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A new method of 2D NMR data collection for time-saving and artifact reduction
JOURKAI.
OF MAGNETIC
RESONANCE
92, 528-537
( 1991)
A New Method of 2D NMR Data Collection for Time-Saving and Artifact Reduction PETER L. RINALDI*-t
AND DANIEL J. IVERSON$
*The Department of Chemistry, Knight Chemical Laboratory, The University oJ’Akron, Akron, Ohw 44325; and $ Varian Instruments, 611 Hansen Way, Palo Alto, California 94303 Received
August
7, 1990: revised
November
12, 1990
In this paper we propose an alternative method for collecting 2D NMR data which involves sequentially sampling one free induction decay for all t, increments before signal averaging, rather than signal averaging multiple transients for one FID at a time. When the number of transients averaged is smaller than the number oft, increments this method results in a dramatic reduction in experiment times. For some types of 2D NMR experiments the new method also drastically reduces certain artifacts which are present in standard experiments. 0 1991 Academic Press, Inc
In this paper, a new method of 2D NMR data collection which considerably shortens the time required to collect a 2D NMR spectrum, when sample quantity does not limit the required signal-to-noise level, is proposed. This new method of data collection has the added advantage of reducing many of the artifacts which complicate the interpretation and obscure important information in certain 2D NMR spectra. Over the past 5-10 years, two-dimensional NMR techniques have become a vital means of providing structure and dynamics information about chemical and biological systems. Computer technology has evolved to the point where many of the most useful techniques can be set up and run by novices, and a good spectrum of a reasonably complex molecule is straightforward to interpret. The length of these experiments and the presence of artifacts are the two major factors which are detriments to their utility. Despite considerable improvements in NMR instrument hardware, the presence of spectral artifacts is currently an obstacle to routine use of 2D NMR spectra. Under the best of circumstances, an experienced spectroscopist can recognize and ignore the artifacts which might obscure important spectral information. A serious situation occurs when an inexperienced person misassigns a structure on the basis of an artifact. In an attempt to reduce artifacts we are proposing a new method for 2D NMR data collection which eliminates the artifacts which remain after all instrument hardware problems are corrected. The method also provides the benefit of much shorter experiment times in cases where sample quantity and signal-to-noise are not limiting factors. Previous workers have extensively described some of the sources of artifacts in 2D NMR spectra and methods for their elimination. Most of these reports have dealt with instrument hardware (l-3) and post-acquisition processing (4, 5) as sources of artifacts. t To whom 0022-236419
correspondence
should
I $3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
be addressed. 528
NEW
METHOD
FOR
2D
DATA
COLLECTION
529
These problems have been resolved by careful instrument design, proper instrument maintenance, and attention to the details of data processing. NMR researchers working in the field of 2D NMR spectral artifact reduction have been prolific. Techniques for removing t, ridges by a number of baseline correction procedures (4, 6-8) and by reduction of the intensities of the large diagonal peaks responsible for the ridges (9-12) have been described. These latter methods remove only baseline offset artifacts and do not affect the discrete artifacts described below. Image processing techniques ( 13, 14) have been suggested for the reduction of some artifacts, but these techniques are not commonly used because of the long computation times required. Symmetrization techniques (1.5) have been the most frequently used methods of artifact elimination in homonuclear shift correlation spectra such as COSY and NOESY and in 2D J spectroscopy. These methods are especially unsatisfactory since artifacts and obvious t, ridges from two or more intense diagonal peaks can combine to produce false signals which look real. An experienced spectroscopist can in most cases recognize artifacts in unsymmetrized data; this is not always true with symmetrized spectra. Recent application of principal-component analysis to 2D NMR was successful at removing t, noise from spectra, but is not generally applicable to removing discrete artifacts of the nature produced by residual magnetization ( 16). Even after all possible hardware problems have been controlled numerous spectral artifacts often remain. These artifacts result from the inherent response of the spins to the pulse sequence and the phase cycling used in the experiment. Several recent publications have recognized this factor as a problem ( 17-22). Turner and Patt (20. 21) have identified these contributions to artifacts in COSY spectra and have concluded that they can be minimized, but not totally eliminated, by phase cycling in the correct order. THEORY
The standard method for the collection of COSY 2D NMR data is outlined in Scheme 1; it involves collection of all NT transients in a single t, increment, with appropriate phase cycling for systematic elimination of artifacts, and cancellation of
Standard
Order
(DI-90;-O&90;-AT,)ss, (Dl-90;-O&90;-AT,),,, (Dl-90’:--16-90’:-AT,),,, (Dl-90;--I&90;-AT,)ss, (DI-90;-28-90;-AT,),,, (Dl-90;-26-90;-AT,),,, (Dl-90;--(NI (Dl-90;-(NI
SCHEME
I
for COSY
Data
Acquisition
(Dl-90’:-Oh-90”,-AT,),,, (D1-90::-06-90;-AT,)N,, (D1-90”,-lS-90;-AT,).7, (Dl-901:-lb-90;-AT,),,, (Dl-90;---2690’:---AT,),,, (Dl-90;--26-90;-AT,),,. . . . - 1)6-90;-AT,)ss, - l)b-90;-AT,)ss,
Note. x = 0”, 90”, 180”, 270”; increments; Dl = relaxation delay; averaged; AT = acquisition time.
Dl-90;--(NI Dl-90;-(NI
- 1)6--~O;--AT,)NT. - 1)6-90;-AT&.,-r
y = x + 90”; 6 = l/SW2; NI = number of 2D t, SS = steady-state pulses; NT = number of transients
530
RINALDI
AND
IVERSON
DC offsets which might arise from imbalanced receiver channels. Ideally, the repetition time for each cycle should be 5-10 X T1 of the slowest relaxing proton in the molecule. Using a long relaxation delay (D 1) such as this is impractical since it would require 12-24 h to collect a single spectrum; usually Dl delays of 0- 1 s are used to permit reasonably short experiment times ( l-2 h). The detectable component of the signal ( uobs) from an AB spin system after one 90”--t,--90” cycle can be described using product-operator formalism (23): sobs = Z,,sin( Q&1 )cos( r.0, ) + Zs,sin( fist, )cos( ?r.Zt,) - 2Z,,Z,,sin(~2,t,)sin(~Jt,)
- 2ZAyZ~Bzsin(QtBtl)sin(7rJt,).
