160:, and their presence indicates that two nuclei are coupled which have the two different chemical shifts that make up the cross peak's coordinates. Each coupling gives two symmetrical cross peaks above and below the diagonal. That is, a cross-peak occurs when there is a correlation between the signals of the spectrum along each of the two axes at these values. An easy visual way to determine which couplings a cross peak represents is to find the diagonal peak which is directly above or below the cross peak, and the other diagonal peak which is directly to the left or right of the cross peak. The nuclei represented by those two diagonal peaks are coupled.
84:(RF) pulses with delay periods in between them. The timing, frequencies, and intensities of these pulses distinguish different NMR experiments from one another. Almost all two-dimensional experiments have four stages: the preparation period, where a magnetization coherence is created through a set of RF pulses; the evolution period, a determined length of time during which no pulses are delivered and the nuclear spins are allowed to freely precess (rotate); the mixing period, where the coherence is manipulated by another series of pulses into a state which will give an observable signal; and the detection period, in which the
251:
the detection period are called "inverse" experiments. This is because the low natural abundance of most heteronuclei would result in the proton spectrum being overwhelmed with signals from molecules with no active heteronuclei, making it useless for observing the desired, coupled signals. With the advent of techniques for suppressing these undesired signals, inverse correlation experiments such as HSQC, HMQC, and HMBC are actually much more common today. "Normal" heteronuclear correlation spectroscopy, in which the heteronucleus spectrum is recorded, is known as HETCOR.
187:, which cause only signals from double-quantum coherences to give an observable signal. This has the effect of decreasing the intensity of the diagonal peaks and changing their lineshape from a broad "dispersion" lineshape to a sharper "absorption" lineshape. It also eliminates diagonal peaks from uncoupled nuclei. These all have the advantage that they give a cleaner spectrum in which the diagonal peaks are prevented from obscuring the cross peaks, which are weaker in a regular COSY spectrum.
1416:
260:
164:
417:
useful for analysing molecules for which the 1D-NMR spectra contain overlapping multiplets as the J-resolved spectrum vertically displaces the multiplet from each nucleus by a different amount. Each peak in the 2D spectrum will have the same horizontal coordinate that it has in a non-decoupled 1D spectrum, but its vertical coordinate will be the chemical shift of the single peak that the nucleus has in a decoupled 1D spectrum.
1428:
210:
405:, because ROESY has a different dependence between the correlation time and the cross-relaxation rate constant. In NOESY the cross-relaxation rate constant goes from positive to negative as the correlation time increases, giving a range where it is near zero, whereas in ROESY the cross-relaxation rate constant is always positive.
365:
measurable NOEs to the resonance of interest but takes much less time than the full 2D experiment. In addition, if a pre-selected nucleus changes environment within the time scale of the experiment, multiple negative signals may be observed. This offers exchange information similar to the EXSY (exchange spectroscopy) NMR method.
416:
Unlike correlated spectra, resolved spectra spread the peaks in a 1D-NMR experiment into two dimensions without adding any extra peaks. These methods are usually called J-resolved spectroscopy, but are sometimes also known as chemical shift resolved spectroscopy or δ-resolved spectroscopy. They are
176:. In COSY-45 a 45° pulse is used instead of a 90° pulse for the second pulse, p2. The advantage of a COSY-45 is that the diagonal-peaks are less pronounced, making it simpler to match cross-peaks near the diagonal in a large molecule. Additionally, the relative signs of the coupling constants (see
320:
In HMBC, this difficulty is overcome by omitting one of these delays from an HMQC sequence. This increases the range of coupling constants that can be detected, and also reduces signal loss from relaxation. The cost is that this eliminates the possibility of decoupling the spectrum, and introduces
225:
In the case of oligosaccharides, each sugar residue is an isolated spin system, so it is possible to differentiate all the protons of a specific sugar residue. A 1D version of TOCSY is also available, and by irradiating a single proton the rest of the spin system can be revealed. Recent advances in
303:
step can then optionally be used to decouple the signal, simplifying the spectrum by collapsing multiplets to a single peak. The undesired uncoupled signals are removed by running the experiment twice with the phase of one specific pulse reversed; this reverses the signs of the desired but not the
250:
Heteronuclear correlation spectroscopy gives signal based upon coupling between nuclei of two different types. Often the two nuclei are protons and another nucleus (called a "heteronucleus"). For historical reasons, experiments which record the proton rather than the heteronucleus spectrum during
364:
The NOESY experiment can also be performed in a one-dimensional fashion by pre-selecting individual resonances. The spectra are read with the pre-selected nuclei giving a large, negative signal while neighboring nuclei are identified by weaker, positive signals. This only reveals which peaks have
316:
HMBC detects heteronuclear correlations over longer ranges of about 2–4 bonds. The difficulty of detecting multiple-bond correlations is that the HSQC and HMQC sequences contain a specific delay time between pulses which allows detection only of a range around a specific coupling constant.
