Some paradigmatic examples
Typical 1H NMR Spectrum
Ab
so
rba
nc
e
Valore medio: 2 MUSD/anno
K1 T2 L3 T4 L5 E6 A7 A8 L9 R10 N11 A12 W13 L14 R15 E16 V17 G18 L19 K20
500 MHz 1H NMR
Ubiquitin
76 amino acids, 8,5 kDa
Protein 1H NMR spectrum: a “real spectrum”
Fourier Transformation
The NMR signal in the time domain
Free Induction Decay
A short pulse will excite all spinsAll spins will relax (all together) during time AQThe FT of FID gives the NMR spectrum
1D experiment
Could be nice but...
..Too crowded..
What do we learn?
Chemical shifts relaxation rates
Not enough to get a structure
STRUTTURE IN SOLUZIONE VIA NMR
The need for multidimensional NMR
Cosa è un esperimento bidimensionale ?
Dopo un impulso a 90° il segnale è pronto per essere acquisito
Facciamo l’acquisizione ma NON terminiamo l’esperimento 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
Eccito (impulso a 90°)-Acquisisco (t1)- Perturbo (trasferisco)- Acquisisco (t2)
Se la perturbazione non ha effetto e se non c’è 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
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 l’informazione sugli accoppiamenti scalari attivi nel sistema
M (I t1) (St2)
Acquisisco (t1)- Perturbo (trasferisco)- Acquisisco (t2)
EXAMPLEN
H H
CC
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 afterThe J coupling
Spectrum beforeThe J coupling
EXAMPLEN
H H
CC
O
N
H H
C
Spectrum afterThe J coupling
Spectrum beforeThe J coupling4 ppm9 ppm
Signal!This indicates that there is a scalar coupling
between Hn and Hc
EXAMPLEN
H H
CC
O
N
H H
C
Spectrum afterThe J coupling
Spectrum beforeThe J coupling4 ppm9 ppm
Signal!This indicates that there is a scalar coupling
between Hn and Hc
Hn Hn
Hc
J-coupling
EXAMPLEN
H H
CC
O
Spectrum afterThe J coupling
Spectrum beforeThe J couplingHc Hc
Hn
J-coupling
If you begin from Hc , the situation is the same !
EXAMPLEN
H H
CC
O
Spectrum afterThe J coupling
Spectrum beforeThe J coupling
Hc Hc
Hn
J-coupling
Therefore, if I consider only this system
Hn Hn
Hc
J-coupling
The first dimension = t1
The second dimension = t2
the series of pulses that I have to apply to my system = PULSE SEQUENCE
example
t1 t2
t1 dimensionOr F1
t2 dimensionOr F2
Usually t1 is also defined as indirect dimension
t2 is also defined as direct dimension
the series of pulses that I have to apply to my system = PULSE SEQUENCE
example
t1 t2
t1 dimensionOr F1
t2 dimensionOr F2
F1
F2
Definitions
Cross peak Two different frequencies are observed in the two dimensions
Diagonal peakThe same frequency is observed in both dimensions
CROSS PEAK= Yes, There is a COUPLING between the two frequencies
Accoppiamento scalare
L’accoppiamento scalare puo’ comunque essere osservato attraverso esperimenti NMR bidimensionali, quali il COSY
Example: COSY
Through-bond connectivities
COSY= COrrelation SpectroscopY
H4-H5
H4’-H5’
Example: COSY
Through-bond connectivities
COSY= COrrelation SpectroscopY
H4-H5
H4’-H5’
1
2
3
4
5
6
Beyond COSYCOSY 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-couplingIs THROUGH SPACE
COUPLING
Accoppiamento dipolareL’accoppiamento 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
L’entità dell’effetto dipende dal campo magnetico e dalle dimensioni della molecola. Nel caso di spin 1H, l’accoppiamento dipolare si trasferisce per spin che si trovano a distanze inferiori ai 5 A.
