edited by
JAMES P. BIRK Arizona State University
computer series, 149
Tempe, AZ 85281
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NMR Spectroscopy: An Introduction to the Operation of Modern Instrumentation Using Commercial Acquisition and Analysis Software Giving Students Experience with a Graphical Workstation and Notefile Michael G. Prais Norlhern Illinois University, Dekalb, IL 60115 Advances in Laboratow Instruments In the past decade almost all new laboratory instrumentation has incor~oratedanalog-todi&al converters, video displays, and &croprocessor~. An -analog-to-digital converter allows data to be stored without manual intervention and manipulated at a later time. A video display allows data to be quickly displayed and redisplayed on a varietv of scales. A video disolav also allows the varietv of options presented to an operator to change over time and ample explanatory information to be presented with these options. A microprocessor allows complex procedures for the acquisition, manipulation, and presentation of data to be extended and automated. These procedures include to stored data without rescanningones that can be amlied *. or manually converting data on a recorder trace to numbers. Raw data stored as Dart of acauisition is reveatedly smoothed or integrated to'give the optimum presintatioi The ~ e a kin s a svectrum are automatically identified, and thei; locations recorded. Successive s p e c k are superimposed and compared. Time-dependent data are quickly and simply fit to a variety of rate laws.
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Comparison with Previous Technology This is a significant change over instrumentation that permanently records data on chart paper, displays instrument settings with few digital readouts, and drives a scanned variable onlv in a linear fashion. Instrumentation that does not incorporate the changes mentioned above can be driven and extended with an inexpensive,microprocessor-based acquisition and control station. These chanees certainlv benefit instrument o~eratorsbv making theirwork muchkasier: Most details about the a;quisition, analysis, and presentation of the data are controlled by the instrument and hidden from the operator. However, the lack of information about these procedures is also a lack of knowledge about what the data represents. This can be especially true when the operator does not understand the steps that are used to analyze and reduce the data. Experience in Operating Modern Instrumentation To give students insight into the operation of modern instrumentation, an experiment was developed to exhibit the steps necessary to acquire, analyze, and present data from an NMR spectrometer controlled by a personal computer. To develop these procedures we used, as a platform,
a wntinuous-wave spectrometer (Varian EM-360A) connected to an IBM PCIAT with a data-acquisition and instrumenkontrol adapter (Data Translation, DT2805; 100 h c k e Drive, Marlborn, MA01752). Although this equipment is not as powerful as a commercial NMR data station, it does give the student the oppurtunity to use a graphical workstation in the laboratory, and it does show many of the underlying numerical operations. In addition, the software chosen for this experiment introduces students to the use of an electronic notebook. The procedures developed for this exercise should represent a complete set of the fundamental operations used in instrumental analysis. The operations include acquisition of static spectra signal averaging, smwthing, and integration automation presentation comparison calihratian peak identification acquisition of dynamic spectra kinetic analysis
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Most of these procedures are described below. This exercise also includes some basic computing operations. Although this experment is a directivi exercise, in the last section students are encouraged to examine the results of changing the parameters selected, including the points measured per scan the scans used in averaging the acquisition rate the cutofffrequency in smwthing the thresholds in the automated peak identification the scan profile for the dynamic system Students are also encouraged to use the fast Fourier transform to study the noise of a baseline. Connecting a PGControlled Spectrometer The personal computer is connected to the spectrometer
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to control the magnetic field in the spectrometer to record the signal output from it
The connections hetween the terminal strip for the acquisition and control adapter and the pins of a DB25 connector (P41 at the back of the EM-360A are made for the student. Volume 70 Number 5 May 1993
381
One outnut channel of the dipital-to-analog converter on the adapter drives the sweep current in themagnet of the NMR [ACCY SWEEP IN on oinx 23 and 211. The mametic field is proportional to the current in the magnet, wiich is in turn nmnortional to the voltage driving the current. The NMR specira are ohtained by r&ordingihe signal output rRCDR Y MONITOWSPECTRUM AMP OLT on pins 7 and 8)with one input channel of the analog-to-digital converter. The output of the digital-to-analog converter is also connected to a second input channel of the analog-to-digital converter so that the voltage driving the NMR can also be followed bv the nersonal comnuter. It is used to determine the beginning and end of sweep. I t is also convenient to make connections to the SWEEP MONITOR OUT on pins 11 and 13. These connections do not eliminate the function of the sweep controls and the spectrum amplitude controls, which can affect the signal measured by the PC. They also do not eliminate the ability of the internal NMR Recorder to drive the scan, so the spectrometer is still operable in its traditional mode. These procedures were developed for an NMR spectrometer because NMR technique is used by students in organic, nhvsical. inorganic. and analytical chemistry ioursds: Hbwever, other instiurnentation can certainly he used instead. Because NMR snectroscon~experiments are part of these courses, students are alH6 exposed to computer-assisted experimentation while they are exposed to NMR spectroscopy. Providing an Extensive, Easy-to-Use Interface To develop the procedures mentioned above, we used a commercial acquisition and analysis software package called ASYSTANT (Kiethley ASYST, Keithley Metrabytd ASYSTIDAC, 440 Myles Standish Blvd., Tauton, MA 02780).This package gives students an instructive alternative to the simple productioninterfaces that are built on commercial data stations, in which students would need only press a few keys to complete the whole experiment.