217:
The TOCSY experiment is similar to the COSY experiment, in that cross peaks of coupled protons are observed. However, cross peaks are observed not only for nuclei which are directly coupled, but also between nuclei which are connected by a chain of couplings. This makes it useful for identifying
141:
The first and most popular two-dimension NMR experiment is the homonuclear correlation spectroscopy (COSY) sequence, which is used to identify spins which are coupled to each other. It consists of a single RF pulse (p1) followed by the specific evolution time (t1) followed by a second pulse (p2)
376:
In HOESY, much like NOESY is used for the cross relaxation between nuclear spins. However, HOESY can offer information about other NMR active nuclei in a spatially relevant manner. Examples include any nuclei X{Y} or X→Y such as H→C, F→C, P→C, or Se→C. The experiments typically observe NOEs from
420:
For the heteronuclear version, the simplest pulse sequence used is called a Müller–Kumar–Ernst (MKE) experiment, which has a single 90° pulse for the heteronucleus for the preparation period, no mixing period, and applies a decoupling signal to the proton during the detection period. There are
345:
In NOESY, the nuclear
Overhauser cross relaxation between nuclear spins during the mixing period is used to establish the correlations. The spectrum obtained is similar to COSY, with diagonal peaks and cross peaks, however the cross peaks connect resonances from nuclei that are spatially close
241:
of C is only about 1%, only about 0.01% of molecules being studied will have the two nearby C atoms needed for a signal in this experiment. However, correlation selection methods are used (similarly to DQF COSY) to prevent signals from single C atoms, so that the double C signals can be easily
133:
242:
resolved. Each coupled pair of nuclei gives a pair of peaks on the INADEQUATE spectrum which both have the same vertical coordinate, which is the sum of the chemical shifts of the nuclei; the horizontal coordinate of each peak is the chemical shift for each of the nuclei separately.
307:
Heteronuclear multiple-quantum correlation spectroscopy (HMQC) gives an identical spectrum as HSQC, but using a different method. The two methods give similar quality results for small to medium-sized molecules, but HSQC is considered to be superior for larger molecules.
167:
H COSY spectrum of progesterone. The spectrum that appears along both the horizontal and vertical axes is a regular one dimensional H NMR spectrum. The bulk of the peaks appear along the diagonal, while cross-peaks appear symmetrically above and below the
263:
H–N HSQC spectrum of a fragment of the protein NleG3-2. Each peak in the spectrum represents a bonded N–H pair, with its two coordinates corresponding to the chemical shifts of each of the H and N atoms. Some of the peaks are labeled with the
104:. A single two-dimensional experiment is generated as a series of one-dimensional experiments, with a different specific evolution time in successive experiments, with the entire duration of the detection period recorded in each experiment.
317:
This is not a problem for the single-bond methods since the coupling constants tend to lie in a narrow range, but multiple-bond coupling constants cover a much wider range and cannot all be captured in a single HSQC or HMQC experiment.