NON si osservano doppietti
L’accoppiamento dipolare da luogo ad un trasferimento di magnetizzazione da uno spin all’altro. Questo effetto va sotto il nome di effetto NOE
Nuclear Overhauser Effect
Perturbo A Aumenta la intensità di B
Accoppiamento dipolare
L’accoppiamento dipolare è “indipendente dall’accoppiamento scalare2 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
L’effetto 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
Through space AND throuhg bonds
Through space
Through bond
Example:Nuclear Overhauser Effect SpectroscopY
NOESYNOE Effect:
If two spins that are close in space are excited out of equilibrium, they will mutually transfer their magnetization
AAAA ABAB
Example:
Cross peaks: A and B Cross peaks: A and B are closeare close
Diagonal peakDiagonal peak
The real The real case:case:Some 1500 Some 1500 peaks are peaks are observed observed for a for a protein of protein of 75 75 aminoacidaminoacidss
AAAA ABAB
NOESY experiment
2D NOESY Spectrum
Distance constraints
NOESY volumes are proportional to the sixth power of the interproton distance and to the correlation time for the dipolar coupling
BB00
II
JJ
rr
6IJ
cIJ r
The “old times” approachNOESYNOESY
CCOOSYSY
Identify through space connectivitiesIdentify through space connectivitiesHN(i)-Ha(i) and HN(i)Ha(i-1)HN(i)-Ha(i) and HN(i)Ha(i-1)
Identify through bond connectivitiesIdentify through bond connectivitiesHN(i)-Ha(i)HN(i)-Ha(i)
NOESY conn.NOESY conn.
COSCOSY connY conn
1J couplings for
backbone resonances
1J couplings for
backbone resonances
The 2D Hetcor experiment
Two dimensional Heteronuclear correlation Experiment
The 2D Hetcor experimentTwo dimensional Heteronuclear correlation Experiment
E’ possibile, in uno stesso esperimento mandare impulsi su nuclidi diversi (Es: 1H, 13C)
’ 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
E’ possibile, in uno stesso esperimento mandare impulsi su nuclidi diversi (Es: 1H, 13C)
Inoltre possiamo combinare tutto cio’ con quello che sappiamo sugli esperimenti bidimensionali
Eccito (impulso a 90°) 1H Acquisisco (t1) 1H –Perturbo (Trasferisco la magnetizzazione da 1H a 13C utilizzando l’accoppiamento scalare 1JHC
Acquisisco (t2) 13C
2D HETCOR Expriment
2D HETCOR Expriment
2D HETCOR Expriment
Prima dimensione
2D HETCOR Expriment
Prima dimensione
2D HETCOR Expriment
Prima dimensione
Seconda dimensione
2D HETCOR Expriment
Prima dimensione
Seconda dimensione
Esempio
COSY
Esempio
COSY
N.B. In questo caso non si osserva solo l’accoppiamento 3J ma si osserva una “propagazione” dell’informazione attraverso gli accoppiamenti scalari
EsempioHETCOR
514
32
Heteronuclear Single Quantum coherence
2D HSQC Experiment
2D HSQC Expriment
Heteronuclear Single Quantum coherence
2D HSQC Experiment
Prima dimensione
Seconda dimensione
Heteronuclear Single Quantum coherence
2D HSQC Expriment
Prima dimensione
Heteronuclear Single Quantum coherence
2D HSQC Experiment
Seconda dimensione
Prima dimensione
Heteronuclear Single Quantum coherence
2D HSQC Experiment
Heteronuclear Single Quantum coherence
Seconda dimensione
Prima dimensione
E’ possibile progettare esperimenti per trasferire la magnetizzazione da un nucleo all’altro anche indipendentemente dall’acquisizione
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 l’accoppiamento scalare ed alla fine osservo 1H
Eccito (impulso a 90°) 1H Trasferisco la magnetizzazione da 1H a 13C utilizzando l’accoppiamento scalare 1JHC
Acquisisco (t1) 13C –
Perturbo -Trasferisco la magnetizzazione da 13C a 1H utilizzando l’accoppiamento scalare 1JHC
Acquisisco (t2) 1H
2D HSQC Experiment
Questo tipo di esperimento si chiama anche Out and backSignifica 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 l’esperimento sia molto piu’ selettivo
Osservo solo 1H e 13C che sono accoppiati tra di se per effetto di 1J
The HSQC experiment
Caratteristiche dell’esperimento HSQC
Non esiste la diagonaleNon esiste la diagonale
La magnetizzazione viene trasferita da 1H al 13C ad esso accoppiato
Successivamente si acquisisce, nella dimensione indiretta, 13C
Infine si ri-trasferisce su 1H e si osserva 1H
Tutti gli Tutti gli 11H che non sono accoppiati a H che non sono accoppiati a 1313C NON si osservanoC NON si osservano
Heteronuclear NMR
OBSERVE 13C during t1
Transfer the information to all 1H coupledOBSERVE 1H during t2
1H
13C
No more diagonal
Each peak indicate A different H-C pair
Heteronuclear NMROBSERVE 13C during t1
Transfer the information to all 1H coupledObserve 1H during t2
1H
13C
No more diagonal
Two protons are bound to the same carbon
CH2
The HSQC experiment
Heteronuclear cases
The scheme of 1J scalar couplings
The 1H- 15N HSQC experiment
Heteronuclear Single Quantum Coherence
The HSQC experiment
In 5 minutes you may know….if your protein is properly foldedif 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 1H experiment (although you are
observing 15N)but much larger resolution
if you can do the job (whatever is your job)
Heteronuclear NMR in proteinsexample: 15N labelled proteins
Heteronuclear NMR in proteinsexample: 15N labelled proteins
The HSQC experiment
In 5 minutes you may know….if your protein is properly foldedif 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 1H experiment (although you are
observing 15N)but much larger resolution
Ca2+
Apo Cb @ 3.3 M GdmCl
Loss of secondary structure elements: unfolded protein
Refolding
Ca2Cb @ 3.3 M GdmCl
The role of metal cofactor in protein unfolding
Metal triggered protein folding
7795
5935
26
60
45 96 7
33
20
34
27
75
15 6
62
65
9472
90
137812
80
100
8
8846
4286
87
81
8568
102
582
5254 55
71
56 91
29
66
36
328
38101
2558
98 5376
1674
84
30
21
3969
40
9767
37
41
99 1443
6489
2448
5147
11
23
9357 19
4 10
83
22
1779
HN1 83
HN 28
HN 32
Apo vs holo protein, mapping the environment of the Apo vs holo protein, mapping the environment of the metal ionmetal ion
15N
15H
The need for multidimensional NMR
Troppi segnali 1H ?
