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Some paradigmatic examples. Typical 1 H NMR Spectrum Absorbance.

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Presentazione sul tema: "Some paradigmatic examples. Typical 1 H NMR Spectrum Absorbance."— Transcript della presentazione:

1 Some paradigmatic examples

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3 Typical 1 H NMR Spectrum Absorbance

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11 Valore medio: 2 MUSD/anno

12 K1 T2 L3 T4 L5 E6 A7 A8 L9 R10 N11 A12 W13 L14 R 15 E16 V17 G18 L19 K MHz 1 H NMR

13 Ubiquitin 76 amino acids, 8,5 kDa

14 Protein 1 H NMR spectrum: a real spectrum Fourier Transformation The NMR signal in the time domain Free Induction Decay A short pulse will excite all spins All spins will relax (all together) during time AQ The FT of FID gives the NMR spectrum

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18 1D experiment Could be nice but.....Too crowded.. What do we learn? Chemical shifts relaxation rates Not enough to get a structure

19 STRUTTURE IN SOLUZIONE VIA NMR

20 The need for multidimensional NMR

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22 Cosa è un esperimento bidimensionale ? Dopo un impulso a 90° il segnale è pronto per essere acquisito Facciamo lacquisizione ma NON terminiamo lesperimento ed applichiamo ancora uno o piu impulsi in modo da perturbare ulterioremente il sistema Attraverso una combinazione di impulsi e delays noi facciamo in modo che ci sia uno scambio di magnetizzazione tra spin accoppiati SUCCESSIVAMENTE, acquisiamo il segnale una seconda volta, Registrando il segnale NMR che rimane sul piano xy dopo la perturbazione

23 Eccito (impulso a 90°)-Acquisisco (t1)- Perturbo (trasferisco)- Acquisisco (t2) Se la perturbazione non ha effetto e se non cè trasferimento di alcun tipo, Ottengo lo stesso spettro in ciascuna delle 2 dimensioni tempo (t1 e t2) Dopo la trasformate di Fourier io otterro uno spettro dove i segnali appaiono su una diagonale di una matrice quadrata

24 Se durante la perturbazione una parte della magnetizzazione si traferisce da un nucleo ad un altro, per esempio per effetto di accoppiamento scalare, allora lo spettro della dimensione t2 sarà diverso da quello della dimensione t1. Il risultato è che avro dei segnali fuori dalla diagonale. Ciascun segnale fuori dalla diagonale darà linformazione sugli accoppiamenti scalari attivi nel sistema M ( I t 1 ) ( S t 2 ) Acquisisco (t1)- Perturbo (trasferisco)- Acquisisco (t2)

25 EXAMPLE N H H C C O We make a 1H experiment and we acquire. Then all signals transfer the information because of scalar coupling N H H C Then I observe Hc I observe Hn I consider the first and the second acquisition as two indpendent dimensions Spectrum after The J coupling Spectrum before The J coupling

26 EXAMPLE N H H C C O N H H C Spectrum after The J coupling Spectrum before The J coupling 4 ppm9 ppm Signal! This indicates that there is a scalar coupling between Hn and Hc

27 EXAMPLE N H H C C O N H H C Spectrum after The J coupling Spectrum before The J coupling 4 ppm9 ppm Signal! This indicates that there is a scalar coupling between Hn and Hc Hn Hc J-coupling

28 EXAMPLE N H H C C O Spectrum after The J coupling Spectrum before The J coupling Hc Hn J-coupling If you begin from Hc, the situation is the same !