136:
In standard COSY, the preparation (p1) and mixing (p2) periods each consist of a single 90° pulse separated by the evolution time t1, and the resonance signal from the sample is read during the detection period over a range of times
91:
The two dimensions of a two-dimensional NMR experiment are two frequency axes representing a chemical shift. Each frequency axis is associated with one of the two time variables, which are the length of the evolution period (the
671:
Buddrus, J. and Bauer, H. (1987), Direct
Identification of the Carbon Skeleton of Organic Compounds using Double Quantum Coherence 13C-NMR Spectroscopy. The INADEQUATE Pulse Sequence. Angew. Chem. Int. Ed. Engl., 26: 625-642.
278:
HSQC detects correlations between nuclei of two different types which are separated by one bond. This method gives one peak per pair of coupled nuclei, whose two coordinates are the chemical shifts of the two coupled atoms.
107:
The end result is a plot showing an intensity value for each pair of frequency variables. The intensities of the peaks in the spectrum can be represented using a third dimension. More commonly, intensity is indicated using
226:
this technique include the 1D-CSSF (chemical shift selective filter) TOCSY experiment, which produces higher quality spectra and allows coupling constants to be reliably extracted and used to help determine stereochemistry.
294:
pulse sequence; this first step is done because the proton has a greater equilibrium magnetization and thus this step creates a stronger signal. The magnetization then evolves and then is transferred back to the
153:
and H.) Diagonal peaks correspond to the peaks in a 1D-NMR experiment, while the cross peaks indicate couplings between pairs of nuclei (much as multiplet splitting indicates couplings in 1D-NMR).
149:, most commonly hydrogen (H) along both axes. (Techniques have also been devised for generating heteronuclear correlation spectra, in which the two axes correspond to different isotopes, such as
441:
3D and 4D experiments can also be done, sometimes by running the pulse sequences from two or three 2D experiments in series. Many of the commonly used 3D experiments, however, are
368:
NOESY experiments are important tool to identify stereochemistry of a molecule in solvent whereas single crystal XRD used to identify stereochemistry of a molecule in solid form.
180:) can be elucidated from a COSY-45 spectrum. This is not possible using COSY-90. Overall, the COSY-45 offers a cleaner spectrum while the COSY-90 is more sensitive.
329:
These methods establish correlations between nuclei which are physically close to each other regardless of whether there is a bond between them. They use the
273:
291:
321:
phase distortions into the signal. There is a modification of the HMBC method which suppresses one-bond signals, leaving only the multiple-bond signals.
920:
1454:
646:
1138:
1071:
1016:
985:
183:
Another related COSY technique is double quantum filtered (DQF) COSY. DQF COSY uses a coherence selection method such as phase cycling or
68:, a professor at the Université Libre de Bruxelles, in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and
172:
COSY-90 is the most common COSY experiment. In COSY-90, the p1 pulse tilts the nuclear spin by 90°. Another member of the COSY family is
980:
385:
ROESY is similar to NOESY, except that the initial state is different. Instead of observing cross relaxation from an initial state of
350:
which do not provide extra information and can be eliminated through a different experiment by reversing the phase of the first pulse.
218:
the larger interconnected networks of spin couplings. This ability is achieved by inserting a repetitive series of pulses which cause
57:
provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a
393:
axis and then spin-locked by an external magnetic field so that it cannot precess. This method is useful for certain molecules whose
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1171:
1033:
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during the mixing period. Longer isotropic mixing times cause the polarization to spread out through an increasing number of bonds.
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34:
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1111:
1056:
1006:
486:
Aue, W. P.; Bartholdi, E.; Ernst, R. R. (1976). "Two-dimensional spectroscopy. Application to nuclear magnetic resonance".
466:
421:
several variants on this pulse sequence which are more sensitive and more accurate, which fall under the categories of
37:(NMR) methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include
196:
703:
Wu, Bin; Skarina, Tatiana; Yee, Adelinda; Jobin, Marie-Claude; DiLeo, Rosa; Semesi, Anthony; et al. (June 2010).
