y Spin echo ½πI - -πi y - : as needed, no correlaed wih 1/J. Funcions: 1. refocusing; 2. decoupling. Chemical shif evoluion is refocused by he spin-echo. Heeronuclear J-couplings evoluion are refocused by a spin-echo. Because only one spin eperiences a 180 pulse. Homonuclear couplings evoluion are no refocused by a spin-echo. Because boh spins eperiences a 180 pulse. I s also used for decoupling H 1 -N 15 by refocusing he H 1 magneizaion.
Hahn echo In 1950, Erwin Hahn firs deeced echoes in NMR, he applied wo successive 90 pulses separaed by a shor delay ime. This was furher developed by Carr and Purcell who used a 180 refocusing pulse o replace he second pulse. Spin echoes are someimes also called Hahn echoes. Hahn, E.L. (1950). "Spin echoes". Physical Review. 80: 580 594. Carr, H. Y.; Purcell, E. M. (1954). "Effecs of Diffusion on Free Precession in Nuclear Magneic Resonance Eperimens". Physical Review. 94: 630 638. Advanages: 1. Baseline disorions can be removed. (i s originally inroduced by Rance and Byrd.) 2. Waer suppression was significanly improved due o he refocusing properies of he 180 o pulse, which reduces he effec of inhomogeneous broadening a he base of he residual waer peak.
2D NOESY Reurns spins o y plane and creaes observable ransverse magneizaion 1 m 2 Frequencylabels he spins and inverse hem o z ais Allows ransfer of magneizaion beween proons dipolar-coupled, wihin ~5Å in space, by dipolar crossrelaaion, during he miing ime m Afer he final (90 ), a Hahn echo sequence (-Δ 1 πi y - Δ 2 ) can be added for improvemen of flaness of baseline. Φ 1 Φ 2 Φ 3 Φ 4 1 m 1 2 2 1 = 2 2 90 /π - gae
JR-NOESY: NOESY wih a jump-reurn observe pulse Φ 1 Φ 2 Φ 3 Φ 4 1 m 2 Much more peaks near he waer peak can be shown up han presa mehod. delay (~100us) and he lengh of he las pulse need o be opimized. Relayed NOESY Φ 1 Φ 2 Φ 3 Φ 4 m 1 1 2 2 DIPSI2rc Isoropic miing is normally 20-30 ms. For sequenial assignmen purpose, no for NOE disance informaion.
INEPT Insensiive Nucleus Enhanced by Polarizaion Transfer 1 H ±y = 1/(4* 1 J CH ) = 1/(4*212Hz) = 1.18ms 13 C ± INEPT sequence: ransfer of populaion differences from 1 H o X (X: 13 C, 15 N ec. 1 H and X are J-coupling ineracion), (by inversion of populaions of proon, changing populaions of spin X). I can enhance signal inensiy of X by γ H /γ X ( 13 C, ~4; 15 N, ~10), and is widely used in NMR eperimens.
1 H Refocused INEPT and Produc operaor analysis: righ hand rule y decouple 13 C ½πI - -π(i +S ) - - ½π(I y +S ) - - π(i +S ) - afer INEPT, ge ani-phase componen -2I z S y -2I z S y cos(πj) +S sin(πj) πi πi +2I z S y cos(πj) +S sin(πj) πs πs -2I z S y cos(πj) +S sin(πj) -2I z S y cos(πj) cos(πj) +S cos(πj) sin(πj) +S sin(πj) cos(πj) 2I z S z +2I z S y sin(πj) sin(πj) If = 1/(4J), +2I -2I z S z S y y +S In-phase componen
Reverse INEPT --- he reverse ransfer is achieved. 1 H 13 C ±y ± = 1/(4* 1 J CH ) = 1/(4*212Hz) = 1.18ms In real 2D or 3D ep., he firs 90 pulse on he 13 C is no needed because he aniphase magneizaion is already presen afer 1 evoluion, he reverse INEPT looks like he following in HSQC: 1 H ±y ± 13 C
HSQC Heeronuclear Single-Quanum Coherence Refocusing of 1 H J & δ Transfer of 15 N magneizaion o 1 H 1 H y 15 N 1 /2 1 /2 decouple To remove J NH double fine srucure, and double S/N raio Afer INEPT, SQ coherence ( 2I z S y ) generaes Reverse INEPT reverses he ani-phase 1 H magneizaion ino in-phase 1 H magneizaion Evoluion of 15 N chemical shif
HSQC produc operaor analysis: Afer INEPT sequence, we go: 2I z S y -½ 1 - πi -½ 1 - ½ 1 ½ 1 +2I z S y cos(ω s ½ 1 ) cos(ω s ½ 1 ) -2I z S y cos(ω s ½ 1 ) +2I z S sin(ω s ½ 1 ) πi πi +2I z S y cos(ω s ½ 1 ) -2I z S sin(ω s ½ 1 ) ½ 1 ½ ½ 1 1 ½ 1-2I z S cos(ω s ½ 1 ) sin(ω s ½ 1 ) -2I z S sin(ω s ½ 1 ) cos(ω s ½ 1 ) Sar of 1 cos(2α) = cos 2 α -sin 2 α; sin(2α) = 2sin(α)cos(α) = sin(α)cos(α) + cos(α)sin(α) -2I z S y sin(ω s ½ 1 ) sin(ω s ½ 1 ) +2I z S y [cos 2 (Ω s ½ 1 ) - sin 2 (Ω s ½ 1 )] -2I z S 2sin(Ω s ½ 1 )cos(ω s ½ 1 ) +2I z S y cos(ω s 1 ) Here is he end of 1. -2I z S sin(ω s 1 ) PEP-HSQC keep his erm oo, increase sensiiviy by up o 2.