29 EXAMPLE N H H C C O Spectrum after The J coupling Spectrum before The J coupling Hc Hn J-coupling Therefore, if I consider only this system Hn Hc J-coupling

30 The first dimension = t 1 The second dimension = t 2 the series of pulses that I have to apply to my system = PULSE SEQUENCE example t1t1 t2t2 t 1 dimension Or F1 t 2 dimension Or F2

31 Usually t 1 is also defined as indirect dimension t 2 is also defined as direct dimension the series of pulses that I have to apply to my system = PULSE SEQUENCE example t1t1 t2t2 t 1 dimension Or F1 t 2 dimension Or F2

32 F1 F2 Definitions Cross peak Two different frequencies are observed in the two dimensions Diagonal peak The same frequency is observed in both dimensions CROSS PEAK= Yes, There is a COUPLING between the two frequencies

33 Accoppiamento scalare Laccoppiamento scalare puo comunque essere osservato attraverso esperimenti NMR bidimensionali, quali il COSY

34 Example: COSY Through-bond connectivities COSY= COrrelation SpectroscopY H4-H5

35 Example: COSY Through-bond connectivities COSY= COrrelation SpectroscopY H4-H

36 Beyond COSY COSY is not the only 2D experiment It is possible to transfer the information from spin A to spin B via several possible mechanisms The most important routes, which is COMPLEMENTARY TO J-coupling Is THROUGH SPACE COUPLING

37 Accoppiamento dipolare Laccoppiamento dipolare si ha tra due spin che sono vicini nello spazio Si tratta della interazione tra due dipoli magnetici, tra i quali, quando essi sono vicini nello spazio, si ha uno scambio di energia Lentità delleffetto dipende dal campo magnetico e dalle dimensioni della molecola. Nel caso di spin 1H, laccoppiamento dipolare si trasferisce per spin che si trovano a distanze inferiori ai 5 A. NON si osservano doppietti Laccoppiamento dipolare da luogo ad un trasferimento di magnetizzazione da uno spin allaltro. Questo effetto va sotto il nome di effetto NOE Nuclear Overhauser Effect Perturbo A Aumenta la intensità di B

38 Accoppiamento dipolare Laccoppiamento dipolare è indipendente dallaccoppiamento scalare 2 spin possono essere accoppiati : -Scalarmente E dipolarmente se sono vicini nello spazio e legati da legami chimici -scalarmente ma non dipolarmente se sono legati da legami chimici ma non vicini nello spazio -dipolarmente ma non scalarmente se sono spazialmente vicini ma non legati da legamei chimici Pensate a degli esempi, per favore Leffetto NOE è osservabile in un esperimento NMR bidimensionale, detto NOESY (in realtà si puo anche osservare in esperimenti monodimensionle (1D NOE) di cui pero non parleremo

39 Through space AND throuhg bonds Through space Through bond

40 Example: Nuclear Overhauser Effect SpectroscopY NOESY NOE Effect: If two spins that are close in space are excited out of equilibrium, they will mutually transfer their magnetization AA AB

41 Example: Cross peaks: A and B are close Diagonal peak The real case: Some 1500 peaks are observed for a protein of 75 aminoacid s AAAB NOESY experiment

42 2D NOESY Spectrum

43 Distance constraints NOESY volumes are proportional to the sixth power of the interproton distance and to the correlation time for the dipolar coupling B0B0B0B0 I J r

44 The old times approachNOESY COSY Identify through space connectivities HN(i)-Ha(i) and HN(i)Ha(i-1) Identify through bond connectivities HN(i)-Ha(i) NOESY conn. COSY conn

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47 1 J couplings for backbone resonances

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49 The 2D Hetcor experiment Two dimensional Heteronuclear correlation Experiment

50 The 2D Hetcor experiment Two dimensional Heteronuclear correlation Experiment

51 E possibile, in uno stesso esperimento mandare impulsi su nuclidi diversi (Es: 1 H, 13 C) possibile, combinare questa possibilità con ciò che sappiamo a proposito degli accoppiamenti scalari e quindi UTILIZZARE gli accoppiamenti scalari per trasferire la magnetizzazione dauno spin 1H ad uno spin 13C ad esso scalarmente accoppiato

52 E possibile, in uno stesso esperimento mandare impulsi su nuclidi diversi (Es: 1 H, 13 C) Inoltre possiamo combinare tutto cio con quello che sappiamo sugli esperimenti bidimensionali

53 Eccito (impulso a 90°) 1 H Acquisisco (t1) 1 H – Perturbo (Trasferisco la magnetizzazione da 1 H a 13 C utilizzando laccoppiamento scalare 1 J HC Acquisisco (t2) 13 C 2D HETCOR Expriment