1338:
1090:
906:
884:
848:
811:
1343:
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1328:
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408:
ROESY is sometimes called "cross relaxation appropriate for minimolecules emulated by locked spins" (CAMELSPIN).
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1078:
975:
1432:
1085:
990:
397:
falls in a range where the nuclear
Overhauser effect is too weak to be detectable, usually molecules with a
1219:
1066:
955:
442:
88:
signal from the sample is observed as a function of time, in a manner identical to one-dimensional FT-NMR.
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1214:
1183:
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394:
334:
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1307:
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The two-dimensional spectrum that results from the COSY experiment shows the frequencies for a single
1391:
1370:
1011:
330:
50:
1133:
650:
333:(NOE) by which nearby atoms (within about 5 Ă…) undergo cross relaxation by a mechanism related to
237:
INADEQUATE is a method often used to find C couplings between adjacent carbon atoms. Because the
1264:
960:
346:
rather than those that are through-bond coupled to each other. NOESY spectra also contain extra
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876:
869:
533:
232:
177:
157:
100:). They are each converted from a time series to a frequency series through a two-dimensional
61:, particularly for molecules that are too complicated to work with using one-dimensional NMR.
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1023:
937:
184:
20:
495:
85:
8:
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970:
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In these methods, magnetization transfer occurs between nuclei of the same type, through
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807:
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585:
450:
238:
101:
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730:
720:
503:
398:
229:
TOCSY is sometimes called "homonuclear
Hartmann–Hahn spectroscopy" (HOHAHA).
69:
702:
304:
undesired peaks, so subtracting the two spectra will give only the desired peaks.
725:
446:
81:
380:
521:
254:
311:
1448:
402:
803:
233:
Incredible natural-abundance double-quantum transfer experiment (INADEQUATE)
929:
744:
353:
One application of NOESY is in the study of large biomolecules, such as in
109:
841:
One-dimensional and two-dimensional NMR Spectra by Modern Pulse
Techniques
793:
673:
454:
354:
65:
371:
265:
121:
507:
430:
300:
150:
528:
Two-Dimensional NMR Methods for
Establishing Molecular Connectivity
163:
58:
843:. Mill Valley, California: University Science Books. p. 136.
389:-magnetization, the equilibrium magnetization is rotated onto the
132:
340:
245:
146:
898:
115:
381:
Rotating-frame nuclear
Overhauser effect spectroscopy (ROESY)
190:
259:
255:
Heteronuclear single-quantum correlation spectroscopy (HSQC)
64:
The first two-dimensional experiment, COSY, was proposed by
312:
Heteronuclear multiple-bond correlation spectroscopy (HMBC)
204:
377:
protons on X, X{H}, but do not have to include protons.
209:
96:) and the time elapsed during the detection period (the
27:
Two-dimensional nuclear magnetic resonance spectroscopy
866:
357:, in which relationships can often be assigned using
274:
Heteronuclear single-quantum correlation spectroscopy
549:
2D NMR Density Matrix and
Product Operator Treatment
372:
Heteronuclear
Overhauser effect spectroscopy (HOESY)
324:
795:
High-Resolution NMR Techniques in
Organic Chemistry
868:
551:. Englewood Cliffs, New Jersey: PTR Prentice Hall.
525:
282:HSQC works by transferring magnetization from the
127:
485:
1446:
429:. Homonuclear J-resolved spectroscopy uses the
838:
705:"NleG Type 3 Effectors from Enterohaemorrhagic
566:. Cheltenham, UK: Stanley Thornes. p. 273.
547:Mateescu, Gheorghe D.; Valeriu, Adrian (1993).