Isotope labelingFor biomolecules, tipically, 15N or 13C and 15N, or 13C, 15N, 2H
15N Only
A more effective fingerprint-characterization-folding-dynamics
protein size >10000Homonuclear 2D experiments donot have enough resolution
HSQC or HMQCHSQC-NOESY or HSQC TOCSY
Isotope labelingFor biomolecules, tipically, 15N or 13C and 15N, or 13C, 15N, 2H
15N and 13C
Scalar couplings through 13C atoms-triple resonance-assignment-structure
protein size >20000
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 15N labeling in CHO cells can cost $150-250K!
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: http://www.the-scientist.com/yr1997/sept/profile2_970901.html
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
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)
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
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, don’t want to cleave plasmid multiple times
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
Introduction to Isotope Labeling of Proteins For NMR
Overview of Protein ExpressionT7 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.
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 37O
– 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
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
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
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
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 exchangec) Ni column d) heparine) reverse-phase f) 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
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
Introduction to Isotope Labeling of Proteins For NMR 13C and 15N Isotope Labeling of the protein
• cells need to be grown in “minimal media”
• use 13C glucose to achieve ~ 100% uniformed 13C labeling of protein• use 15N NH4Cl to achieve ~ 100% uniformed 15N labeling of protein
glucose and NH4Cl 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 13C glucose and 15N NH4Cl can be added simultaneously
Journal of Biomolecular NMR, 20: 71–75, 2001.
13C and 15N 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
1H-15N HSQC spectra of 13C,15N labeled protein
Introduction to Isotope Labeling of Proteins For NMR
13C and 15N Isotope Labeling of the protein • Can introduce specific amino acid labels• A variety of 13C and 15N labeled amino acids are commercial available
Add saturating amounts of 19 of 20 amino acids to minimal growth media Add 13C and 15N 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.
1H-15N-HSQC of His, Tyr & Gly labeled SH2-Domain
no mechanism to label one specific amino acid i.e Gly-87
Introduction to Isotope Labeling of Proteins For NMR
13C and 15N 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, 5591-5592
15N-labeled
Protein of Interest
Introduction to Isotope Labeling of Proteins For NMR
13C and 15N 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” 13C, 15N 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
Introduction to Isotope Labeling of Proteins For NMR
2H Labeling of the protein • simply requires growing the cells in D2O
severe isotope effect for 1H2H stresses the cell E. coli needs to be acclimated to D2O pass cells into increasing percentage of D2O cell growth slows significantly in D2O (18-60 hrs) level of 2H labeling depends on the percent D2O the cells are grown in aromatic side-chains will be highly protonated if 1H-glucose is used exchange labile N2H to N1H by temperature increase or chemical denaturation of the protein
Introduction to Isotope Labeling of Proteins For NMR
[3,3-2H2]-13C 2-ketobutyrate.
[2,3-2H2]-15N, 13C Val
2H 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 1H-glucose to generate 1H-aromatic side-chains
Metabolic pathway for generating 1H-methyl-Ile
EXPERIMENTAL
The NMR spectrometer
• Magnet
• Probe
• Coils
• Transmitters
• Amplifiers and pre-amplifiers
• Receiver
• ADC
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.
CryomagnetsSuperconducting wirehas a resistance approximately equal to zerowhen it is cooled to a temperature close toabsolute zero (-273.15o C or 0 K) byemersing it in liquid helium. Once current iscaused to flow in the coil it will continue toflow for as long as the coil is kept at liquidhelium temperatures.
