Program. short intro the hardware locking and shimming 1D proton setup presaturation spectra

Transcription

1 Program short intro the hardware locking and shimming 1D proton setup presaturation spectra 13 C spectra, DEPT processing of 1D file transfer and backup 2D homonuclear + 2D processing 2D heteronuclear

2 The NMR signal and the chemical shift Certain nuclei (ydrogen, Carbon-13, Nitrogen-15) posses a nuclear spin (I=1/2). Once these nuclei are placed into a static magnetic field, a magnetic moment is introduced. This magnetic moment causes the spins to orient along the static field. The spins precess about the magnetic field axis with a characteristic frequency, which is called the lamor frequency. This is the signal frequency that is detected in NMR.

3

4 z J z = + h 2 m= +1/2 + 1/2 h / B E=h J z = - h 2 m= -1/2-1/2 h / B Bo= 0 Bo 0

5 0

6 z I Det x y t T

7 I N S t

8 Longitudinal (T1) Relaxation z z z z z y y y y y x x x x x T1-Relaxation determines the relaxation delay

9 Transverse (T2) Relaxation T2-Relaxation determines the linewidths

10 M x x M x y M z

11 Thermal equilibrium: Bo=z y x y x x y α-state is (slightly) higher populated than β -state» resultant longitudinal component points along + z No phase coherence» resultant transverse component is zero

12 B o =z! y x x x y y

13 Coherent state: Bo=z y x x y x y (usually) α-state is (slightly) higher populated than β -state» resultant longitudinal component points along + z phase coherence» resultant transverse component is non-zero

14 B o=z! y x x x y y

15 B o! 0 B o B o B eff! 1 B 1

16 The transformation into the rotating frame B o! B o! B o! x y! 0 B 1! "! 0! 0

17 B 0 -!/" B eff B B eff B 1 B B eff B eff B 1 B 1! o! o! o

18

19 Important parameters SFO1 or O1 (O1p): Center frequency of the spectrum RG (receiver gain; amplification of the signal) p1,p2... durations of 90 and 180 pulses (depends on solvent; salt etc) ns: number of scans (concentration) sw: spectral width (ppm; swh in z) pulprog: pulseprogram use parameterset (rpar)! Only need to change ns,sw and rg

20 quickly starting experiements clean the NMR tube(!), set the temperature, and insert the sample in magnet read in standard shim set (rsh) and perform gradient shimming (gradshim) activate the lock circuit (lock) tune and match the probe (wobb/atma) read in shim set (rpar) getprosol ( ) set ns, sw and rg to correct values start experiment with zg

21 The Components of a NMR Spectrometer a magnet a shim system a lock system a RF probe for detecting the signals a RF system, consisting of RF transmitter, receiver and amplifier an analog-digital converter (ADC) a computer of data analysis and storage

22 N S COMPUTER (FT) ADC RF RF-Erzeugung Empfänger/Sender Verstärker

23 liq. N 2 Inlet liq. e System Probehead Preamp

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25 room-temperature shim tube liq. N 2 dewar liq. e dewar Main Coil Probehead

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27 The probeheads

28 A cryoprobe system

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30

31

32 The helmholtz coils

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34 The radiofrequency part (synthesizers, modulaters, phase switching etc.)

35 The shim current supplies (BSMS)

36 The VTU (variable temperature unit)

37 The amplifiers

38 The lock channel sweep mode locked

39 The shim system ΔB o ΔB Shim Z z ΔB 0 Z Z

40 Z1 Z2 Z3 Z Z Z Z4 Z5 Z Z

41 Influence of misset shims on the lineshape of signals

42 exponential decay of signal non-exponential decay due to field inhomogenity

43 Gradient Shimming B O Grad

44 Tuning and matching the resonance circuit C E L B ω = 1 LC

45 The receiver system Quadrature Detection a) single detector -ν 0 +ν -ν 0 +ν

46 b) quadrature detector -ν 0 +ν -ν 0 +ν

47 sin ωt FT cos ωt FT Σ

48 90 pase-shifter Splitter F (Mz) sin ωt cos ωt Mixer SFO1 ( Mz) The detector + Amplifier Audio ( 0-Kz) Analog-Digital Converter ADC FT

49 Recording a spectrum spectral width (sw) - sw/2 0 + sw/2 transmitter frequency (sfo1)

50 The Transmitter system ω B o ω B o ω 0 x y ω 0 B 1 ω 1

51 Sampling of the signal DW AQ (td =16)

52

53 AQ D1 t r

54 ω B o ω o=sfo1 ω 1 =(4*(PW90))-1

55 Basic Steps (changing the probehead) setting the correct temperature for measurement correct positioning of the tube in the spinner and insertion of the sample into the magnet recall a shimset with approximately correct shims activation of the field/frequency lock system adjustment of the homogeneity of the magnetic field recalling a parameterset for the desired experiment tuning of the probehead calibration of the pulse lengths determination of the correct receiver gain setting performing a preliminary scan with large spectral width acquisition of the final spectrum with sufficient S/N and optimized spectral width