546:
341:Nuclear Overhauser effect spectroscopy (NOESY)
246:Heteronuclear through-bond correlation methods
914:
770:
768:
758:
756:
754:
436:
16:Set of methods providing two-dimensional data
986:Vibrational spectroscopy of linear molecules
834:
832:
687:
605:
603:
601:
520:
411:
156:Cross peaks result from a phenomenon called
116:Homonuclear through-bond correlation methods
820:
777:
290:nucleus (usually the heteroatom) using the
53:spectroscopy (NOESY). Two-dimensional NMR
19:"COSY" redirects here. For other uses, see
981:Nuclear resonance vibrational spectroscopy
921:
907:
862:
860:
765:
751:
678:
630:
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573:
191:Exclusive correlation spectroscopy (ECOSY)
124:of nuclei connected by up to a few bonds.
80:Each experiment consists of a sequence of
1354:Inelastic electron tunneling spectroscopy
1034:Resonance-enhanced multiphoton ionization
829:
734:
724:
598:
584:(2nd ed.). Wiley. pp. 184–187.
561:
532:. New York: VCH Publishers, Inc. p.
1122:Extended X-ray absorption fine structure
621:
258:
208:
162:
131:
1455:Nuclear magnetic resonance spectroscopy
857:
570:
142:followed by a measurement period (t2).
75:
35:nuclear magnetic resonance spectroscopy
1447:
867:Schraml, Jan; Bellama, Jon M. (1988).
674:https://doi.org/10.1002/anie.198706251
579:
205:Total correlation spectroscopy (TOCSY)
902:
1427:
649:. Queen's University. Archived from
647:"2D: Homonuclear correlation: TOCSY"
467:Two-dimensional correlation analysis
286:nucleus (usually the proton) to the
213:Typical TOCSY values for amino acids
72:, who published their work in 1976.
299:nucleus for observation. An extra
13:
562:Akitt, J. W.; Mann, B. E. (2000).
197:Exclusive correlation spectroscopy
178:J-coupling#Magnitude of J-coupling
14:
1466:
1339:Deep-level transient spectroscopy
1091:Saturated absorption spectroscopy
325:Through-space correlation methods
1426:
1415:
1414:
1344:Dual-polarization interferometry
928:
1359:Scanning tunneling spectroscopy
1334:Circular dichroism spectroscopy
1329:Acoustic resonance spectroscopy
871:Two-Dimensional NMR Spectrocopy
826:Keeler, pp. 273, 297–299.
786:
783:Keeler, pp. 274, 281–284.
709:Are U-Box E3 Ubiquitin Ligases"
696:
693:Keeler, pp. 208–209, 220.
665:
639:
268:residue that gives that signal.
128:Correlation spectroscopy (COSY)
1288:Fourier-transform spectroscopy
976:Vibrational circular dichroism
612:
582:Understanding NMR Spectroscopy
555:
540:
514:
479:
1:
1086:Cavity ring-down spectroscopy
991:Thermal infrared spectroscopy
839:Nakanishi, Koji, ed. (1990).
472:
1220:Inelastic neutron scattering
875:. New York: Wiley. pp.
726:10.1371/journal.ppat.1000960
443:triple resonance experiments
7:
1281:Data collection, processing
1157:Photoelectron/photoemission
488:Journal of Chemical Physics
460:
395:rotational correlation time
10:
1471:
1366:Photoacoustic spectroscopy
1308:Time-resolved spectroscopy
774:Keeler, pp. 215–219.
762:Keeler, pp. 209–215.
684:Keeler, pp. 206–208.
636:Keeler, pp. 223–226.
627:Keeler, pp. 199–203.
609:Keeler, pp. 190–191.
453:, which are often used in
437:Higher-dimensional methods
271:
194:
18:
1410:
1392:Astronomical spectroscopy
1384:
1371:Photothermal spectroscopy
1321:
1280:
1273:
1235:
1207:
1149:
1099:
999:
936:
618:Akitt & Mann, p. 287.
412:Resolved-spectrum methods
331:nuclear Overhauser effect
51:nuclear Overhauser effect
524:; Zekter, A. S. (1988).