[l]
The first two terms are the diagonal signals, and the last two terms correspond to the cross peaks between flA and QB. With short repetition times, longitudinal and transverse magnetization which survives this first 90”--t,-90” cycle gets carried through additional cycles and contributes to the observed signal in subsequent transients. Magnetization from methyl or isolated aromatic protons, which have long relaxation times and produce intense sharp signals, is especially prone to produce artifacts. In fact these protons can produce magnetization components which are carried through many cycles. Murali and Kumar (18) pointed out that multiple-quantum components can contribute to COSY artifacts in a similar fashion. Because a scheme such as Cyclops phase cycling is used, a true steady-state magnetization is never established (20). Residual magnetization components which survive through a second cycle carry a history of their behavior through two t, evolution periods and show additional cross peaks at 20A in thefi dimension: clOhs= Z,,sin(Q,t,)cos(~Jt,)
+ Zs,sin(Qatl)cos(7rJtl)
- 2Z,,ZB,sin(Q2Atl)sin(7r.Zt,)
- 2Z,,Za,sin(RBtl)sin(7r.Zti)
+ Z,,sin(Q,t,)cos(aJt,)
- +2Z,J,ZB,sin(2Q2AtI)sin(27r.Zt,)
+ Zn,sin(Q,t,)cos(aJti)
- $2ZAzZriysin(2QrJ1)sin(27rJti)
- $2Z,, ZB,sin(21;2at,)sin(27rJt,) + terms from double-quantum
- $2Z,,Zr+in(2QAtl)sin(27r.Zt,) coherence developed in previous cycle.
[2]
In general, it can be shown that transverse magnetization from spin A which survives n cycles will produce artifacts at nQ A. Although this residual magnetization could be destroyed by a 90” pulse sandwiched by two field-gradient pulses (24)) this sequence would disrupt a stable lock, producing a different set of artifacts (25). Elimination of the phase cycling which produces the artifacts described above would result in the observation of other undesired signal components. Our proposed alternative method for 2D NMR data collection is outlined in Scheme 2. In this method a single transient of data is collected for all values of t, with the phases held constant. Phases are incremented, using a Cyclops sequence, after one transient is collected for all t, values. This method has the advantage of requiring
NEW
METHOD
FOR
2D
DATA
SCHEME Alternative
Order
,Vote. All terms
as defined
2
for COSY
{(Dl-90;-06-90;--AT&, (DI-90”,--1690;--AT,), . . . (Di-90;--(NI (Dl-900,-O&-90;-AT,)ss, (DI-90”,-O&--90;-AT,), . . . (Dl-90;-(NI in Scheme
531
COLLECTION
Data
Acquisition
(Dl-90;--06--90;-AT,), (Dl-90;-26-90;-AT,), - 1)6-90;--AT,). (Dl-90;--la--90;-AT,). (Dl-90”,-26--90;-AT,), - 1)&90;--AT,)},, 1.
considerably fewer steady-state cycles when signal-to-noise is not the determining factor influencing experiment time (i.e., when NI > NT). In this latter sequence, product-operator calculations yield the following expression for magnetization which persists through two 90”--t,-90” cycles of a COSY experiment: cobs = Z,,sin(Q,t,)cos(7rJtl)
+ ZB,sin(Q2BtI)cos(7r.Ztl)
- 2Z,,ZB,sin(0,tl)sin(~Jt,)
- 2ZAyZBBrsin(DBtl)sin(7rJt,)
- Z,,sin( QAd)cos( aJ6) - ZBXsin(OB6)cos( aJ6) + 2Z,,Z,,sin(
Q,S)sin(7rJG) + 2Z,,ZBzsin( L@)sin(?r.Z6).
[3]
Since 6 = 1/SW2 is a fixed delay, the additional terms in Eq. [ 31 produce axial peaks which can be cancelled with phase cycling in successive transients which are averaged. A third alternative for data collection, outlined in Scheme 3, in which the real and imaginary FIDs are sequentially sampled for all t, increments before signal averaging is accomplished, might also be envisioned. Although this method might be preferable for other 2D NMR experiments, in the COSY experiment, it suffers from the same disadvantages as the standard method of 2D NMR data collection in that 90” phase shifts occur between FID samplings. It is this phase shift which contributes to artifacts in the standard mode of data collection.
SCHEME Undesirable
Alternative
Order
{(DI-90;-O&90”,--AT,),,, (DI-90;-06-90;-AT,), (Dl-90;-I&--90;-AT,), (Dl-90;-26-90;--AT,), . . . (Dl-90;-(NI Note.
All terms
for Sequential
COSY
Data
Acquisition
(Dl-90;-OS--90;-AT,), (Dl-90;-16--90;-AT,), (Dl-90;-28-90;-AT.,).
- I)&--90;-AT,), as defined
3
in Scheme
(Dl-90;--(NI 1.
- l)&-90;-AT,)}..
