended to be followed in order, thus, descriptions given in a previous exercise will not be repeated. The Virtual Spectrometer is an add-on to the NUTS NMR data processing software. It consists of a set of commands which simulate an FT-NMR spectrometer, producing realistic data corresponding to acquisition parameters set by the user. It is designed to mimic a "vanilla" NMR spectrometer, rather than imitating any actual manufacturer's instrument. In addition to the functions which are specific to the Virtual Spectrometer, all of the processing capabilities of NUTS are also available. As with the rest of the NUTS program, most commands can be executed either from the menu or as keyboard commands, usually 2-letter commands which are executed without typing <Enter>. The virtual NMR sample is a text file containing some parameters and a list of NMR frequencies and their intensities. Several sample files are provided, which are listed below. The file must contain certain keywords which allow the Virtual Spectrometer to interpret correctly the data being read from the file. The file is most easily created from within the NUTS spin simulation subroutine (NS), but can also be created from scratch or edited using any text editor. The following sample files are provided, although not all are used in the exercises: pyridine.fs (pyridine) ethoh1.fs (1 mM ethanol) mek.fs (methyethyl ketone) ethoh2.fs (2 mM ethanol) vinyl.fs (vinyl acetate) odcb.fs (orthodichlorobenzene) eth_ace.fs (ethyl acetate) pur.fs (purine) nitrbenz.fs (3-nitrobenzaldehyde) The user must set values for several NMR acquisition parameters as well as select the sample whose spectrum will be acquired. The sample is chosen using the GS command or by selecting Get Sample from the vSpec menu. This opens a standard dialog box for specifying the name of a file to be opened. The file itself is not shown. To set acquisition parameters, type VP or select Parameters from the vSpec menu. This opens a dialog box for input of parameters -- see description. Once parameters are entered, the dialog box is closed and data acquisition initiated by typing ZG or selecting Zero and Go from the vSpec menu. Messages are displayed on the screen as acquisition proceeds, and the accumulating FID is displayed. If the user's PC is 2
  • equipped for sound, the FID will be "played" through the PC's speakers. Upon completion of data acquisition, the data can be processed using the NUTS processing capabilities. Commands specific to the Virtual Spectrometer GS Get Sample. Specifies the file from which frequencies will be read. VP Virtual Parameters. Opens a dialog box to set acquisition parameters. ZG Zero & Go. Reads the file containing frequencies and generates the NMR data with the acquisition parameters provided. Virtual Sample The following is an example of a text file which is a "virtual NMR sample"; input for the Virtual Spectrometer. It specifies several parameters of the sample and contains a list of NMR frequencies and their intensities. Sample files for input into the Virtual Spectrometer are most easily created from within the NUTS spin simulation subroutine. This allows input of chemical shifts and coupling constants corresponding to real NMR data and calculates the NMR spectrum. To create a virtual sample file, enter the simulation subroutine by typing NS or choosing NMR Simulation from the Tools menu. Type A or choose Add/Edit Simulation Data from the Edit menu. Enter the number of spins to simulate, along with the chemical shifts and coupling constants in the appropriate boxes. Provide a value for the linewidth. Choose Accept and Recalculate. Now type V or choose Create virtual sample file from the File menu. Fill in a value for the sample concentration (in mM), the sensitivity (to simulate different fields and probes), the 90 degree pulse width and the sample name (optional). Choose Save file and provide a file name. It is recommended to use the same extension for all virtual files so they can be easily distinguished from other file types - such as real data. All virtual files provided with this program end in the extension .fs (to designate “flight simulator”). The result of the calculation is a list of frequency and intensity values. This list can also be created in any text editor, using the following format. NUTSsimulation Sample Name = purine Spectrometer Frequency = 361.005981 MHz Concentration = 30.000000 EBsensitivity = 120.000000 PW90 = 10.000000 Intensity_per_spin = 32.0000 Number Transitions(Hz) Transitions(PPM) Intensities 1 3971.1106 11.0001 32.000 2 3317.6821 9.1901 32.000 3 3245.4805 8.9901 32.000 4 3133.5674 8.6801 32.000 5 1732.8486 4.8001 125.000 6 0.0000 0.0000 10.000 End NUTS Simulation File 3