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55 Prima dimensione

56 2D HETCOR Expriment Prima dimensione

57 2D HETCOR Expriment Prima dimensione Seconda dimensione

58 2D HETCOR Expriment Prima dimensione Seconda dimensione

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60 Esempio COSY

61 Esempio COSY N.B. In questo caso non si osserva solo laccoppiamento 3J ma si osserva una propagazione dellinformazione attraverso gli accoppiamenti scalari

62 Esempio HETCOR

63 Heteronuclear Single Quantum coherence 2D HSQC Experiment

64 2D HSQC Expriment Heteronuclear Single Quantum coherence

65 2D HSQC Experiment Prima dimensione Seconda dimensione Heteronuclear Single Quantum coherence

66 2D HSQC Expriment Prima dimensione Heteronuclear Single Quantum coherence

67 2D HSQC Experiment Seconda dimensione Prima dimensione Heteronuclear Single Quantum coherence

68 2D HSQC Experiment Heteronuclear Single Quantum coherence Seconda dimensione Prima dimensione E possibile progettare esperimenti per trasferire la magnetizzazione da un nucleo allaltro anche indipendentemente dallacquisizione In questo esperimento il primo spin che viene eccitato è 1H, la magnetizzazione viene trasferita da 1H a 13C PRIMA della acquisizione della prima dimensione, che quindi è 13C. SOLO i 13C che sono accoppiati ad 1H possono essere osservati! Successivamente la magnetizzazione e di nuovo trasferita 1H utilizzando sempre laccoppiamento scalare ed alla fine osservo 1H

69 Eccito (impulso a 90°) 1 H Trasferisco la magnetizzazione da 1 H a 13 C utilizzando laccoppiamento scalare 1 J HC Acquisisco (t1) 13 C – Perturbo -Trasferisco la magnetizzazione da 13 C a 1 H utilizzando laccoppiamento scalare 1 J HC Acquisisco (t2) 1 H 2D HSQC Experiment

70 Questo tipo di esperimento si chiama anche Out and back Significa che parto da 1H, trasferisco da 1H a 13C (out), acquisisco 13C nella prima dimensione e poi torno (back) sullo stesso nucleo da cui sono partito 2D HSQC Experiment Il doppio trasferimento fa si che lesperimento sia molto piu selettivo Osservo solo 1H e 13C che sono accoppiati tra di se per effetto di 1 J

71 The HSQC experiment

72 Caratteristiche dellesperimento HSQC Non esiste la diagonale La magnetizzazione viene trasferita da 1 H al 13 C ad esso accoppiato Successivamente si acquisisce, nella dimensione indiretta, 13 C Infine si ri-trasferisce su 1 H e si osserva 1 H Tutti gli 1 H che non sono accoppiati a 13 C NON si osservano

73 Heteronuclear NMR OBSERVE 13 C during t 1 Transfer the information to all 1 H coupled OBSERVE 1 H during t 2 1H1H 13 C No more diagonal Each peak indicate A different H-C pair

74 Heteronuclear NMR OBSERVE 13 C during t 1 Transfer the information to all 1 H coupled Observe 1 H during t 2 1H 13C No more diagonal Two protons are bound to the same carbon CH 2

75 The HSQC experiment

76 Heteronuclear cases The scheme of 1J scalar couplings

77 The 1H- 15N HSQC experiment Heteronuclear Single Quantum Coherence

78 The HSQC experiment In 5 minutes you may know…. if your protein is properly folded if all aminoacids gives rise to an observable peak Each amide NH group gives rise to one peak Detect H-N couplings Same sensitivity of a 1 H experiment (although you are observing 15 N) but much larger resolution if you can do the job (whatever is your job)

79 Heteronuclear NMR in proteins example: 15 N labelled proteins

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81 The HSQC experiment In 5 minutes you may know…. if your protein is properly folded if all aminoacids gives rise to an observable peak Each amide NH group gives rise to one peak Detect H-N couplings Same sensitivity of a 1 H experiment (although you are observing 15 N) but much larger resolution