532
RINALDI
AND
IVERSON
In addition to the time-savings achieved by using fewer steady-state pulses, further savings are realized by averaging the entire 2D data matrix in the acquisition computer memory on our spectrometer. A significant amount of overhead in most 2D NMR experiments comes from disk I/O. Of this disk I/O time, a large fraction is related to disk formatting, seek time, and exchanging instructions for acquisition of the FID for the next f, increment, and only a small fraction of the time is used for writing the data onto the disk. In the sequential-acquisition 2D experiment, data are written to disk once at the end of an entire experiment (or at the end of each 4-l 6 transients) rather than once at the end of each 128-1024 t, increments. For shorter 2D NMR experiments, this results in a substantial time-savings. EXPERIMENTAL
All experimental data were collected on a standard Varian VXR-300 NMR spectrometer with Motorola 68000-based acquisition and processing CPUs, 1 Mbyte of processing memory, and 256 kbyte of acquisition memory. The instrument was thoroughly scrutinized to remove most hardware-related artifacts. Pulse amplitude variation over 1 h was less than 0. l%, and RF phase stability was within lo. We verified that variable disk I/O times were not introducing spurious signals by the observation of identical spectra obtained with 4 and 16 steady-state cycles. Probe and sample temperature were stabilized using a heat exchanger consisting of 100 ft of copper tubing immersed in a 200 liter water bath as described by Allerhand et al. (26). Ail spectra were obtained without sample spinning to eliminate artifacts from spinner modulation, sample vibration, and vortexing. All spectra were obtained on a sample of trans-stilbene in CDC13. Two-dimensional spectra were collected in the phase-sensitive mode using the method of States et al. (27). Data collection parameters were temperature regulation at 25”, 128 X 64 X 2 data table size, spectral window of 300 Hz in both dimensions, 1.O s relaxation delay, 16 transients with Cyclops phase cycling of the receiver and the second pulse, and 16 dummy pulses before data averaging in order to establish steady-state magnetization. The standard instrument software was used to collect normal phase-sensitive COSY spectra. These data (in standard Varian VXR-4000 format) were then transmitted via Ethernet to a Sun 3/l 10 for further processing. Special data-acquisition programs which utilize hardware looping and explicit dataacquisition features of the instrument were employed to collect COSY spectra using the sequential acquisition methods in Schemes 2 and 3. The 2D NMR data collected using these programs cannot be processed using commercial software packages. These data files were transmitted via Ethernet to a Sun 3/ 110, where a C program, written in-house, was used to reorder the data to Varian’s standard VXR-4000 format. COSY spectra from both acquisition methods were converted from VXR-4000 to UNIX file format and processed using Varian’s VNMR software. Data processing parameters were shifted sine-bell weighting in both dimensions, zero filling to 256 X 256 before transformation, and 32-bit floating-point transform. RESULTS
AND
DISCUSSION
Figure 1 shows a COSY spectrum of trans-stilbene obtained under carefully controlled conditions, on a spectrometer that was scrupulously prepared to eliminate
NEW METHOD
FOR 2D DATA
COLLECTION
533
Fl FIG. I. Standard COSY spectrum obtained using the sampling order in Scheme I, on a VXR-300 which was rigorously prepared to eliminate hardware instabilities. Both this spectrum and the one in Fig. 2 were collected using the same sample during the same 30 min period. The circled signals are modulation artifacts (see text ).
hardware imperfections and post-acquisition data processing as sources of artifacts. The spectrum was obtained without sample spinning, with temperature regulation, and under conditions to eliminate artifacts which might arise from the disruption of steady-state conditions caused by variable disk addressing times between the acquisition of FIDs. This spectrum shows a substantial series of artifacts atf2 = 7.2 ppm, the shift of the isolated olefinic protons. Careful examination reveals that these peaks are aliased from fi frequencies corresponding to integral multiples of the diagonal-peak offset from the transmitter frequency, as predicted in Eq. [ 21. The isolated protons responsible for these resonances have long relaxation times and their magnetization components survive through at least four COSY cycles to produce these artifacts. The intensities of these undesired peaks are such that they interfere with the reliable identification of cross peaks along this trace of the spectrum.
534
RINALDI
AND
IVERSON
A spectrum of trans-stilbene obtained with the sequential FID sampling method outlined in Scheme 2 is shown in Fig. 2. Both this spectrum and that displayed in Fig. 1 were obtained within a 30 min period using identical instrument conditions with the exception of the order in which the data were collected. A series of artifacts (circled) in Fig. 1 is completely absent in Fig. 2. Only a small quadrature image is observed in Fig. 2 (circled), which was also present in Fig. 1. With the alternate method for data collection, it is possible to see cross peaks (in boxes), due to long-range coupling interactions between the olefin and ortho-aromatic protons, which were obscured by artifacts in the normal COSY spectrum. Similar improvements have also been obtained from a sample of methyl methacrylate where artifacts from the methyl resonances are suppressed. Sequential FID sampling results in a considerable reduction in experimental time. When signal-to-noise is not a limiting factor, COSY spectra are usually acquired with four transients per FID; however, before signal averaging can be performed, a steady
l”“I”“I”“I”“l”“l”“r”“l”“l”“l 7.9 7.0 7.7
7.6
7.5
7.4
7.3
7.2
FIG, 2. Nonstandard COSY spectrum obtained using the sampling order parameters were identical to those used to collect the spectrum in Fig. 1. which are present in Fig. 1 are eliminated. The weak circled signal is an also present in Fig. I; the signals surrounded by squares are real cross peaks
mm
in Scheme 2; all other acquisition All of the rapid sampling artifacts F, quadrature image, which was arising from long-range couplings.
NEW
METHOD
FOR
2D
DATA
535
COLLECTION
state must be established with two or four dummy scans. Using the new sequential FID sampling technique, four dummy scans are performed for NI FIDs (where NI is usually 128-5 12). On real samples with a large number of FIDs the reduction in data collection time approaches 50%. Additionally, because all of the data are acquired in memory until the entire experiment is completed, fewer disk accessesare required. Most of the overhead associated with disk access involves seek time to identify physical locations: a smaller time is required to perform the actual write operation for a 1 kbyte file. Although the same quantity of data is transferred to disk with the old and new data-acquisition schemes. sequential FID sampling requires only a single disk accessat the end of an experiment. Examples of this time-saving are outlined in Table 1. On our instrument this shortens a 15-20 min COSY experiment to less than 8 min. On older, unreliable instruments it was desirable to perform frequent disk backups during long experiments in order to prevent the loss of all the data in the event of an instrument failure. On newer instruments frequent backup is less essential (we have not lost data from a system crash in the past year of operation). In the event that periodic backup is desired, this can still be done at multiples of transients that complete a phase cycle. Since this alternate method for data collection involves acquiring the whole data table in the acquisition computer memory, and the standard COSY experiment requires saving each FID as acquisition is complete, the only remaining source of artifacts in the latter experiment would be potential variation in the disk addressing between experiments, which might disrupt the steady state. Although we have collected many spectra to indicate that this is not a factor, a standard COSY spectrum was obtained with conditions identical to those used to acquire the spectrum in Fig. 1 with the exception that 16 steady-state transients were averaged and discarded, to completely eliminate this factor as a possible source of artifacts. The resulting spectrum was virtually identical to that shown in Fig. 1. The third method for sequential FID sampling as illustrated in Scheme 3 is undesirable for COSY data acquisition. In this experiment, residual magnetization components which are propagated through the second COSY cycle mix intensity information from the real and imaginary,< signal components, resulting in severej; quadTABLE Experiment
Experimental SS = 4. NT SS = 0. NT SS = 4. NT SS=4.NT=4,DI SS = 4, NT
Times
for Collection of Data Using 2D NMR Sampling Technique
= 4, DI
.Vorc,. All parameters
Standard
Standard COSY (min)
conditions = 4, Dl = 4, DI = I, DI
I
= 0.5 = 0.5 = 0.5 =O.l = 1.0 as defined
28 16 20 16 52 in Scheme
I, AT = 0.426
and New
Alternate COSY (min) 13 13 2.5 6 22 s. and NI = IX.
