Some paradigmatic examples

Typical 1H NMR Spectrum

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

We make a 1H experiment and we acquire.

Then all signals transfer the information because of scalar coupling

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

Spectrum beforeThe J coupling4 ppm9 ppm

Signal!This indicates that there is a scalar coupling

between Hn and Hc

EXAMPLEN

Spectrum beforeThe J coupling4 ppm9 ppm

Signal!This indicates that there is a scalar coupling

between Hn and Hc

J-coupling

EXAMPLEN

Spectrum beforeThe J couplingHc Hc

J-coupling

If you begin from Hc , the situation is the same !

EXAMPLEN

Spectrum beforeThe J coupling

J-coupling

Therefore, if I consider only this system

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 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 dimensionOr F1

t2 dimensionOr 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’

Example: COSY

Through-bond connectivities

COSY= COrrelation SpectroscopY

H4’-H5’

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

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

Prima dimensione

2D HETCOR Expriment

Prima dimensione

2D HETCOR Expriment

Prima dimensione

Seconda dimensione

2D HETCOR Expriment

Prima dimensione

Seconda dimensione

Esempio

N.B. In questo caso non si osserva solo l’accoppiamento 3J ma si osserva una “propagazione” dell’informazione attraverso gli accoppiamenti scalari

EsempioHETCOR

Heteronuclear Single Quantum coherence

2D HSQC Experiment

2D HSQC Expriment

2D HSQC Experiment

Prima dimensione

Seconda dimensione

2D HSQC Expriment

Prima dimensione

2D HSQC Experiment

Seconda dimensione

Prima dimensione

2D HSQC Experiment

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

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

No more diagonal

Two protons are bound to the same carbon

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

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

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

45 96 7

137812

5254 55

98 5376

99 1443

9357 19

HN1 83

Apo vs holo protein, mapping the environment of the Apo vs holo protein, mapping the environment of the metal ionmetal ion

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

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

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)

Very large collection of restriction enzymes that target different DNA sequences

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

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

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.

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

14 liter bioreactor

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

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

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?

1H-15N HSQC spectra of 13C,15N labeled protein

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

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

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

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

[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

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

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!

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.

PreparationDetection

zx90 t

dte)t(f)(F ti

x90 t2

108 Hz

Static:

Rotating (0 B):

x90 t2

103 Hzx’

Det. ADCNMRSignal

0 (reference)

ComputerMemory

500 MHz ± 2500 Hz

500 MHz

± 2500 Hz

Repetition Time

DEP1 = 1/BW

AQ = DW·TDAcquisition Time

Quadrature Phase Detection

Pulse!-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

Digital resolution

Resolution is expressed in Hertz/point

Quadrature Phase Detection

PSD ADC

NMRSignal 0

0° reference

90° reference

ComputerMemory

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

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

Repetition Time

DEP1 = 1/BW

AQ = DW·TDAcquisition Time

Pulses and Phases

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