  • Virtual Spectrometer Parameters Acquisition parameters are input using the Virtual Spectrometer dialog box, which is activated by typing VP or by selecting Parameters from the vSpec menu. A brief explanation of each item is shown below. A more complete description of what each item is and how it affects the final spectrum is available by clicking on the Help button and is also provided in the exercises where appropriate. User: Enter user's name or initials (for information only; does not affect data acquisition) Date: For information only; does not affect data acquisition Experiment: Select from the list of available experiments. Currently the only option is Single Pulse. Scans to do: Enter the number of scans (acquisitions) which will be collected and added to form the final spectrum. Pulse Width: The excitation pulse width in microseconds. Recycle Delay: Delay between repeat scans to allow relaxation (in sec). Receiver Gain: Adjusts the amplitude of the signal going into the receiver. Sweep Width: Frequency range for the spectrum. Offset Frequency: Frequency of the "carrier", or center of the spectrum. Data size: The number of points that will define the final spectrum, must be a power of two (thus, 1K points = 1024). Delay for first point: Delay between the excitation pulse and data collection, in microseconds. The following values are displayed for information only, and cannot be changed directly. Their values will change in response to values of the above parameters. Acquisition Time: The time required to complete a single scan. Digital Resolution: The number of data points per Hertz which will define the final spectrum. Dwell Time: The time between data points, in microseconds. This is determined by the value chosen for Sweep Width. 4
  • Exercise #1. Acquire a spectrum Begin by opening the Virtual Parameter dialog box by typing VP or by selecting parameters from the vSpec menu. Enter your name and the date, then the following values: Scans to do 1 Sweep Width 4000 Pulse Width 10 Offset Frequency 1800 Relaxation Delay 1 Data Size 16384 Receiver Gain 20 Delay for 1st Point 175 Check that Simultaneous Acquisition is selected. Exit the parameter entry box by clicking on OK. Next, select the sample for which a spectrum will be acquired by typing GS or select Get Sample from the vSpec menu. Select the file called eth_ace.fs (in the data subdirectory). Begin acquisition by typing ZG or by selecting Zero & Go from the vSpec menu. When the acquisition is complete, type FT to execute a Fourier transform. Additional processing steps include phasing, setting the chemical shift reference, integrating and generating a list of peaks. Phase: Type QP or choose Phasing-Quick Phase from the Process menu. Reference: Either the solvent, or preferably, a reference compound is used to reference the frequency scale of a spectrum (the chemical shifts). The spectrum can be referenced in one of two ways; the first is general and can always be used, while the second only applies to samples which contain a reference compound (e.g., TMS or TSP, both of which are designated as having a chemical shift of zero). The general method is done outside of all subroutines (press <Enter> a few times to be sure). Choose a peak of known chemical shift, either a solvent peak or a reference compound, align the cursor crosshairs vertically with said peak and type O. This opens a dialog box in which the chemical shift, in ppm, is entered. Please note, it is usually more precise to use Zoom to expand around the peak of interest, then exiting to set the reference. To do this, type ZO or choose Start Zoom Operation from the View menu. Press and hold the left mouse button and highlight the region of interest by wiping across the spectrum and type control-E (for expand). Exit Zoom with <Enter>, and reference the chemical shifts as described above. The second method for referencing is done within Zoom. Enter the Zoom subroutine with ZO, expand around the reference compound (TMS, the most upfield (far right) peak), and type SZ (Set Zero). Type control-F (full) to show the entire spectrum again. Integrate: Next, to integrate the spectrum, enter the integration subroutine by typing ID or choosing Integrate Display from the Process menu. Using the mouse, click to bring a vertical line onscreen. Position this to one side of the peak, and click again. Move to the other side of the peak and click again. Continue for all other sample peaks in the spectrum. To display the integral values, click to bring the vertical cursor onscreen, position it over a peak whose contributing number of protons is known or suspected, and type V (for value). Enter a number in the dialog box, for example 1 for a CH or 3 for a CH3 group, and click OK to exit. Now the integrals are more easily converted to the number of protons contributing to each peak. To display the integrals on the spectrum after exiting the Integrate subroutine (with <Enter>), depress the Tab key. 5