56 Calibration of Pulse Lengths θ = γ B 1 τ p 1 13 C 1/2J(C,)

57 Processing

58 The Fourier Transform Α Β FT t C ν C Β Α

59 Signal processing 1/πT 2 dispersion mode absorption mode d(t) = e πlt e 2πiω 0t f ( ω) = πl ( πl) 2 + (2π(ω ω 0 )) i 2π(ω ω 0 ) 2 πl ( ) 2 + (2π(ω ω 0 )) 2

60 Phase correction PC "0th order" PC "1st order" Re(Δφ) = Im(Δφ = 0)sinφ + Re(Δφ = 0)cosφ Im(Δφ) = Re(Δφ = 0)sinφ + Im(Δφ = 0)cosφ

61 real imaginary

62 t = 0 t = 0

63 A B DE A B B

64 M = Re 2 + Im 2 M = Re 2 + Im 2

65 Resolution (Resolution enhancement)

66 A B C D Signal pre-processing (window functions) E F

67 zero-filling

68 Exponential multiplication: Apodization π lb kδt a k = e (lb = linebroadening) Lorentz-to-Gauss transformation: a = e π lb kδt e gb (kδt )2 k

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70

71 untreated exponential multiplication (lb=5) exponential multiplication (lb=1) sine-bell (ssb=0) sine-bell (ssb=3) sine-bell (ssb=2)

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73 Baseline Correction

74 Linear prediction

75 2D NMR

76 sampling in 1D acquisition is done stroposcopically.. (td =16) DW AQ

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78

79 !! F1! (! 1,! 2 ) (! 1,! 1 )! (! 2,! 2 ) (! 2,! 1 ) d iagonal:! 1 =! 1 F2 anti-diagonal:! 1 = -! 1

80 Resolution in 2D spectra F2 [ppm] F1 [ppm] F2 [ppm] F1 [ppm]

81 COSY TOCSY NOESY ROESY

82 SQC MBC SQC-TOCSY INADEQUATE

83 COSY correlates geminal and vicinal protons one of the most commonly used experiments very sensitive requires high proton density

84 COSY cross peak fine structure J(A,B) ppm F1 J(A,B) ppm F2

85 TCOSY (total correlation spectroscopy) multiple proton-proton transfer depending on the mixing time complete correlations throught the whole spin system may be derived only a single resolved resonance required (carbohydrates) not sensitive for large moelcules

86 COSY vs. TOCSY COSY: single step transfer TOCSY: multiple step transfer cyclosporin

87 NOESY (nuclear Overhauser spectroscopy) correlates protons that are close in space, irrespective of how many bonds are in between strength of NOE is prop. d -6 works the best for large molecules, less for small, badly for mediumsized TE experiment for determining stereochemistry

88 NOESY vs. ROESY cross peaks NOESY ROESY cross peak intensity with respect to diagonal peak 0.5 positive negativ e 1.0 ω 0 τ c 1.0 ω 0 τ c molecule size: smaller larger smaller larger temperature: higher lower higher lower ω 0 τ c = spectrometer frequency = rotational correlation time (proportional to molecular size, temperature dependent)

89 Polarization transfer inverse-gated 13C 1 Decoupling Sensitivity (fully relaxed, 100% isotopic abundance) γ(13c)5/2 13C RD 13C{1} 1 Decoupling γ(13c)5/2 + NOE 13C RD INEPT 1 13C RD Decoupling γ(1)(13c)3/2 SQC 1 RD γ(1)5/2 13C t1 Decoupling

90 The SQC (heteronulear single quantum coherence) experiment correlates protons with their directly bonded carbons via 1 J C, helps to recognize geminal protons is very sensitive and yields carbon chemical shifts of PROTONATED carbons

91 MBC (heteronuclear multiple-bond correlation) correlates protons with carbons at ADJACENT positions via 2 J and 3 J ( 4 J) couplings very useful to assign quarternary carbons ambiguity always exists whether 2 J or 3 J correlations are seen correlations follow a Karplus-type relations and hence the cpoupling may be zero!