Menu-Driven Features and Commands The menu-driven, interactive features of this package simplify the development and use of the procedures because all options are presented on screen and the response to any action is immediate. A large variety of operations are available both through the simple selection of a menu item and throueh the t w e d entw of a simnle command. The use of typez comm&ds to pe;fom an operation gives ASYSTANT the abilitv to store and automate extensive procedures. The limited time and knowledge required by each student to complete this exercise is a realistic alternative to exercises that require the development of programs in BASIC, assembler, C, or Pascal for the same ends. This is especially true because this exercise is primarily a chemistry experiment. Acquiring and Viewing Data ASYSTANT provides several options for acquiring data and for viewing and comparing- i t graphically. I t can quickly present two spectra for comparison on the same screen a t the same time using one or two windows. What is dis~lavedon the screen can also be sent to the nrinter. h a i s Gvalues are plotted on automatically scalid axes, and annotations can be added anvwhere within these windows. The averaged and the integrated spectra of the TMS reference sample are shown in Figure 1.ASYSTANT also provides a scrollable display of the data in an array. The student can also
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Journal of Chemical Education
Figure 1. The averaged and the integrated spectra for the TMS referBnce sample. move the data across the screen at a variety of resolutions acquire information about the datapoints that are selected with a movable cursor extract a segment of the data The student need not interact with DOS except to format a diskette and start ASYSTANT from its directory. Students need not even understand the idea of a drive or a directory because their files are stored on the diskette, and students can use a readily available ASYSTANT menu to manipulate their files. Alternate Sofhvare There are several alternatives to ASYSTANT. Besides operations to acquire, analyze, and graph data, these software packages are menu-driven, and most have the equivalent of an electronic notebook that accepts numerical data without manual transcription. EASYeST for DOS (Kiethley ASYST) has the same operations as ASYSTANT presented on-screen graphicallyrather than textually-with icons to represent operations. Labview for the Apple Macintosh (National Instruments, 12109 Technology Blvd., Austin, TX 78727) allows the creation of virtual instruments on-screen with front panels for user interaction and block diamams for their function. each of these vlnual instruments are cornplctc, exeruulhle modules rrpresented hy icons that can be ~~aphicnlly connecrcd, thus alluwmg the now of doto brrwecn modules. Workbench for the Mac (Strawberry Tree Computers, 150 North Wolfe Road, Sunnyvale, CA 94086) also provides connectable icons for acquisition, analysis, and presentation of data. Superscope for the M a c (GW Instruments, 35 Medford Street, Samerville, MA 02143) converts the Macintosh display into the front panel of a user-defined data-recordinginstrument. .Unkelseopr for DOS tvnkel Soflwarr, Carnhridge. MAI providcs many of the aequlsitian and analysis fcarurrs needed. However, it does not allow cnstomization, and it requires an additional . program to act as a notebook. Measure for DOS (National Instruments) provides acquisition mams to work with Lotus 1-2-3and Symphony, but it lacks readv-made.. hieh-level analysis and manipulation routines. Unkelscope and Measure are text-based packages, and Lotus 1-2-3 with Measure does not have a concurrent electronic notebook Using the Software: ASYSTANT This first section of the experiment gives students some background in the general operation of ASYSTANT that is needed to understand and complete later sections of the
experiment. ASYSTANT is based on a FORTH-like calculator that operates in reverse Polish notation on values in a stack of five values. Numbers are placed on the stack last-in-first-out by typing the number and pressing the Enter key.
Stack Contents
Parameters
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Features and Options
One novel feature of ASYSTANT is that the stack can hold numben and one- or two-dimensionalarrays, thus allowing a variety of array operations. There are several techniques for generating arrays. Numbers and arrays can be stored outside of the volatile stack as Parameters and Variables. Unlike Variables, Parameters can only store numbers and not arrays. Numbers and arrays are easily moved between Variables and Parameters and the stack.