  • Peak Pick: To make a list of all of the peaks in the spectrum, be sure that all subroutines have been exited (<Enter> exits all subroutines). Press and hold the left mouse button to display the crosshair cursor on screen. Position the horizontal line across the peaks in the spectrum to define a threshold and type M (minimum) to set the minimum height for the peak-pick routine. Type PP to choose the peaks; a red line will appear over the selected peaks. To list the peaks on the screen, type CB or control-B. Again, refer to the NUTS manual or on-line Help for more details. The sample selected is a 1H spectrum of ethyl acetate in CDCl3 with TMS as a shift reference. When complete, the processed spectrum should resemble the figure shown below. 2.98 2.89 2.00 10 8 6 4 2 0 ppm Exercise #2. Phasing This exercise explores the origin of spectral phase distortions and how they are corrected. There are two types of phase distortions, referred to as zero- and first-order. The zero-order phase distortion affects the entire spectrum uniformly. It arises from phase lags introduced by the spectrometer’s electronics, and varies from instrument to instrument. The first-order phase distortion is proportional to frequency, varying from zero at the left end of the spectrum and increasing across the spectrum. It arises from imperfect timing during data collection. The digitizer, or Analog-to-Digital Converter (ADC), digitizes the signal coming from the NMR probe at a rate determined by the user's chosen value of Spectral Width. The time between data points is called the Dwell Time. However, the optimum time for the first data point is not at zero time, but after a small delay. This is because the filters used to eliminate frequencies outside the chosen range cause a finite delay in the signal reaching the ADC. By default, most spectrometers (including the Virtual Spectrometer) set the DE value equal to the dwell time, meaning that a delay of 1 dwell time is used. This is usually not equal to the actual 6
  • filter delay, which must be determined empirically. When the DE value does not match the filter delay, baseline distortions (baseline "roll") are seen in the spectrum which make phasing and integration more difficult. If the DE value is too low, the baseline will have a hump ("frown") and the first-order phase value required to phase the spectrum will be negative. If the DE value is too high, the opposite is true: the baseline will have a dip ("smile") and the first-order phase value required to phase the spectrum will be positive. At the optimum DE value, the baseline will be flat and the first-order phase value required to phase the spectrum will be zero. The first- order phase correction, unlike zero-order, is not applied to the entire spectrum uniformly, but is a linear function of frequency. The first-order correction is zero at the left end of the spectrum (pivot point) and increases across the spectrum. This means the zero-order phase should be adjusted first, phasing downfield peaks, and the first-order phase adjusted second, using upfield peaks. Note that neither phase value should exceed 360 degrees. If large values are obtained, all phasing can be removed by typing ZP (Zero Phase) and phasing begun again. Phase Full Spectrum: Open the Virtual Parameter dialog box (VP). Type in the following parameters: Scans to do 1 Sweep Width 4000 Pulse Width 10 Offset Frequency 1800 Relaxation Delay 1 Data Size 16384 Receiver Gain 10 Delay for 1st Point 175 Type GS and select the file vinyl.fs. Type ZG. Fourier transform the spectrum with FT. Now the spectrum is ready to be phased. In exercise #1, the autophase routine was used. This time, manual phasing routines will be used. See the User’s Guide or on-line Help for details on phasing. This spectrum has negligible first-order phase distortion. To phase, type PH (Phasing by Mouse). The screen will change as this routine uses a reduced data point display for speed. Using the mouse, press the left button and drag to the right or left to phase the peaks in the spectrum (this adjusts only the zero-order phase). <Enter> to exit the routine. Type TP (Total Phase) to see what the zero order phase (called Phase A in NUTS) value is and note it. There should not be any first order phase (called Phase B). This is a spectrum of vinyl acetate in CD2Cl2 with TMS. The spectrum should look like the figure below. Phase Expanded: Now, type ZP (Zero Phase) to remove the applied phase values, so a second phasing procedure can be applied. It is possible to achieve perfect phasing using Phasing by mouse, but in general, expanding around and phasing peaks at opposite ends of the spectrum makes fine adjustments easier. To do this (either before of after initial phasing with PH), enter the Zoom subroutine and highlight a region around the downfield peaks, type 1. Now highlight the upfield methyl peak and type 2. <Enter> to exit Zoom. Type PE (Phasing Expanded). Hold down the left mouse button and drag right or left to phase the downfield peak, then hold down the right mouse button and drag right or left to phase the upfield peak. <Enter> to exit this routine. Type TP (Total Phase) to see what the zero and first order phase values are and note them. There should be very little first order phase. 7