536
RINALDI
AND
IVERSON
rature images. Artifacts and images which are a nuisance in standard COSY experiments create insurmountable problems using this method. One drawback of the technique is its demand for acquisition memory, which at minimum requires containment of half the data set (i.e., all FIDs for one phase in the States method for phase-sensitive 2D data collection). On careful consideration this requirement is not severely restrictive. On a 300 MHz instrument an additional I Mbyte acquisition memory board (to provide a total of 1.25 Mbytes of acquisition memory), at an additional cost of $2000 on a $350,000 instrument, removes memoryimposed limitations. With an instrument configured in this way, we have been able to include the entire 3 kHz proton spectral window in a COSY spectrum with a collection of 1024 X 256 X 2 data sets and zero filling to 2048 X 2048 before processing. Currently marketed NMR instruments based on personal workstation technology include up to 2 Mbytes of acquisition-dedicated memory as a standard feature. While all 2D NMR experiments will not be beneficially affected by the use of this alternate method of data acquisition, many experiments will provide better-quality spectra and should be performed in this manner. From our preliminary investigations, this alternative order of data collection is preferable to the standard method for COSY experiments. The ideal method of performing other 2D NMR experiments will depend on the nature and the number of pulses in the sequence and their effect on the spins. This alternative method of acquisition does not require that chemists learn new experiments or interpret new forms of data display. As always, they will decide whether a COSY or some other experiment is the best one to provide the structural information they need. Once this decision is made the instrument should be programmed to automatically collect data using the appropriate method. We are currently investigating further applications of this technique to the collection of 3D NMR data where it should yield considerable time-savings. ACKNOWLEDGMENTS We thank the University for an Academic Challenge
of Akron for a Faculty Research Grant Grant that provided financial support
and the State of Ohio of this research.
Board
of Regents
REFERENCES
Reson. 58,3 15 ( 1984). J. Magn. Reson. 56,207 ( 1984). 3. K. NAGAYAMA, P. BACHMANN, AND K. WUTHRICH, J. Magn. Reson. 31, 133 ( 1978). 4. G. OTTING, H. WIDMER, G. WAGNER, AND K. W~THRICH, J. Magn. Reson. 66, 187 ( 1986). 5. J. COOPER, in “Transform Techniques in Chemistry” (P. R. Griffiths, Ed.), Chap 4, Plenum Press, New
1. A. F. MEHLKOPF, D. KORBEE, T. A. TIGGELMAN, 2. G. WIDER, S. MACURA, A. KUMAR, R. R. ERNST,
6
York, 1978. R. E. KLEVIT, J. Magn.
7. 8. 9. IO.
S. 1. H. G.
Ii. 12.
W. E. P. P.
13. 14.
Reson.
AND R. FREEMAN, AND K. W~THRICH,
J. Magn.
62, 551 (1985).
GLASER AND H. R. KALBITZER, J. Magn. Reson. 68,350 ( 1986). L. BAARSUKOV AND A. S. ARSENIEV, J. Magn. Reson. 73, 148 ( 1987). SANTOS, D. L. TURNER, AND A. V. XAVIER, J. Magn. Resort 55,463 (1983). S. HARBISON, J. FEIGON, D. J. RUBEN, J. HERZFELD, AND R. G. GRIFFIN, J. Am. 5567 (1985). DENK, G. WAGNER, R. P. ZUIDERWEG, K. H. BOLTON, J. Magn. H. BOLTON, J. Magn.
Sot. 107,
AND K. W~THRICH, J. Magn. Reson. 62,350 (1985). AND E. T. OLEJNICZAK, J. Magn. Reson. 70, 336 ( 1986). 67, 391 (1986).
M. RANCE, HALLENGA,
Reson. Reson.
Chem.
64, 352 (1985).
NEW
METHOD
FOR
2D
DATA
537
COLLECTION
I.(. (u) R. BAUMANN, G. WIDER, R. R. ERNST, AND K. WOTHRICH, .I. Magn. Reson. 44,402 ( 1981); (h) R.BAUMANN, A.KuMAR,R.R.ERNsT, ANDK. WUTHRICH, J Magn.Reson.44,76( 1981). 16. (a) P. L.RINALDI ANDJ.K. HARDY, J. Magn. Reson. 88,320(1990);(b) D.E. BROWN ANDT. W. CAMBELL, J. Magn. Reson. 89,255 ( 1990). 17. N.MOLLER,G.BODENHAUSEN,K.WOTHRICH, AND R.R. ERNST, J. Magn. Reson.65,531 (1985). 1X. N.MuRALIANDA.KuMAR,C~~~.P~~~. Left. 128,58(1986). 1Y. M. L. GYNDELL, J. Mugn. Reson. 81,474 (1989). 20. C.J.TURNERANDS. L.PATT. J. Magn.Reson.85,492( 1989). 21. C. J. TURNER. J. Magn. Reson. 86,420 (1990). ?? A.E.DERoMEANDM.P. WILLIAMSON, J.Magn.Reson.88, 177(1990). 23. 0. W. SORENSEN.G. W. EICH, M. H. LEVITT. G. BODENHAUSEN, AND R. R. ERNST. Ptqq. NMR Spcctrosc.
16, 163 (1983).
34. W.KREIBICHANDA.SCWWENK, J Magn.Reson. 77,308(1988). -75. S. R. MAPLEANDA. ALLERHAND, J. Magn. Reson. 75, 147(1987). 26. A.ALLERHAND, R.E.ADDLEMAN,ANDD.OSMAN, J.Am.Chem.Soc. -77 D.J. STATES,R. A.HABERKORN, AND D.J.RuBEN, J. Mugn.Reson.48,286(
107,5809(1985).