82 Ca 2+ Apo 3.3 M GdmCl Loss of secondary structure elements: unfolded protein Refolding Ca M GdmCl The role of metal cofactor in protein unfolding Metal triggered protein folding

83 HN 1 83 HN 28 HN 32 Apo vs holo protein, mapping the environment of the metal ion 15 N 15 H

84 The need for multidimensional NMR

85 Troppi segnali 1 H ?

86 Isotope labeling For biomolecules, tipically, 15 N or 13 C and 15 N, or 13 C, 15 N, 2 H 15 N Only A more effective fingerprint -characterization -folding -dynamics protein size >10000 Homonuclear 2D experiments do not have enough resolution HSQC or HMQC HSQC-NOESY or HSQC TOCSY

87 Isotope labeling For biomolecules, tipically, 15 N or 13 C and 15 N, or 13 C, 15 N, 2 H 15 N and 13 C Scalar couplings through 13 C atoms -triple resonance -assignment -structure protein size >20000

88 Overview of Protein Expression Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein. Introduction to Isotope Labeling of Proteins For NMR Many recombinant proteins can be expressed to high levels in E. coli systems. most common choice for expressing labeled proteins for NMR Yeast (Pichia pastoris, Saccaromyces cerevisiae) is an alternative choice for NMR protein samples issues with glycosolyation of protein, which is not a problem with E. coli. choice between E. coli and yeast generally depend on personal experience. Insect cells (Baculovirus) and mammalian cell lines (CHO) are very popular expression systems that are currently not amenable for NMR samples no mechanism to incorporate isotope labeling or the process is cost prohibitive 15 N labeling in CHO cells can cost $ K!

89 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression First step of the process involves the insertion of the DNA coding region of the protein of interest into a plasmid. plasmid - small, circular pieces of DNA that are found in E. coli and many other bacteria generally remain separate from the bacterial chromosome carry genes that can be expressed in the bacterium plasmids generally replicate and are passed on to daughter cells along with the chromosome Plasmids are highly infective, so many of the bacteria will take up the particles from simple exposure. – Treating with calcium salts make membranes permeable and increase uptake of plasmids Plasmids used for cloning and expressing proteins are modified natural vectors - more compact and efficient - unnecessary elements removed Some Common plasmids - pBR322 - pUC19 - pBAD large collections of plasmids with unique features and functions - see:

90 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Basic Features of a Plasmid Defined region with restriction sites for inserting the DNA Gene that provides antibiotic resistance (ampicillin resistance in this case) replication is initiated

91 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Restriction Enzymes Recognizes and cuts DNA only at particular sequence of nucleotides blunt end – cleaves both ends sticky ends – cleaves only one strand Complimentary strand from DNA insert will match sticky end and insert in plasmid followed by ligation of the strands (T4 DNA Ligase)

92 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Restriction Enzymes Very large collection of restriction enzymes that target different DNA sequences

93 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Restriction Enzymes Restriction Map of plasmid showing the location where all restriction enzymes will cleave. allows determination of where & how to insert a particular DNA sequence – want a clean insertion point, dont want to cleave plasmid multiple times

94 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Next step of the process involves getting E. coli to express the protein from the plasmid. this occurs by the position of a promoter next to the inserted gene two common promoters are lac complex promoter T7 promoter lac complex promoter: Transcription is simply switched on by the addition of IPTG (isopropyl β- D-thiogalactoside) to remove LacI repressor protein. IPTG binds LacI which no longer binds the promoter region allowing transcription to occur

95 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression T7 promoter: Again, transcription is switched on by the addition of IPTG to remove LacI repressor protein. IPTG binds LacI which no longer binds the promoter region allowing transcription/production of T7 RNA polymerase to occur. T7 RNA polymerase binds the T7 promoter in the plasmid to initiate expression of the protein two-step process leads to an amplification of the amount of gene product - produce very high quantities of protein.