-Calcxllator Functions next
store pedit status print
dup
drop swap roll
pi
sin
asin sinh asinh
inv
cos
acos wsh acosh neg
Numbers and Arrays
tan
atan tanh atanh
abs
To make ASYSTANT easier to use, seven-character labels can be attached to the Parameters and the Variables. For this experiment students are asked to generate the labels shown in Figure 2 to track their data. The labels are also used later in expressions.
exp
In
sqrt
Parameters
Variables
10exp log
-Main Menu OptionAcquire
Graphics
Functions
File Proc
File I10
Polys
#POINTS
R:
RAMP
Wave Proc
Curve Fit
Diff Eqs
B:
#SCANS
S:
DATAPTS
Wave Gen
Slats
SaveIExit
C:
ACQRATE
T:
AVERAGE
D:
DURAT'N
U:
SMOOTH
E:
BASELIN
V:
INTEGRL
F:
CHCI@PT
W:
COMPARE
G:
TMS@PT
X:
H:
#CALPTS
Y: Z:
OONC
Figure 2. Labels Attached to Parameters and Variables. Windows
ASYSTANT provides a screen with five windows, as shown in Figure 3. The stack contents, the Parameters, and the Variables are displayed in separate windows. The calculator functions and the main menu options are displayed in other windows. Calculator Functions: Operations that typically act on the contents of the stack. The contents of the stack are changed by selecting an operation f m the displayed menus typing the name of the operation pressing the Enter key or typing a list of operations and arguments pressing the Enter key Main Menu Options: Complicated sets of operations that require several parameters and other selections to define their operations. The Calculator Functions Menu: Actually one menu in a sequence of four menus: Calculator Functions Array Operations Conversions and Special hctirms Wave and Matrix Operations
R=
.oooo
S=
.OOOO
U=
.oooo .oooo
X= W=
.OOOO
Z= Y=
.OOOO
T=
~~~~
A:
I:
Variables-
.oooo .oooo
Figure 3. Main ASYSTANT Screen Selecting next on any of these menus displays the next menu. The "C0MMAND:"F'rompt: When numbers or letters are typed, a window with a "COMMAND:" prompt drops down over the main screen. The window then accepts instructions in reverse Polish notation. Alternatively, typing a Backslash calls up a COMMAND: Window that interprets regular algebraic notation, including parentheses. A calculator window is also available a t the press of a key from almost any screen. It displays the stack contents and a "COMMAND:" prompt. A window with a menu of DOS commands is similarly available. Help is available from any screen by typing a Question Mark. Acquiring Time-Independent Spectra This section illustrates the basic operations for
controlling an instrument through an array of chosen values collectingthe response of a sample for each stimuli Once the array of control values for several scans is constructed, ASYSTANT simply acquires the NMR spectra by automatically driving and recording several scans of the spectrum. The reference sample (12%TMS in CHC13)is used in all procedures described here except for the kinetics measurements. Once the system is calibrated, other samples can be used. Three scans, which comprise 1,024 points and cover approximately 10 ppm, are made for each static sample. The Acquisition Rate
To produce an acquisition rate comparable to the EM360Ain its Integrate mode, the system is set to change the magnetic field and measure the sample response 16 times per second (Hz), for a scan time of 64 s. The EM-360A is designed to integrate a 4,096-measurement scan in 60 s. At its fastest speed, the EM-360A can record a 4,096-meaVolume 70 Number 5
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surement scan a t 136.533 Hz for each scan, for a minimum scan time of 30 s ( I ) . Although it is not shown in this experiment, ASYSTANT can surpass the ability of the typical integral chart recorder and human operator. This can be done using the same inexpensive acquisition and control adapter and the same personal computer that are used here. Generating a Sequence of Scans
The ASYSTANT Function Generator, which drives the digital-to-analog converter, uses an array of values in a variahle (RAMP)to generate each scan. The Function Generator cycles through the values in the variahle several times to give a sequence of scans. The minimum and maximum values in the array produce the predefined minit mum and maximum o u t ~ uvoltaws. ., All other values oroduce scaled, Intermediate output voltages. The number of values in the arrav iftP01NTS1sets the resolution of a scan. The total nugbe; of measurements taken is the product of the number of values in the array and the number of scans (#SCANS).