1982).
OF MAGNETIC
RESONANCE
92, 528-537
( 1991)
A New Method of 2D NMR Data Collection for Time-Saving and Artifact Reduction PETER L. RINALDI*-t
AND DANIEL J. IVERSON$
*The Department of Chemistry, Knight Chemical Laboratory, The University oJ’Akron, Akron, Ohw 44325; and $ Varian Instruments, 611 Hansen Way, Palo Alto, California 94303 Received
August
7, 1990: revised
November
12, 1990
In this paper we propose an alternative method for collecting 2D NMR data which involves sequentially sampling one free induction decay for all t, increments before signal averaging, rather than signal averaging multiple transients for one FID at a time. When the number of transients averaged is smaller than the number oft, increments this method results in a dramatic reduction in experiment times. For some types of 2D NMR experiments the new method also drastically reduces certain artifacts which are present in standard experiments. 0 1991 Academic Press, Inc
In this paper, a new method of 2D NMR data collection which considerably shortens the time required to collect a 2D NMR spectrum, when sample quantity does not limit the required signal-to-noise level, is proposed. This new method of data collection has the added advantage of reducing many of the artifacts which complicate the interpretation and obscure important information in certain 2D NMR spectra. Over the past 5-10 years, two-dimensional NMR techniques have become a vital means of providing structure and dynamics information about chemical and biological systems. Computer technology has evolved to the point where many of the most useful techniques can be set up and run by novices, and a good spectrum of a reasonably complex molecule is straightforward to interpret. The length of these experiments and the presence of artifacts are the two major factors which are detriments to their utility. Despite considerable improvements in NMR instrument hardware, the presence of spectral artifacts is currently an obstacle to routine use of 2D NMR spectra. Under the best of circumstances, an experienced spectroscopist can recognize and ignore the artifacts which might obscure important spectral information. A serious situation occurs when an inexperienced person misassigns a structure on the basis of an artifact. In an attempt to reduce artifacts we are proposing a new method for 2D NMR data collection which eliminates the artifacts which remain after all instrument hardware problems are corrected. The method also provides the benefit of much shorter experiment times in cases where sample quantity and signal-to-noise are not limiting factors. Previous workers have extensively described some of the sources of artifacts in 2D NMR spectra and methods for their elimination. Most of these reports have dealt with instrument hardware (l-3) and post-acquisition processing (4, 5) as sources of artifacts. t To whom 0022-236419
correspondence
should
I $3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
be addressed. 528
NEW
METHOD
FOR
2D
DATA
COLLECTION
529
These problems have been resolved by careful instrument design, proper instrument maintenance, and attention to the details of data processing. NMR researchers working in the field of 2D NMR spectral artifact reduction have been prolific. Techniques for removing t, ridges by a number of baseline correction procedures (4, 6-8) and by reduction of the intensities of the large diagonal peaks responsible for the ridges (9-12) have been described. These latter methods remove only baseline offset artifacts and do not affect the discrete artifacts described below. Image processing techniques ( 13, 14) have been suggested for the reduction of some artifacts, but these techniques are not commonly used because of the long computation times required. Symmetrization techniques (1.5) have been the most frequently used methods of artifact elimination in homonuclear shift correlation spectra such as COSY and NOESY and in 2D J spectroscopy. These methods are especially unsatisfactory since artifacts and obvious t, ridges from two or more intense diagonal peaks can combine to produce false signals which look real. An experienced spectroscopist can in most cases recognize artifacts in unsymmetrized data; this is not always true with symmetrized spectra. Recent application of principal-component analysis to 2D NMR was successful at removing t, noise from spectra, but is not generally applicable to removing discrete artifacts of the nature produced by residual magnetization ( 16). Even after all possible hardware problems have been controlled numerous spectral artifacts often remain. These artifacts result from the inherent response of the spins to the pulse sequence and the phase cycling used in the experiment. Several recent publications have recognized this factor as a problem ( 17-22). Turner and Patt (20. 21) have identified these contributions to artifacts in COSY spectra and have concluded that they can be minimized, but not totally eliminated, by phase cycling in the correct order. THEORY
The standard method for the collection of COSY 2D NMR data is outlined in Scheme 1; it involves collection of all NT transients in a single t, increment, with appropriate phase cycling for systematic elimination of artifacts, and cancellation of
Standard
Order
(DI-90;-O&90;-AT,)ss, (Dl-90;-O&90;-AT,),,, (Dl-90’:--16-90’:-AT,),,, (Dl-90;--I&90;-AT,)ss, (DI-90;-28-90;-AT,),,, (Dl-90;-26-90;-AT,),,, (Dl-90;--(NI (Dl-90;-(NI
SCHEME
I
for COSY
Data
Acquisition
(Dl-90’:-Oh-90”,-AT,),,, (D1-90::-06-90;-AT,)N,, (D1-90”,-lS-90;-AT,).7, (Dl-901:-lb-90;-AT,),,, (Dl-90;---2690’:---AT,),,, (Dl-90;--26-90;-AT,),,. . . . - 1)6-90;-AT,)ss, - l)b-90;-AT,)ss,
Note. x = 0”, 90”, 180”, 270”; increments; Dl = relaxation delay; averaged; AT = acquisition time.
Dl-90;--(NI Dl-90;-(NI
- 1)6--~O;--AT,)NT. - 1)6-90;-AT&.,-r
y = x + 90”; 6 = l/SW2; NI = number of 2D t, SS = steady-state pulses; NT = number of transients
530
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DC offsets which might arise from imbalanced receiver channels. Ideally, the repetition time for each cycle should be 5-10 X T1 of the slowest relaxing proton in the molecule. Using a long relaxation delay (D 1) such as this is impractical since it would require 12-24 h to collect a single spectrum; usually Dl delays of 0- 1 s are used to permit reasonably short experiment times ( l-2 h). The detectable component of the signal ( uobs) from an AB spin system after one 90”--t,--90” cycle can be described using product-operator formalism (23): sobs = Z,,sin( Q&1 )cos( r.0, ) + Zs,sin( fist, )cos( ?r.Zt,) - 2Z,,Z,,sin(~2,t,)sin(~Jt,)
- 2ZAyZ~Bzsin(QtBtl)sin(7rJt,).