96 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Next step of the process involves growing the E. coli cells Shake Flask cells are place in a growth media that provides the required nutrients to the cell - amino acids, vitamins, growth factors, etc shake the flask at a constant temperature of 37 O – keeps homogenous mixture – increases oxygen uptake grow cells to proper density (OD ~ 0.7 at 600nm) Cell growth in a Shake flask LB Broth Recipe (Luria-Bertani) 10 g tryptone 5 g of yeast extract 10 g of NaCl

97 Overview of Protein Expression Next step of the process involves growing the E. coli cells Bioreactors more efficient higher production volumes – can be 100s of liters in size Can grow cells to a higher density – better control of pH – better control of oxygen levels – better control of temperature – better control of mixing – sterile conditions Introduction to Isotope Labeling of Proteins For NMR 14 liter bioreactor

98 Introduction to Isotope Labeling of Proteins For NMR Biotechnology Letters (1999) 12,1131 Overview of Protein Expression Next step is to harvest and lysis the cells and purify the protein Now that E. coli is producing the desired protein, need to extract the protein from the cell and purify it. the amount of protein that can be obtained from an expression system is highly variable and can range from g to mg to even g quantities. it depends on the behavior of the protein, expression level, method of fermentation and the amount of cells grown over-expressed protein

99 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Cell Lysis A number of ways to lysis or break open a cell Gentle Methods Osmotic – suspend cells in high salt Freeze-thaw – rapidly freeze cells in liquid nitrogen and thaw Detergent – detergents (DSD) solubilize cellular membranes Enzymatic – enzymatic removal of the cell wall with lysozyme Vigorous Methods Sonication – sonicator lyse cells through shear forces French press – cells are lysed by shear forces resulting from forcing cell suspension through a small orifice under high pressure. Grinding – hand grinding with a mortar and pestal Mechanical homogenization - Blenders or other motorized devices to grind cells Glass bead homogenization - abrasive actions of the vortexed beads break cell walls French Press

100 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Protein Purification - A large number of ways to purify a protein protocols are dependent on the protein chromatography is a common component of the purification protocol where typically multiple columns are used: a) size-exclusion b) ion exchange c) Ni columnd) heparin e) reverse-phasef) affinity column dialysis for buffer exchange and removal of low-molecular weigh impurities To increase the ease of purifying a protein generally include a unique tag sequence at the N- or C-terminus HIS tag – add 6 histidines to the N- or C- terminus - preferentially binds Ni column FLAG tag – DYKDDDDK added to terminus - preferentially binds M1 monoclonal antibody affinity column glutathione S-transferase (GST) tags – fusion protein - readily purified with glutathione-coupled column

101 Introduction to Isotope Labeling of Proteins For NMR Overview of Protein Expression Some Common Problems protein is not soluble and included in inclusion bodies insoluble aggregates of mis-folded proteins inclusion bodies are easily purified and can be solubilized using denaturing conditions How to re-fold the Protein? Finding a re-folding protocol may take significant effort (months-years?) and involve numerous steps something to be avoided if possible protein is toxic to cell find a different expression vector or use a similar protein from a different organisim proper protein fold proper disulphide bond formation – may need to re-fold the protein presence of tag may inhibit proper folding – may need to remove the tag low expression levels try different plasmid constructs try different protein sequences

102 Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein cells need to be grown in minimal media use 13 C glucose to achieve ~ 100% uniformed 13 C labeling of protein use 15 N NH 4 Cl to achieve ~ 100% uniformed 15 N labeling of protein glucose and NH 4 Cl are sole source of carbon and nitrogen in minimal media E. coli uses glucose and NH4Cl to synthesize all amino-acids protein added prior to expressing protein of interest both 13 C glucose and 15 N NH4Cl can be added simultaneously Journal of Biomolecular NMR, 20: 71–75, 2001.