  • 8 7 6 5 4 3 2 1 0ppm Effect of Delay for First Point: Open the Virtual Parameters Dialog box. Change Delay for first point to 250 sec (the default value, which is equal to the dwell time), and click on OK. Acquire a spectrum with ZG and Fourier transform it. Type PS (Phase Same) to phase the spectrum using the same values for the zero- and first-order phases as used in the previous acquisition. Now use the Phase Expanded routine to touch up the phase. When complete, type TP to see what the phase values are and note them. Q2-1: How has increasing the delay for the first point (referred to as DE on most spectrometers) affected the zero- and first-order phase values? Q2-2: How has it affected the baseline? (Increase the vertical scale if necessary, to see the baseline well enough.) Now change DE to 100 sec. Acquire and Fourier transform a spectrum, and phase by one of the methods described above. Type TP to see the phase corrections applied. Q2-3: How has decreasing the delay affected the zero- and first-order phases? Q2-4: How has it affected the baseline? Exercise #3: Multiple Scans The signal to noise ratio (s/n) of a spectrum can be increased by repeating scans and adding the data. Signal from the sample will add with each repeat scan. Noise, which is random in phase, will add at a slower rate. Therefore the signal to noise ratio increases as the square root of the number of scans; e.g., four times the number of scans is required to double the signal to noise ratio. 8
  • In this exercise the effect of adding multiple acquisitions together to increase s/n in the final spectrum is evaluated. Acquire and process a spectrum of the sample file etoh1.fs (this is a 1 mM sample of ethanol in D2O), with the following parameters. The spectrum should look like the figure below. Scans to do 1 Sweep Width 4000 Pulse Width 10 Offset Frequency 1800 Relaxation Delay 1 Data Size 16384 Receiver Gain 10 Delay for 1st Point 175 5 4 3 2 1 0 ppm Expand around the region of the ethanol quartet, leaving an appreciable amount of baseline on either side of the peak. Calculate s/n by typing SN (Signal to Noise). This command calculates s/n of the tallest peak in the displayed region to the RMS noise of the baseline. Note the value. Now change the number of scans to 4, and re-acquire a spectrum. Do as before to calculate s/n. Q3-1: By how much has s/n increased? Once again, change the number of scans to 16 and repeat the acquisition and s/n calculation. Q3-2: Now by how much has s/n increased? Q3-3: A typical NMR sample volume is 600 L. How many mg of ethanol were used to make this 1 mM sample? Now, get the sample etoh2.fs (a 2 mM sample of ethanol). Collect and process a spectrum with 4 scans and calculate s/n. Q3-4: What is the difference in s/n for this sample and etoh1.fs, each collected with 4 scans? 9
  • Exercise #4: Pulse Width Measure the 90 degree pulse width: The 90 degree tip angle (pulse width) will be measured and a comparison of s/n for spectra collected with various tip angles will be made. A short discussion on relaxation and relaxation delay effects will also be given. Relaxation effects have not yet been incorporated into this version of the Virtual Spectrometer, therefore, practice exercises cannot be done. Maximum signal is obtained when the excitation pulse is turned on for exactly the length of time necessary to rotate the net magnetization vector 90 degrees, referred to as a 90-degree pulse. This rotates the magnetization from its initial position aligned along the magnetic field (designated as the z-axis) to a position perpendicular to the z-axis, in the x-y plane. The pulse length necessary to do this is dependent on the instrument's hardware, such as the transmitter power and the efficiency of the detector coil in the NMR probe. It must therefore be determined empirically and will vary slightly from day to day. Typical 1H values are 5-15 s on high power. The NMR operator must decide what fraction of the 90-degree pulse, commonly referred to as the "tip angle", will be used to generate the NMR signal. For a single-pulse experiment, as is used to acquire a basic spectrum, there is no value in exceeding a 90-degree pulse. So the choice for values of pulse width are from zero up to the 90-degree pulse. The obvious question is: Why not always use a full 90-degree pulse? More is better, right? The answer is, not always. For a concentrated sample, a smaller pulse angle might be necessary to keep the signal from overloading the receiver, which leads to artifacts in the spectrum. Some of the most valuable information which can be obtained from NMR is quantitative. For this data to be reliable, the spectrum must be obtained under conditions which result in peaks whose integrated area is proportional to the number of nuclei which each peak represents. Another complication is different nuclei relax at different rates. Any nuclei which have not fully relaxed by the time the next excitation pulse is applied will give a signal with somewhat reduced intensity. The result is a spectrum with distorted integration. Therefore, the factors which must be considered are the concentration of the sample and the relaxation time of the nuclei under observation. As with many aspects of NMR, the choice is a tradeoff and requires consideration of which factors are most important for the case at hand. The easiest way to determine the 90 degree pulse length is to determine a 180 degree pulse length and divide by 2. A 180 pulse will give zero signal, and is very sensitive to small offsets from the exact value. It is much easier to determine zero signal than to determine