The length of superconducting wire in themagnet is typically several miles.
The NMR spectrometer
Det. ADCNMRSignal
0 (reference)
ComputerMemory
500 MHz ± 2500 Hz
500 MHz
± 2500 Hz
The ProbeThe 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
B0 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.
Set up an experiment. What to do?
• Shimming the magnet
• Lock
• Tune
• 90° Pulse
The NMR experiment: what do we need?The NMR experiment: what do we need?
1. The magnet:1. The magnet:Requires control of field homogeneity SHIMSHIM
Requires stabilization of main field LOCKLOCK
SHIM:SHIM:additional coils with special field distribution,
e.g. Z, Z2, Z3, X, Y, X3....
We have cryo shims and room temperature shims
LOCKLOCK1.contineously determine frequency of 2H 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. 2H signal also used for shimming
Problem: how to keep B0 constant throughout the NMR
sample?
B0 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.
The effect of shim coils
The effect of z2
The effect of z4
The effect of z1
The effect of z3
SHIMMINGSHIMMING
line shape distortions from on-axis shims:
OK Z4Z2 Z3Z1
Effect of B0 inhomogeneity in the NMR spectrum
BEFORE
AFTER
Problem: how to keep B0 constant during an experiment?
The NMR experiment: what do we need?The NMR experiment: what do we need?
1. The magnet:1. The magnet:Requires control of field homogeneity SHIMSHIM
Requires stabilization of main field LOCKLOCK
SHIM:SHIM:additional coils with special field distribution,
e.g. Z, Z2, Z3, X, Y, X3....
We have cryo shims and room temperature shims
LOCKLOCK1.contineously determine frequency of 2H 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. 2H signal also used for shimming
The Lock: How does it work?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:
= Bo but: Bo is not stable
= (Bo+Ho) (Bo+Ho) = const.
Regulator
amplitude,frequencyTransmitter 2H
ProbeProbe Receiver 2H
Ho
Shim systemShim system
Problem: how to optimized the sensitivity of the receiving coil
with respect to the observed frequency?
Tuning
The tuning circuit
Problem: how to give a 90° pulse in real life?
Precession in the laboratory frame
dM/dt=M^B dM/dt=M^(B-)
L.F.R.F. at freq.
If = 0 dM/dt=0
dM/dt=M^B1
If = 0+B1
dM/dt=M^(B0 +B1 -0)
Rotation!
B1
Pulse:=1t=B1t
Pulse:=/2=B1t
The 90° pulse
Calibration of pulse lenght
Performing an NMR experiment
The practical application of the rotating frame of reference….
FTrelax.
x90
PreparationDetection
x
y
zx90 t
2 0
dte)t(f)(F ti
A B C
x90 t2
x
y
z
108 Hz
Static:
Rotating (0 B):
x
y
z
x
y
z
x90 t2
x’
y’
z
103 Hzx’
y’
z
x’
y’
z
0 B
Det. ADCNMRSignal
0 (reference)
ComputerMemory
500 MHz ± 2500 Hz
500 MHz
± 2500 Hz
D1
Repetition Time
DEP1 = 1/BW
PL1
AQ = DW·TDAcquisition Time
RG
x
yt
My
x
y
x
yt
Mx
x
y
Quadrature Phase Detection
Pulse!-y -y
-y -y
-y -y
-y-y
-y
y
The rotation of magnetization under the effect of 90° pulses according to the convention
of Ernst et al..
The phase of an NMR signal
Phase Correction
)()()( iDAF )()(exp)( iDAiF instr
)()(Re AF
)(sin)(cos)(Im ADF instrinstr
)(sin)(cos)(Re DAF instrinstr
)()(Im DF
DEAQ = DW·TD
Acquisition Time
FT
Digital resolution
Resolution is expressed in Hertz/point
Quadrature Phase Detection
PSD ADC
PSD ADC
NMRSignal 0
0° reference
90° reference
ComputerMemory
A
ComputerMemory
B
Fourier Pairs
dte)t(f)(F ti
1D-NMR with/without removal of water
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 t t
Sensibilità dell’Esperimento NMR
S/N N 5/2 B03/2
N = Numero di spins che contribuiscono al segnale
rapporto giromagnetico del nuclide studiato
Camp magnetico utlizizzato
Signal to noise
Signal to noiseScans S/N1 1.00 80 8.94 8 2.83 800 28.28 16 4.00
D1
Repetition Time
DEP1 = 1/BW
PL1
AQ = DW·TDAcquisition Time
RG
Pulses and Phases
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