103 13 C and 15 N Isotope Labeling of the protein Usually isotope labeling does not negatively impact protein expression Some Common Problems with Isotope Labeling Problems minimal media stresses cells slower growth typically lower expression levels isotope labeling of All proteins minimal isotope affect may affect enzyme activities isotope labeling of expressed protein may affect protein properties solubility? proper folding? Introduction to Isotope Labeling of Proteins For NMR 1 H- 15 N HSQC spectra of 13 C, 15 N labeled protein

104 Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein Can introduce specific amino acid labels A variety of 13 C and 15 N labeled amino acids are commercial available Add saturating amounts of 19 of 20 amino acids to minimal growth media Add 13 C and 15 N labeled amino acid prior to protein expression media is actually very rich and the cells grow very well cells exclusively use the supplied amino-acids to synthesize proteins all of the occurrences of the amino-acid are labeled in the protein may be some additional labeled residues if the labeled amino acid is a precursor in the synthesis of other amino acids. 1 H- 15 N-HSQC of His, Tyr & Gly labeled SH 2 -Domain no mechanism to label one specific amino acid i.e Gly-87

105 Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein Can label specific segment in protein use peptide splicing element intein (Protozyme) inteins are insertion sequences which are cleaved off after translations preceding and succeeding fragments are ligated extein J. Am. Chem. Soc. 1998, 120, N-labeled Protein of Interest

106 Introduction to Isotope Labeling of Proteins For NMR 13 C and 15 N Isotope Labeling of the protein Can also label only one component of a complex simply mix unlabeled and labeled components to form the complex greatly simplifies the NMR spectra only see 13 C, 15 N NMR resonances for labeled component of complex can see interactions (NOEs) between labeled and unlabeled compoents J. OF BIOL. CHEM. (2003) 278(27), 25191–25206

107 Introduction to Isotope Labeling of Proteins For NMR 2 H Labeling of the protein simply requires growing the cells in D 2 O severe isotope effect for 1 H 2 H stresses the cell E. coli needs to be acclimated to D 2 O pass cells into increasing percentage of D 2 O cell growth slows significantly in D 2 O (18-60 hrs) level of 2 H labeling depends on the percent D 2 O the cells are grown in aromatic side-chains will be highly protonated if 1 H-glucose is used exchange labile N 2 H to N 1 H by temperature increase or chemical denaturation of the protein

108 Introduction to Isotope Labeling of Proteins For NMR [3,3- 2 H 2 ]- 13 C 2-ketobutyrate. [2,3- 2 H 2 ]- 15 N, 13 C Val 2 H Labeling of the protein As we have seen, deuterium labeling a protein removes a majority of protons necessary for protein structure calculation can introduce site specific protonation to regain some proton based distance constraints label the methyl groups of Leu, Ile, and Val by adding to the growth media. use 1 H-glucose to generate 1 H-aromatic side-chains Metabolic pathway for generating 1 H-methyl-Ile

109 EXPERIMENTAL

110 The NMR spectrometer Magnet Probe Coils Transmitters Amplifiers and pre-amplifiers Receiver ADC

111 The Magnet A cutted magnet History First magnets were built using ferromagnetic material= permanent magnet Then Electromagnets: i.e. field was generated by wiring of conducting material Now: cyomagnets: i.e. electromagnets made of superconducting wire.

112 Cryomagnets Superconducting wire has a resistance approximately equal to zero when it is cooled to a temperature close to absolute zero ( o C or 0 K) by emersing it in liquid helium. Once current is caused to flow in the coil it will continue to flow for as long as the coil is kept at liquid helium temperatures. The length of superconducting wire in the magnet is typically several miles.

113 The NMR spectrometer

114 Det.ADC NMR Signal 0 (reference) Computer Memory 500 MHz ± 2500 Hz 500 MHz ± 2500 Hz

115 The Probe The sample probe is the name given to that part of the spectrometer which accepts the sample, sends RF energy into the sample, and detects the signal emanating from the sample. It contains the RF coil, sample spinner, temperature controlling circuitry, and gradient coils. Picture an axial cross section of a cylindrical tube containing sample. In a very homogeneous Bo magnetic field this sample will yield a narrow spectrum

116 B 0 homogeneity In a more inhomogeneous field the sample will yield a broader spectrum due to the presence of lines from the parts of the sample experiencing different Bo magnetic fields.