[l]
The first two terms are the diagonal signals, and the last two terms correspond to the cross peaks between flA and QB. With short repetition times, longitudinal and transverse magnetization which survives this first 90”--t,-90” cycle gets carried through additional cycles and contributes to the observed signal in subsequent transients. Magnetization from methyl or isolated aromatic protons, which have long relaxation times and produce intense sharp signals, is especially prone to produce artifacts. In fact these protons can produce magnetization components which are carried through many cycles. Murali and Kumar (18) pointed out that multiple-quantum components can contribute to COSY artifacts in a similar fashion. Because a scheme such as Cyclops phase cycling is used, a true steady-state magnetization is never established (20). Residual magnetization components which survive through a second cycle carry a history of their behavior through two t, evolution periods and show additional cross peaks at 20A in thefi dimension: clOhs= Z,,sin(Q,t,)cos(~Jt,)
+ Zs,sin(Qatl)cos(7rJtl)
- 2Z,,ZB,sin(Q2Atl)sin(7r.Zt,)
- 2Z,,Za,sin(RBtl)sin(7r.Zti)
+ Z,,sin(Q,t,)cos(aJt,)
- +2Z,J,ZB,sin(2Q2AtI)sin(27r.Zt,)
+ Zn,sin(Q,t,)cos(aJti)
- $2ZAzZriysin(2QrJ1)sin(27rJti)
- $2Z,, ZB,sin(21;2at,)sin(27rJt,) + terms from double-quantum
- $2Z,,Zr+in(2QAtl)sin(27r.Zt,) coherence developed in previous cycle.
[2]
In general, it can be shown that transverse magnetization from spin A which survives n cycles will produce artifacts at nQ A. Although this residual magnetization could be destroyed by a 90” pulse sandwiched by two field-gradient pulses (24)) this sequence would disrupt a stable lock, producing a different set of artifacts (25). Elimination of the phase cycling which produces the artifacts described above would result in the observation of other undesired signal components. Our proposed alternative method for 2D NMR data collection is outlined in Scheme 2. In this method a single transient of data is collected for all values of t, with the phases held constant. Phases are incremented, using a Cyclops sequence, after one transient is collected for all t, values. This method has the advantage of requiring
NEW
METHOD
FOR
2D
DATA
SCHEME Alternative
Order
,Vote. All terms
as defined
2
for COSY
{(Dl-90;-06-90;--AT&, (DI-90”,--1690;--AT,), . . . (Di-90;--(NI (Dl-900,-O&-90;-AT,)ss, (DI-90”,-O&--90;-AT,), . . . (Dl-90;-(NI in Scheme
531
COLLECTION
Data
Acquisition
(Dl-90;--06--90;-AT,), (Dl-90;-26-90;-AT,), - 1)6-90;--AT,). (Dl-90;--la--90;-AT,). (Dl-90”,-26--90;-AT,), - 1)&90;--AT,)},, 1.
considerably fewer steady-state cycles when signal-to-noise is not the determining factor influencing experiment time (i.e., when NI > NT). In this latter sequence, product-operator calculations yield the following expression for magnetization which persists through two 90”--t,-90” cycles of a COSY experiment: cobs = Z,,sin(Q,t,)cos(7rJtl)
+ ZB,sin(Q2BtI)cos(7r.Ztl)
- 2Z,,ZB,sin(0,tl)sin(~Jt,)
- 2ZAyZBBrsin(DBtl)sin(7rJt,)
- Z,,sin( QAd)cos( aJ6) - ZBXsin(OB6)cos( aJ6) + 2Z,,Z,,sin(
Q,S)sin(7rJG) + 2Z,,ZBzsin( L@)sin(?r.Z6).
[3]
Since 6 = 1/SW2 is a fixed delay, the additional terms in Eq. [ 31 produce axial peaks which can be cancelled with phase cycling in successive transients which are averaged. A third alternative for data collection, outlined in Scheme 3, in which the real and imaginary FIDs are sequentially sampled for all t, increments before signal averaging is accomplished, might also be envisioned. Although this method might be preferable for other 2D NMR experiments, in the COSY experiment, it suffers from the same disadvantages as the standard method of 2D NMR data collection in that 90” phase shifts occur between FID samplings. It is this phase shift which contributes to artifacts in the standard mode of data collection.
SCHEME Undesirable
Alternative
Order
{(DI-90;-O&90”,--AT,),,, (DI-90;-06-90;-AT,), (Dl-90;-I&--90;-AT,), (Dl-90;-26-90;--AT,), . . . (Dl-90;-(NI Note.
All terms
for Sequential
COSY
Data
Acquisition
(Dl-90;-OS--90;-AT,), (Dl-90;-16--90;-AT,), (Dl-90;-28-90;-AT.,).
- I)&--90;-AT,), as defined
3
in Scheme
(Dl-90;--(NI 1.
- l)&-90;-AT,)}..