the maximum, as it is fairly broad, making it difficult to distinguish among values that are close to the maximum. So, start with a small pulse length and increase it, viewing the signal after each pulse. The signal amplitude will go through a maximum and back to zero as the pulse length is increased from 0 degrees to 180 degrees. It is important to allow enough time for complete relaxation between each scan, so that the initial condition is the same for each measurement. Failure to do so will yield an incorrect value for the 90 degree pulse. The relaxation delay is the delay between the end of acquisition of a scan and the next excitation pulse. For nuclei with long relaxation times, more time must be allowed between successive excitations to permit the magnetization to return to equilibrium. Distortions of the spectrum, including distorted integration, results from too short a relaxation delay. On the other hand, one does not want to waste time in acquiring data. Another trade-off. Relaxation times can vary 10
  • widely. However, in general, a few to several seconds is enough for protons, since relaxation also occurs during the acquisition time. Often a less than 90 degree tip angle coupled with a shorter relaxation delay is the most efficient way to collect multiple scan acquisitions while still allowing for relaxation. Acquire a spectrum of pyridine.fs. This is a spectrum of pyridine in D2O with TSP as a reference. The spectrum should look like the figure below. 10 8 6 4 2 0 ppm Collect a spectrum with a Pulse Width of 1 (much less than 90 degrees for most spectrometers) and process it, being sure to phase it purely absorptive. Now increase the Pulse Width in increments of a few sec and determine the value that results in a null signal. Process each successive acquisition with PS, this way the phase is always in relation to the purely absorptive spectrum collected and phased with a tip angle known to be less than the 90 degrees. Q4-1: What are the 180 and 90 degree tip angles? Now, increase the Pulse Width by 10 sec more. Q4-2: What happened when a greater than 180 degree tip angle was applied? Now that the 90 degree tip angle is known, set the Pulse Width to this value and collect and process a spectrum. Calculate s/n for the tallest peak in the spectrum. Note the value. Now change the Pulse Width to a 45 degree tip angle and repeat. Finally, determine s/n for the spectrum collected with a 115 degree tip angle. Q4-3: Now, from this experience, why is the 90 degree tip angle important? 11
  • Exercise #5: Receiver Gain Setting In this exercise, the optimum Receiver Gain setting will be determined for a sample, and distortions introduced into the spectrum by overloading the ADC will be considered. The Receiver Gain controls an amplifier through which the signal passes just before it reaches the receiver (analog-to-digital converter, or ADC). This should be adjusted to the maximum amplitude that the ADC can handle without overloading it. If the signal amplitude is too low, small signals will not be discernible. If the ADC is overloaded, the signal will be truncated, or "clipped", resulting in distortion of the resulting spectrum. This can have the appearance of an undulating baseline, in the case of a small degree of clipping, or spurious signals in the case of more severe clipping. The optimum setting can be determined by increasing the gain until clipping is observed and then reducing it from that value. In the case of NUTS, the cutoff occurs at about +/- half of full screen. Note that receiver gain affects signal and noise equivalently, so will not affect the signal/noise ratio. Acquire a spectrum of mek.fs, using a Pulse Width of 6 and a Receiver Gain of 5. This is a spectrum of methylethyl ketone in D2O. The spectrum should look like the figure below. 5 4 3 2 1 0 ppm Note the shape of the FID and process the spectrum. Now optimize the Receiver Gain setting as described above. Q5-1 What is the optimal setting? Q5-2 At what setting does clipping occur? Collect a spectrum at the optimal setting and process it to verify there are no distortions. If the baseline is not flat then the setting chosen is too high. Now increase the setting until clipping is noticeable and acquire and process a spectrum. 12
  • Q5-3 What has happened to the processed spectrum? Exercise #6...Setting The Spectral Window (sweep width and frequency offset) This sets the range of frequencies which will be observed. In the case of NUTS, this is the size of the entire range, from one end to the other. (Note that some instruments set this parameter to be +/- on either side of center, thus +/- one-half of the range.) The spectral window must be set large enough to include all peaks of interest, but too large a setting reduces digital resolution and results in "wasting" data by acquiring regions which include only noise. Peaks which fall outside the spectral window, or "width", will be partially filtered out by the spectrometers filters, but not totally. They will appear "folded" or "aliased" into the spectrum; meaning they appear at frequencies which are not their correct values. This can be detected by