½π(I + S ) - - π(i + S ) - +2I z S y cos(ω s 1 ) -2I z S sin(ω s 1 ) ½πI ½πI -2I y S y cos(ω s 1 ) +2I y S sin(ω s 1 ) ½πS ½πS -2I y S z cos(ω s 1 ) +2I y S sin(ω s 1 ) -2I y S z cos(ω s 1 ) +I cos(ω s 1 ) +2I y S sin(ω s 1 ) E/2 cos(πj) sin(πj) cos(πj) πi πi πi +2I y S z cos(ω s 1 ) +I cos(ω s 1 ) -2I y S sin(ω s 1 ) cos(πj) sin(πj) cos(πj) πs πs πs -2I y S z cos(ω s 1 ) +I cos(ω s 1 ) -2I y S sin(ω s 1 ) cos(πj) sin(πj) cos(πj) -2I y S z cos(ω s 1 ) +I +I cos(πj) cos(ω s 1 ) cos(ω s 1 ) +2I y S z cos(ω s 1 ) -2I cos(πj) sin(πj) y S sin(ω s 1 ) cos(πj) cos(πj) cos(πj) sin(πj) sin(πj) sin(πj) If = 1/(4J), -I y S z cos(ω s 1 ) +I y S z cos(ω s 1 ) +I cos(ω s 1 )
+I cos(ω s 1 ) The chemical shif of spin S cosine modulaes he ampliude of peak I. 1. HSQC and HMQC provide single-bond heeronuclear shif correlaions, he correlaion daa are equivalen for boh. 2. Hisorically, HSQC is favored by biological communiy, and presens 1 H- 15 N correlaions in proein molecules; HMQC is favored by chemical communiy, and presens 1 H- 13 C correlaions in small organic molecules. 3. Boh HSQC and HMQC have he following 3 feahers: From known proon assignmens, ge o know he correlaed heeronucleus assignmens. Proon peaks disperse according o he heeronucleus shif. Can idenify diasereoopic geminal pairs. 4. Only difference beween HSQC and HMQC is during 1 period: HSQC, only heeronulear ransverse SQ magneizaion (-2I z S y ) evolves, HMQC, 1 H- 13 C MQ coherence (-2I S y ) evolves. 5. In HSQC, homonuclear 1 H- 1 H couplings do no influence heeronuclear X (S y ) magneizaion evoluion signals do no conain homonuclear 1 H- 1 H couplings along f 1 improve resoluion in f 1 his is he principle advanage of HSQC over HMQC for small organic molecules. Bu HSQC use more pulses, especially 180 pulses on heeronuclears promoing inensiy losses from pulse miscalibraion, rf inhomogeneiy
PEP-HSQC --- Preservaion of Equivalen Pahways developed by Rance and coworkers addiional addiional Afer he firs INEPT, I z -2I z S y, afer 1 evoluion ( 1 /2 - π(i +K ) - 1 /2), -2I z S y 2I z S y cos(ω s 1 ) - 2I z S sin(ω s 1 ); hese wo orhogonal erms can be preserved, and sensiiviy can be improved by a facor up o 2. When processing hese kinds of 2D or 3D specra, one should choose Rance-Kay as ymode or zmode in nmrpipe process macro.
S 3 CT : spin-sae-selecive coherence ransfer I spin: 180 180 S spin: 90 180 90 y I spin: = 1/(4J IS ) S spin: y The S 3 CT elemen can conver ZQ and DQ coherences o SQ coherence.