532
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In addition to the time-savings achieved by using fewer steady-state pulses, further savings are realized by averaging the entire 2D data matrix in the acquisition computer memory on our spectrometer. A significant amount of overhead in most 2D NMR experiments comes from disk I/O. Of this disk I/O time, a large fraction is related to disk formatting, seek time, and exchanging instructions for acquisition of the FID for the next f, increment, and only a small fraction of the time is used for writing the data onto the disk. In the sequential-acquisition 2D experiment, data are written to disk once at the end of an entire experiment (or at the end of each 4-l 6 transients) rather than once at the end of each 128-1024 t, increments. For shorter 2D NMR experiments, this results in a substantial time-savings. EXPERIMENTAL
All experimental data were collected on a standard Varian VXR-300 NMR spectrometer with Motorola 68000-based acquisition and processing CPUs, 1 Mbyte of processing memory, and 256 kbyte of acquisition memory. The instrument was thoroughly scrutinized to remove most hardware-related artifacts. Pulse amplitude variation over 1 h was less than 0. l%, and RF phase stability was within lo. We verified that variable disk I/O times were not introducing spurious signals by the observation of identical spectra obtained with 4 and 16 steady-state cycles. Probe and sample temperature were stabilized using a heat exchanger consisting of 100 ft of copper tubing immersed in a 200 liter water bath as described by Allerhand et al. (26). Ail spectra were obtained without sample spinning to eliminate artifacts from spinner modulation, sample vibration, and vortexing. All spectra were obtained on a sample of trans-stilbene in CDC13. Two-dimensional spectra were collected in the phase-sensitive mode using the method of States et al. (27). Data collection parameters were temperature regulation at 25”, 128 X 64 X 2 data table size, spectral window of 300 Hz in both dimensions, 1.O s relaxation delay, 16 transients with Cyclops phase cycling of the receiver and the second pulse, and 16 dummy pulses before data averaging in order to establish steady-state magnetization. The standard instrument software was used to collect normal phase-sensitive COSY spectra. These data (in standard Varian VXR-4000 format) were then transmitted via Ethernet to a Sun 3/l 10 for further processing. Special data-acquisition programs which utilize hardware looping and explicit dataacquisition features of the instrument were employed to collect COSY spectra using the sequential acquisition methods in Schemes 2 and 3. The 2D NMR data collected using these programs cannot be processed using commercial software packages. These data files were transmitted via Ethernet to a Sun 3/ 110, where a C program, written in-house, was used to reorder the data to Varian’s standard VXR-4000 format. COSY spectra from both acquisition methods were converted from VXR-4000 to UNIX file format and processed using Varian’s VNMR software. Data processing parameters were shifted sine-bell weighting in both dimensions, zero filling to 256 X 256 before transformation, and 32-bit floating-point transform. RESULTS
AND
DISCUSSION
Figure 1 shows a COSY spectrum of trans-stilbene obtained under carefully controlled conditions, on a spectrometer that was scrupulously prepared to eliminate
NEW METHOD
FOR 2D DATA
COLLECTION
533
Fl FIG. I. Standard COSY spectrum obtained using the sampling order in Scheme I, on a VXR-300 which was rigorously prepared to eliminate hardware instabilities. Both this spectrum and the one in Fig. 2 were collected using the same sample during the same 30 min period. The circled signals are modulation artifacts (see text ).
hardware imperfections and post-acquisition data processing as sources of artifacts. The spectrum was obtained without sample spinning, with temperature regulation, and under conditions to eliminate artifacts which might arise from the disruption of steady-state conditions caused by variable disk addressing times between the acquisition of FIDs. This spectrum shows a substantial series of artifacts atf2 = 7.2 ppm, the shift of the isolated olefinic protons. Careful examination reveals that these peaks are aliased from fi frequencies corresponding to integral multiples of the diagonal-peak offset from the transmitter frequency, as predicted in Eq. [ 21. The isolated protons responsible for these resonances have long relaxation times and their magnetization components survive through at least four COSY cycles to produce these artifacts. The intensities of these undesired peaks are such that they interfere with the reliable identification of cross peaks along this trace of the spectrum.
534
RINALDI
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A spectrum of trans-stilbene obtained with the sequential FID sampling method outlined in Scheme 2 is shown in Fig. 2. Both this spectrum and that displayed in Fig. 1 were obtained within a 30 min period using identical instrument conditions with the exception of the order in which the data were collected. A series of artifacts (circled) in Fig. 1 is completely absent in Fig. 2. Only a small quadrature image is observed in Fig. 2 (circled), which was also present in Fig. 1. With the alternate method for data collection, it is possible to see cross peaks (in boxes), due to long-range coupling interactions between the olefin and ortho-aromatic protons, which were obscured by artifacts in the normal COSY spectrum. Similar improvements have also been obtained from a sample of methyl methacrylate where artifacts from the methyl resonances are suppressed. Sequential FID sampling results in a considerable reduction in experimental time. When signal-to-noise is not a limiting factor, COSY spectra are usually acquired with four transients per FID; however, before signal averaging can be performed, a steady
l”“I”“I”“I”“l”“l”“r”“l”“l”“l 7.9 7.0 7.7
7.6
7.5
7.4
7.3
7.2
FIG, 2. Nonstandard COSY spectrum obtained using the sampling order parameters were identical to those used to collect the spectrum in Fig. 1. which are present in Fig. 1 are eliminated. The weak circled signal is an also present in Fig. I; the signals surrounded by squares are real cross peaks
mm
in Scheme 2; all other acquisition All of the rapid sampling artifacts F, quadrature image, which was arising from long-range couplings.
NEW
METHOD
FOR
2D
DATA
535
COLLECTION
state must be established with two or four dummy scans. Using the new sequential FID sampling technique, four dummy scans are performed for NI FIDs (where NI is usually 128-5 12). On real samples with a large number of FIDs the reduction in data collection time approaches 50%. Additionally, because all of the data are acquired in memory until the entire experiment is completed, fewer disk accessesare required. Most of the overhead associated with disk access involves seek time to identify physical locations: a smaller time is required to perform the actual write operation for a 1 kbyte file. Although the same quantity of data is transferred to disk with the old and new data-acquisition schemes. sequential FID sampling requires only a single disk accessat the end of an experiment. Examples of this time-saving are outlined in Table 1. On our instrument this shortens a 15-20 min COSY experiment to less than 8 min. On older, unreliable instruments it was desirable to perform frequent disk backups during long experiments in order to prevent the loss of all the data in the event of an instrument failure. On newer instruments frequent backup is less essential (we have not lost data from a system crash in the past year of operation). In the event that periodic backup is desired, this can still be done at multiples of transients that complete a phase cycle. Since this alternate method for data collection involves acquiring the whole data table in the acquisition computer memory, and the standard COSY experiment requires saving each FID as acquisition is complete, the only remaining source of artifacts in the latter experiment would be potential variation in the disk addressing between experiments, which might disrupt the steady state. Although we have collected many spectra to indicate that this is not a factor, a standard COSY spectrum was obtained with conditions identical to those used to acquire the spectrum in Fig. 1 with the exception that 16 steady-state transients were averaged and discarded, to completely eliminate this factor as a possible source of artifacts. The resulting spectrum was virtually identical to that shown in Fig. 1. The third method for sequential FID sampling as illustrated in Scheme 3 is undesirable for COSY data acquisition. In this experiment, residual magnetization components which are propagated through the second COSY cycle mix intensity information from the real and imaginary,< signal components, resulting in severej; quadTABLE Experiment
Experimental SS = 4. NT SS = 0. NT SS = 4. NT SS=4.NT=4,DI SS = 4, NT
Times
for Collection of Data Using 2D NMR Sampling Technique
= 4, DI
.Vorc,. All parameters
Standard
Standard COSY (min)
conditions = 4, Dl = 4, DI = I, DI
I
= 0.5 = 0.5 = 0.5 =O.l = 1.0 as defined
28 16 20 16 52 in Scheme
I, AT = 0.426
and New
Alternate COSY (min) 13 13 2.5 6 22 s. and NI = IX.