Integrating - Spectra Structural and auantitative information about the sample is available fr& the comparison of the peak areas. The peak areas are determined by intenation. This section ihows students ~
the ease and flexibility of digital integration a problem with baseline selection one of its solutions The EM-360A is built to integrate a spectrum electronically and to display the result on the recorder. The scan time of this integration is limited to 60 s (3).ASYSTANT can be used to integrate a t a variety of scan times and to precisely choose a partial scan to be reintegrated. ASYSTANT uses Simpsonk rule for integration. The acauired dataooints must be offset before interntion beca&e a zero d'ata value does not correspond to"the baseline. A nonzero baseline is a constant com~onentthat. when integrated, produces a sloped component. The haseline value to he subtracted can he chosen bv -----~ right by averaging the whole spectrum by clipping the peaks and averaging the remainder by averaging a segment of the spectrum without peaks
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~d
The XY Recorder
The XY Recorder option of ASYSTANT is used to acquire static spectra because a single set of data is simply presented across the screen and because it has a rescan feature that is useful for setting the Sweep Offset. The data can be stored to disk at cornoletion. The XY Recorder ooerates a t rates up to 600 Hz, hLt it must store less than 4:000 datapoints. The XY Recorder is significantly faster than the EM360A recorder. However. without additional electronics. this particular data acquisition and control adapter is ca: Dable of onlv balfthe control resolution: The inout rawe of the ~ ~ - 3 allows 6 b ~ the digital-to-analog cbnvert'r to drive the EM-360Ausing half of its range (2,048 measurements over d to 0 V, rather than 4,096 measurements over -5 to +5 V). The signal from the NMR is used as the Y input, and the signal from the digital-to-analog convertor as recorded by the analog-to-digitalconvertor is used as the X input. The datapoints from the three scans of the spectrum are stored in a single Variable (DATAPTS) for further processing. These scans are then averaged to remove noise and saved as another Variable (AVERAGE). Smoothing Spectra This section shows the student another method, in addition to signal averaging, for removing additional noise from any spectrum after it is recorded and stored. This stands in sharp contrast to what can be done with spectra recorded on paper. Smoothing in ASYSTANT is done by convolution of the datapoints lpk,k=l, ...,Ni with a Blackman Window (2). qk=Pk-nWo+
...+
PkWn+
...+
Pk+nWzn
The Blackman Window weights the respective points in a group of 2n + 1points with values given by the following expression. wj = 0.42 + 0.50 * c o d n (j - nUn1 + 0.08 * cos[ 2%(j - nUn I The weight is a maximum a t the center of the interval, and it drops to zero a t the ends. A moving window that brings together as many as 33 adjacent datapoints is used to produce each output data point. Input components that oscillate from point to point with a period less than the width of the convolution window are eliminated from the output of the convolution. The cutoff frequency for these oscillations is thus the reciprocal of the width in seconds of the convolution window. 384
Journal of Chemical Education
ASYSTANT provides a scrollable display of the data in an array to extract a segment of the data either graphically or numerically. A segment of the signal without peaks can be expected to randomly oscillate about the baseline. Such a portion of a full scan is selected by inspection and then averaged. The average baseline value is subtracted from the smoothed spectrum, and this offset spectrum is integrated. Automating Operations This section provides students with experience automating a sequence of simple operations. These operations are very different from those found in traditional programming languages. ASYSTANT does not have the ability for full automation. Although it does not have a looping structure, used to repeat a set of operations a given number of times, ASYSTANT can repeat a calculation for all the elements in an array. In addition, a small sequence of instructions can be placed in queue to he activated by the press of a keyboard function key. Associating a sequence of instructions with a key is useful when the instructions are extensive or needed more than once. The following sequence of instructions (in reverse Polish notation) for the reduction of a spectrum dictates the averaging, smoothing, and integrating of a set of B scans of A points in Variable S with baseline value E. It illustrates both a procedure associated with a function key and the type of commands used by ASYSTANT in lieu of the menus. S B A W R E S H A P E AVG STORE T 0.20 SET.FREQUENCY
Calibrating the Sweep Drive This section gives the student experience
serolline throueh data on-screen selertmg datapomts for lntprrogatlon ~drnufylng knoun peaks as part ofthe general experience of cal:hatmg an mstrurnent for further peak tdenrlficauon The student can move the data across the screen a t a variety of resolutions and get information about the datapoints selected with a movable cursor. This simulates the most sophisticated data stations.
The precise locations of the peaks are identified by comparing the height of the peak location with the adjacent locations to the right and leR of the peak location. The position on a scan can be identified as the location of a point on the ramp driving the digital-to-analog convertor. The locations of the TMS and CHCls peaks are stored as parameters TMS@FTand CHCL@PT,respectively. To precisely locate a peak relative to the TMS peak, it is necessary to know the increment in pprn that is associated with two-adjacent datapoints. The p&s per pprn is calculated from the ratio of differences between the two peaks from the reference sample. PPM/PP = (7.27 ppm - 0.00 ppm)/(CHCL@PT- TMS@PT) The position of a peak in pprn relative to the position of the TMS peak is calculated from its position PT along the ramp that generated the scan. PPM@PT= (PPM/PT)* (PT - TMS@FT)
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Automatina Peak Identification The identification of more than a few peaks is timeconsumina if dune manually as described in the previous section. This section give; students experience automating the identification of an arbitrary number of peaks. This requires considering successive points in th