117 Set up an experiment. What to do? Shimming the magnet Lock Tune 90° Pulse

118 The NMR experiment: what do we need? 1. The magnet: SHIM Requires control of field homogeneitySHIM LOCK Requires stabilization of main fieldLOCKSHIM: additional coils with special field distribution, e.g. Z, Z2, Z3, X, Y, X3.... We have cryo shims and room temperature shimsLOCK 1.contineously determine frequency of 2 H signal of the solvent (deuterated solvents) 2. add a small extra field to the main field of the magnet to keep the overall field constant 3. 2 H signal also used for shimming

119 Problem: how to keep B 0 constant throughout the NMR sample?

120 B 0 homogeneity In a more inhomogeneous field the sample will yield a broader spectrum due to the presence of lines from the parts of the sample experiencing different Bo magnetic fields.

121 The effect of shim coils The effect of z 2 The effect of z 4 The effect of z 1 The effect of z 3

122 SHIMMING line shape distortions from on-axis shims: OKZ4Z2Z3 Z1

123 Effect of B 0 inhomogeneity in the NMR spectrum BEFORE AFTER

124 Problem: how to keep B 0 constant during an experiment?

125 The NMR experiment: what do we need? 1. The magnet: SHIM Requires control of field homogeneitySHIM LOCK Requires stabilization of main fieldLOCKSHIM: additional coils with special field distribution, e.g. Z, Z2, Z3, X, Y, X3.... We have cryo shims and room temperature shimsLOCK 1.contineously determine frequency of 2 H signal of the solvent (deuterated solvents) 2. add a small extra field to the main field of the magnet to keep the overall field constant 3. 2 H signal also used for shimming

126 The Lock: How does it work? The lock channel can be understood as a complete indepenant spectrometer within the spectrometer: The resonance condition of NMR: = B o but: B o is not stable = (B o +H o ) (B o +H o ) = const. Regulator amplitude, frequency Transmitter 2 H Probe Receiver 2 H HoHo Shim system

127 Problem: how to optimized the sensitivity of the receiving coil with respect to the observed frequency?

128 Tuning

129 The tuning circuit

130 Problem: how to give a 90° pulse in real life?

131 Precession in the laboratory frame dM/dt=M^ BdM/dt=M^ (B- ) L.F. R.F. at freq. If = 0 dM/dt=0 dM/dt=M^ B 1 If = 0 +B 1 dM/dt=M^ (B 0 +B ) Rotation! B1B1 Pulse: = 1 t= B 1 t Pulse: = /2= B 1 t

132 The 90° pulse Calibration of pulse lenght

133 Performing an NMR experiment The practical application of the rotating frame of reference…. FT relax. PreparationDetection x y z t2t2 0

134 A B C t2t2 x y z 10 8 Hz Static: Rotating ( 0 B ): x y z x y z t2t2 x y z 10 3 Hz x y z x y z 0 B

135 Det.ADC NMR Signal 0 (reference) Computer Memory 500 MHz ± 2500 Hz 500 MHz ± 2500 Hz

136 D1 Repetition Time DE P1 = 1/BW PL1 AQ = DW·TD Acquisition Time RG

137 x y t MyMy x y x y t MxMx x y

138 Quadrature Phase Detection

139

140 Pulse! -y y The rotation of magnetization under the effect of 90° pulses according to the convention of Ernst et al..

141 The phase of an NMR signal

142 Phase Correction

143 DE AQ = DW·TD Acquisition Time FT

144 Digital resolution Resolution is expressed in Hertz/point

145 Quadrature Phase Detection PSDADC PSDADC NMR Signal 0 0° reference 90° reference Computer Memory A Computer Memory B

146 Fourier Pairs

147 1D-NMR with/without removal of water

148 Free Induction Decay (FID) Observed NMR signal in the time domain Resonance frequencies are acquired as a function of time Common case of observed FIDs t tt

149 Sensibilità dellEsperimento NMR S/N N 5/2 B 0 3/2 N = Numero di spins che contribuiscono al segnale rapporto giromagnetico del nuclide studiato Camp magnetico utlizizzato

150 Signal to noise

151 ScansS/N

152 D1 Repetition Time DE P1 = 1/BW PL1 AQ = DW·TD Acquisition Time RG

153 Pulses and Phases


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