536
RINALDI
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rature images. Artifacts and images which are a nuisance in standard COSY experiments create insurmountable problems using this method. One drawback of the technique is its demand for acquisition memory, which at minimum requires containment of half the data set (i.e., all FIDs for one phase in the States method for phase-sensitive 2D data collection). On careful consideration this requirement is not severely restrictive. On a 300 MHz instrument an additional I Mbyte acquisition memory board (to provide a total of 1.25 Mbytes of acquisition memory), at an additional cost of $2000 on a $350,000 instrument, removes memoryimposed limitations. With an instrument configured in this way, we have been able to include the entire 3 kHz proton spectral window in a COSY spectrum with a collection of 1024 X 256 X 2 data sets and zero filling to 2048 X 2048 before processing. Currently marketed NMR instruments based on personal workstation technology include up to 2 Mbytes of acquisition-dedicated memory as a standard feature. While all 2D NMR experiments will not be beneficially affected by the use of this alternate method of data acquisition, many experiments will provide better-quality spectra and should be performed in this manner. From our preliminary investigations, this alternative order of data collection is preferable to the standard method for COSY experiments. The ideal method of performing other 2D NMR experiments will depend on the nature and the number of pulses in the sequence and their effect on the spins. This alternative method of acquisition does not require that chemists learn new experiments or interpret new forms of data display. As always, they will decide whether a COSY or some other experiment is the best one to provide the structural information they need. Once this decision is made the instrument should be programmed to automatically collect data using the appropriate method. We are currently investigating further applications of this technique to the collection of 3D NMR data where it should yield considerable time-savings. ACKNOWLEDGMENTS We thank the University for an Academic Challenge
of Akron for a Faculty Research Grant Grant that provided financial support
and the State of Ohio of this research.
Board
of Regents
REFERENCES
Reson. 58,3 15 ( 1984). J. Magn. Reson. 56,207 ( 1984). 3. K. NAGAYAMA, P. BACHMANN, AND K. WUTHRICH, J. Magn. Reson. 31, 133 ( 1978). 4. G. OTTING, H. WIDMER, G. WAGNER, AND K. W~THRICH, J. Magn. Reson. 66, 187 ( 1986). 5. J. COOPER, in “Transform Techniques in Chemistry” (P. R. Griffiths, Ed.), Chap 4, Plenum Press, New
1. A. F. MEHLKOPF, D. KORBEE, T. A. TIGGELMAN, 2. G. WIDER, S. MACURA, A. KUMAR, R. R. ERNST,
6
York, 1978. R. E. KLEVIT, J. Magn.
7. 8. 9. IO.
S. 1. H. G.
Ii. 12.
W. E. P. P.
13. 14.
Reson.
AND R. FREEMAN, AND K. W~THRICH,
J. Magn.
62, 551 (1985).
GLASER AND H. R. KALBITZER, J. Magn. Reson. 68,350 ( 1986). L. BAARSUKOV AND A. S. ARSENIEV, J. Magn. Reson. 73, 148 ( 1987). SANTOS, D. L. TURNER, AND A. V. XAVIER, J. Magn. Resort 55,463 (1983). S. HARBISON, J. FEIGON, D. J. RUBEN, J. HERZFELD, AND R. G. GRIFFIN, J. Am. 5567 (1985). DENK, G. WAGNER, R. P. ZUIDERWEG, K. H. BOLTON, J. Magn. H. BOLTON, J. Magn.
Sot. 107,
AND K. W~THRICH, J. Magn. Reson. 62,350 (1985). AND E. T. OLEJNICZAK, J. Magn. Reson. 70, 336 ( 1986). 67, 391 (1986).
M. RANCE, HALLENGA,
Reson. Reson.
Chem.
64, 352 (1985).
NEW
METHOD
FOR
2D
DATA
537
COLLECTION
I.(. (u) R. BAUMANN, G. WIDER, R. R. ERNST, AND K. WOTHRICH, .I. Magn. Reson. 44,402 ( 1981); (h) R.BAUMANN, A.KuMAR,R.R.ERNsT, ANDK. WUTHRICH, J Magn.Reson.44,76( 1981). 16. (a) P. L.RINALDI ANDJ.K. HARDY, J. Magn. Reson. 88,320(1990);(b) D.E. BROWN ANDT. W. CAMBELL, J. Magn. Reson. 89,255 ( 1990). 17. N.MOLLER,G.BODENHAUSEN,K.WOTHRICH, AND R.R. ERNST, J. Magn. Reson.65,531 (1985). 1X. N.MuRALIANDA.KuMAR,C~~~.P~~~. Left. 128,58(1986). 1Y. M. L. GYNDELL, J. Mugn. Reson. 81,474 (1989). 20. C.J.TURNERANDS. L.PATT. J. Magn.Reson.85,492( 1989). 21. C. J. TURNER. J. Magn. Reson. 86,420 (1990). ?? A.E.DERoMEANDM.P. WILLIAMSON, J.Magn.Reson.88, 177(1990). 23. 0. W. SORENSEN.G. W. EICH, M. H. LEVITT. G. BODENHAUSEN, AND R. R. ERNST. Ptqq. NMR Spcctrosc.
16, 163 (1983).
34. W.KREIBICHANDA.SCWWENK, J Magn.Reson. 77,308(1988). -75. S. R. MAPLEANDA. ALLERHAND, J. Magn. Reson. 75, 147(1987). 26. A.ALLERHAND, R.E.ADDLEMAN,ANDD.OSMAN, J.Am.Chem.Soc. -77 D.J. STATES,R. A.HABERKORN, AND D.J.RuBEN, J. Mugn.Reson.48,286(
107,5809(1985).
1982).