92 SQC-TOCSY correlates prtons with their directly bonded carbons additionally displays correlations to protons on NEIGBOURING carbons in principle gives information similar to COSY, but with increased resolution is much less sensitive (transfer via 13 C)

93 INADEQUATE directly correlates carbon nuclei is very useful when the molecule contains only few protons extremely insensitive

94 Setup of the experiments 1) record 1D spectrum determine sufficient region 2) read in parameters rpar parametersetname 3) set pulses to correct lengths getprosol ) eda (2D parameters) SW1, SW2, check NS in the Pulseprogram comment section (edcpul)

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96 Phase cycling: coherence pathway selection multipulse NMR sequence F1 different coherence pathways phase cycle other resonances crowded spectra only one coherence pathway is of interest select only the wanted coherence

97 edcpul: pulse program, comment section. example, pulse program: cosydfph.. ;pl1 : f1 channel - power level for pulse (default) ;p1 : f1 channel - 90 degree high power pulse ;d0 : incremented delay (2D) [3 usec] ;d1 : relaxation delay; 1-5 * T1 ;d13: short delay [4 usec] ;in0: 1/(1 * SW) = 2 * DW ;nd0: 1 ;NS: 8 * n ;DS: 16 ;td1: number of experiments ;FnMODE: States-TPPI, TPPI, States or QSEC

98 Setup of the experiments: channel routing edasp (RF-channel routing) logical channel physical channel amplifier router preamplifier

99 Basic processing commands 2D commands 1D commands 1) edp and set the processing parameters (window functions, phasing mode ) 2) xfb -2D Fourier transformation 3) sref - reference spectrum 4) pk (apks) - phase correction 5) abs1, abs2 - baseline correction edp ft sref phase abs

100 edp: processing parameters F 2 : SI 0.5TD - TD F 1 : SI TD - 2TD td: number of real points (acquisition) si: number of complex points (processing) si = td zero filling MC2 = FnMODE sign determination / phase cycling (QF, TPPI, States-TPPI, echoantiecho ) WDW SSB 0 or 2 SINE / QSINE Window function on FID, shift sine bell

101 edp: more processing parameters P_mod F 2 F 1 magnitude: no mc phase sensitive: pk pk Mode for phase correction: no, yes, magnitude mode, power spectrum BC_mod F 2 F 1 DQD: no no other: quad no Mode for baseline correction: no, general b.c. (quad), polynomial water deconvolution (qpol), other water deconvolution (qfil) ME_mod no Mode for linear prediction (fc, fr) ABSF1/ to Width of the baseline correction

102 2D-Phasing

103 referencing of 2D spectra reference the corresponding 1D spectrum via TMS or the solvent signal extract the frequency of 0 ppm by typing 2s sf transfer that value into the 2D data set for homonuclear spectra 2s sf and 1s sf for heteronuclear spectra (e.g. SQC) use the SF of the carbon spectrum as 1s sf or calculate the sf via the sf of the proton frequencies according to sf( 13 C) = sf( 1 ) γ( 13 C)/γ( 1 )

104 Indirect referencing o X o X X = tabulated resonance frequency at a field where the proton freq. is 100 Mz = 100 Mz X o o = frequency of 0 ppm of the X nucleus = frequenz of 0 ppm for protons r 13 C 15 N (rel. to N 3 ) TMS DSS TSP

105 Artefacts in 2D NMR

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107 - quadrature images Quad. images are due to incomplete quadrature detection. The intensity of quad. images is proportional to the intensity of the main signal, and hence, usually only quad. images of diagonal signals are observed. Quad. images are located on the anti-diagonal. - t1 noise t1 noise is manifested as vertical strips (noise bands), which are running along F1 and is mainly observed for intense signals, e.g. strong singlet signals (methyl groups). The intensity of the noise band is proportional to the signal height. t1 noise comes from random instabilities of the lock signal, which may originate from instable room or probe temperature, amplitude or phase modulations of the signals due to sample spinning, instabilities in the magnetic field homogeneity caused by magnetic disturbances of the environment (traffic), or from digitization errors. Instable temperature is the major source for t1 noise.

108 - F1 or F2 folding Folding is observed when the spectral width is not chosen sufficiently large enough to cover all signals. The intensity of signals, which are folded in F2 is largely diminished through audio-filters. owever, folded signals in F1 are not attenuated in intensity. Signals are folded along the near edge (real acquisition) or along the distant edge (complex acquisition). Folded signals cannot always be phased.

109 - aaxial peaks Axial peaks arise from magnetization which has relaxed during the evolution period. These signals are therefore not frequency labeled and behave as if they would have zero frequency. Depending on the F1 quad. detection mode, they are on a line running parallel to the F2 axis through the middle of the spectrum (magnitude mode spectra, States quad. detection) or are located at the bottom of the spectrum (TPPI, States-TPPI quad. detection). MBC (TPPI) SQC STATES

110 - repetition-rate artifacts Repetition rate artefacts do occur, when the relaxation delay is not chosen sufficiently long enough and arise from magnetization which is "left over" from the previous scan. Such artefacts may give rise to cross peaks at various frequencies along F1, such as the double-quantum frequency etc... Such artefacts can sometimes be recognized when there is no corresponding signal at the F1 frequency of the cross peak, or when additional signals on diagonal lines of higher steepness are observed.

111 - errors from symmetrization of spectra (processing) Symmetrization is a mathematical tool to improve spectral quality by removing all signals, which are not symmetrical about the diagonal. When two diagonal signals have strong noise bands, symmetrization leaves a signal at the cross peak position: Sym