39:correlation spectroscopy
1376:Pump–probe spectroscopy
1265:Ferromagnetic resonance
1057:Laser-induced breakdown
804:10.1016/c2015-0-04654-8
445:; examples include the
423:gated decoupler methods
335:spin–lattice relaxation
1072:Glow-discharge optical
1052:Raman optical activity
966:Rotational–vibrational
580:Keeler, James (2010).
269:
214:
185:pulsed field gradients
169:
158:magnetization transfer
138:
1293:Hyperspectral imaging
262:
212:
166:
135:
112:or different colors.
47:exchange spectroscopy
21:Cosy (disambiguation)
1045:Coherent anti-Stokes
1000:UV–Vis–NIR "Optical"
653:on 27 September 2011
86:free induction decay
76:Fundamental concepts
1349:Hadron spectroscopy
1139:Conversion electron
1100:X-ray and Gamma ray
1007:Ultraviolet–visible
500:1976JChPh..64.2229A
1397:Force spectroscopy
1322:Measured phenomena
1313:Video spectroscopy
1017:Cold vapour atomic
451:HNCOCA experiments
359:sequential walking
270:
215:
170:
139:
1442:
1441:
1406:
1405:
1298:Spectrophotometry
1225:Neutron spin echo
1199:Beta spectroscopy
1112:Energy-dispersive
591:978-0-470-74608-0
564:NMR and Chemistry
427:spin-flip methods
239:natural abundance
102:Fourier transform
1462:
1430:
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1417:
1278:
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1189:phenomenological
938:Vibrational (IR)
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877:28–33, 49–50, 65
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433:pulse sequence.
399:molecular weight
220:isotropic mixing
70:Richard R. Ernst
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719:(6): e1000960.
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1172:Angle-resolved
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713:PLOS Pathogens
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494:(5): 2229–46.
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98:detection time
94:evolution time
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43:J-spectroscopy
33:) is a set of
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1029:Near-infrared
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110:contour lines
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28:
22:
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1399:(a misnomer)
1385:Applications
1303:Time-stretch
1247:
1194:paramagnetic
1012:Fluorescence
930:Spectroscopy
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655:. Retrieved
651:the original
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522:Martin, G. E
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49:(EXSY), and
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42:
38:
30:
26:
25:
971:Vibrational
455:protein NMR
355:protein NMR
348:axial peaks
66:Jean Jeener
1177:Two-photon
1079:absorption
961:Rotational
473:References
266:amino acid
122:J-coupling
1255:Terahertz
1236:Radiowave
1134:Mössbauer
431:spin echo
301:spin echo
168:diagonal.
1449:Category
1421:Category
1150:Electron
1117:Emission
1067:emission
1024:Vibronic
798:. 2016.
745:20585566
461:See also
59:molecule
41:(COSY),
1433:Commons
1260:ESR/EPR
1208:Nucleon
1036:(REMPI)
736:2891834
657:26 June
496:Bibcode
403:daltons
174:COSY-45
147:isotope
55:spectra
1274:Others
1062:Atomic
883:
847:
810:
743:
733:
588:
31:2D NMR
1215:Alpha
1184:Auger
1162:X-ray
1129:Gamma
1107:X-ray
1040:Raman
951:Raman
946:FT-IR
292:INEPT
881:ISBN
845:ISBN
808:ISBN
741:PMID
659:2011
586:ISBN
449:and
447:HNCA
425:and
1243:NMR
800:doi
731:PMC
721:doi
504:doi
137:t2.
1451::
1248:2D
1167:UV
879:.
859:^
831:^
806:.
767:^
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739:.
729:.
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600:^
572:^
534:59
502:.
492:64
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45:,
922:e
915:t
908:v
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723::
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661:.
594:.
536:.
510:.
506::
498::
391:x
387:z
297:I
288:S
284:I
151:C
29:(
23:.
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