the appearance of peaks at anomalous frequencies and by the fact that folded peaks are often out of phase when all other peaks are phased correctly (If the DE value has been optimized such that there is negligible first- order phase distortion, aliased peaks will phase as well.). The most foolproof way to determine the correct spectral width is to start with a value that is much larger than estimated, to determine where the peaks are, then reduce the value to encompass all peaks. There are two parameters to vary when setting the spectral window. The carrier frequency and the sweep width. The spectral window is centered at the frequency of the NMR transmitter, referred to as the "carrier" frequency. The carrier frequency is the sum of the Spectrometer Frequency (in MHz) and the Offset Frequency (in Hz). On many spectrometers, the Offset Frequency varies depending on the deuterated solvent being used for the Field-Frequency lock, and therefore changes from sample to sample. If the Offset Frequency is set incorrectly, some peaks of interest may fall outside the Spectral Window, which will then appear folded. How large a frequency range is observed, is referred to as the sweep width. This can be set in Hz or ppm. Think of it as a window on a frequency axis, which can be shifted along the frequency axis (carrier frequency) and narrowed or widened horizontally (sweep width). Note, in NUTS, the carrier frequency is called the Offset Frequency. Acquire, process and plot a spectrum of nitrbenz.fs with Sweep Width of 4000 and Offset Frequency of 1800. This is a spectrum of 3-nitrobenzaldehyde in CDCl3 and should look like the figure below. Collect and process a spectrum with a Sweep Width of 2400. Q6-1: How has the appearance of the spectrum changed? What happened? Set the Sweep Width back to 4000 and set Offset Frequency to 2200. Q6-2: What has happened to the spectrum? In what direction did the peaks move? Did any of the peaks fold, and what is the evidence? Change the Offset Frequency to 1000 and answer the same questions. 13
  • 12 10 8 6 4 2 0 ppm Exercise #7. Data size The Digital Resolution is the quotient: Spectral width / number of data points, expressed as Hz/pt. The digital resolution must be great enough (Hz/pt value small enough) compared to the width of the lines being observed, to define and resolve narrow peaks. Usually, the spectral width is fixed by the range of frequencies being observed. Therefore, the only parameter which can be varied is the number of data points. Acquiring more data points requires more time. We again have a trade-off between enough points to adequately digitize the NMR signal, but not so many points that time and disk space are wasted. The data size is the number of data points which will be acquired. The algorithm used to perform a Fourier Transform requires the number of data points in the time domain function (FID) to be a power of two. For this reason, most spectrometers, including the Virtual Spectrometer, limit the choices for the data size to be a power of two. If a value is entered which is not a power of two, the value will be changed to the next higher power of two. To accomplish quadrature detection (which provides the ability to distinguish positive and negative frequencies in relation to the carrier frequency) data are acquired in two channels, related by a 90 degree phase shift. Therefore, the data exist as two halves, usually referred to as real (in phase) and imaginary (90 degrees out of phase). Because these two halves are really complex pairs of data points, the NUTS program expresses the number of data points as complex pairs, but that is not true of all spectrometers. Some spectrometers give the data size as the total number of points (the sum of the number of points in the two channels). In that case, the number of points which will define the final spectrum will be half of the total data size. For example, starting with 4K (4096) total points (2K real and 2K imaginary) yields 2K points in the final spectrum. By contrast, NUTS considers this size of data to be 2K complex. It is important to know how a particular spectrometer’s software reports this value to calculate the data size required to yield the desired digital resolution. 14
  • The larger the data size, the more data points collected, and thus, the better the digital resolution. However, the acquisition time will also be increased, as will the actual data size of the file. Thus, a compromise must be reached. The digital resolution must be good enough to provide the desired information (chemical shifts and couplings) without either taking too long or the file being too large. Larger files also take longer to process. Acquire and process another spectrum of nitrbenz.fs. Use the Zoom routine to expand around the aromatic peaks (8.2 to 8.6 ppm). Note the appearance of the multiplets. Now change data size to 8192 and acquire another spectrum. Repeat with data size of 4096 and 32768. Q7-1: What happened when the data size was reduced? Q7-2: What were the acquisition time and digital resolution for each of the spectra? What data size is adequate to define the couplings? 15