NMR_FINAL Flashcards
In your studies, you hypothesize a protein of interest undergoes an unfolding transition through a molten globule intermediate (I). You also suspect that the transition between F, U,
and I is salt dependent. Describe how you would map out the phase diagram, using methods described in this lecture. What nucleus would you employ in your analysis? Assuming the transition to “I” is fast, how would you propose to observe it by NMR?
Signature spectrum of molten globule in HSQC. Inept periods mean because molten globule T2 is short, it is difficult to understand relative populations.
19F NMR is better due to sensitivity so we can add fluorine tags so look at difference in chemical shifts of folded vs. non folded.
If we have slow exchange we see two distinct peaks in fluorine spectrum because rate < difference in frquencies. The intergration of such peaks gives ratio of the two and the equilibrium constant can then be determined from this. DeltaG= -RTln(K)
If we have fast exchange, we see a weighted average signal with a wide width associated with T2. We can see a profile with a CPMG experiment as it fives Kfolded vs. Kunfolded and can give fraction of each in the states. Keq is related to forward and reverse rates so we can look at the difference in energy of these two.
If we have much more complicated spectra, just look at one transition at a time. By using known concentrations of salt, we can study NMR at different temperatures to generate phase diagrams.
The key here is timescale where we need to create a scenario such that the exchange is slow. Molten globules have 2o structures (weak 3o structures), thermodynamically stable. Dry molten globules are dynamic but water is sequestered from them. Tag the protein (eg: 5-fluorotryptophan) and disrupt the folded state so that it unfolds. If transition is slow, we get distinct signals. In the case of fast exchange, If you have state A and state B, you can record cross peaks in a 2D NMR. This measures population and transition rate from A to B and vice versa which gives us info on KAB and KBA. This gives us Keq and from there I can measure the free energy from B to A and A to B. Studying that in terms of temperature and pressure, I get enthalpic and entropic data, specific volume, etc.
How would you propose to study the entropy, enthalpy, free energy, and specific volume differences between F, I, and U?
. Then measure temperature (enthalpic data) and pressure (specific volume data) dependence between those 2 states. We can also get heat capacity difference if there is curvature to the temperature dependence.
- How would you propose to study the heat capacity differences between F, I, and U?
Can look at pressure dependence
Same method as the question above, We can also get heat capacity difference if there is curvature to the temperature dependence.
Suppose you had an extensive disordered protein (FUS) believed to establish LLPS at appropriate concentrations, pH, [salt], and Temp.
- How would you use NMR to establish a phase diagram that differentiated the low and high density regions and the regions of two phase coexistence?
- How might you use NMR to identify the sizes of these domains?
- Could you map a phase diagram by 19F NMR using NaF as a probe?
- To get a phase diagram, you want to know how much stress granules you have and how much granules are in the bulk phase. If you have a disordered protein, you can label it in some way and observe chemical shift differences (eg: fluorine tag) based on changing parameters like [salt], pH, temperature. Stress granules have large sizes, so it would be more difficult for a protein to diffuse in and out of a stress granule to achieve fast exchange.
- We can use our spectra to determine the fraction and of such domains and the amount of molecules in them. In doing so, we can then estimate the area of those domains.
- Yes, you could dose your granules with NaF and observe the difference in chemical shifts as we vary [salt]. We also want to observe a point where one of the peaks totally disappears so we know we have entered a new phase. We can also measure the ratio of areas under each peak to determine the concentration ratio of each state.
- What are the smallest possible sample volumes available through commercial NMR probes and what are the advantages in terms of S/N, mixture analysis, and high throughput chemistry?
30microliters (triple resonance cryoprobe).
Going to smaller sample volumes per unit probe, S/N goes up 14x due to the coil wrapping around sample more closely.
Less salt sensitive
Signal increases per unit volume. Bigger coils are always better in terms of sensitivity.
1.7mm probe
S/N = 196 x
- Of what value would supercritical CO2 be in the analysis of biological samples?
Low viscosity means low tumbling time of protein (assuming it can stabilized in micelle). scCO2 also solubilizes a wider range of small molecules.
- If you were to consider two macromolecules of comparable specific volume, Vs, whose masses differ by 2, by what factor would you expect the linewidth to change?
2
- By what factor would you expect linewidths to improve for macromolecules in sc CO2?
Inversely proportional to viscosity difference
- Describe a method of detection of precessing nuclear spin magnetization other than induced EMF? What are the advantages and principles of an optical diamond NV center used to measure NMR signal? (nitrogen vacancy centers in diamond, polarization of nuclear spins at high field and transfer to detect volume, e- triplet state and laser irradiation of field dependent splitting, photoluminescence,…) resolution .65 Hz, sample volume picolitre (single cell spectroscopy), microfluidics
Like the Faraday effect. Using a coil, look at induced DMF using diamond lattice with nitrogen valancies. Holds onto paramagnetic species in magnetic field - luminesence and spin perturbation by throwing in 1H RF pulse and looking at purturbation in luminesence. Resolution of 0.6Hz.
Nitrogen valency allows for triplet state and zeeman splitting depends on magnetic field. This is modulated by 1H spin evolution using 90 degree pulse. We can detect via 1H relaxation.
2 stable unpaired e-, triple state, non degenerate in magnetic field
1.5T magnets generate nuclear spin polarization
13mT detection with Helmholtz coil
Atomic lattice stabilizes 2 unpaired e- in the NV center
Microwave stimulation in 2 allowed photoluminescent transitions
Zeeman splitting on field strength
Advantages: Allows for detection when mass is limited, 0.65Hz resolution
- What is dissolution DNP and what is needed to achieve effective 13C or 1H NMR?
lowT, high field glass with radical and NMR sample. microwave irradiation to polarize 1H, CP to transfer to 1H. Dissolution, transfer, run NMR
“Dissolution-DNP is a method to create solutions of molecules with nuclear spin polarization close to unity”
Polarizes nuclear spin at low T (1-2K) in a 3-7T field
E- spin polarization unity
Microwave irradiation close to paramagnetic resonances causes redistribution of spin populations and approach e- spin polarization
Dissolves sample preserved in liquid state
Method used to make solutions of molecules with nuc spin polarization close to unity
To achieve 13C or 1H, freeze sample, microwave irradiation at 1-2K to polarize CP. Rapidly melt sample by auto injecting it into a heated buffer sol’n. Inject in animal and observe hyperpolarization of NMR signal. (relies on long T1 of species)
Uses low magnetic field and a nitroxide (paramagnet) which is polarized at low T. By incubating this sample with your 13C metabolite, you can build up magnetization (polarization) of the metabolite. As you say CP to 1H if needed. Then transfer sample to animal and do in vivo MRS. relies on long T1 of species
Use 13C pyruvate for long T. Low magentic field at ~10 kelvin and coincubate with nitroxide spin label and heat sample then widthdraw. Only 13C species go into animal then polarize. At low temp we have cross polarization as there is a change in B1 field and RF pulse then gives effective transfer.
- Using dissolution DNP describe how we detect cancer markers, monitor metabolic states, or evaluate myocardial infarcts
We can assess aerobic and anaerobic pathways.
Infarcts usually have alternate metabolic pathways due to lack of oxygenation. Similarly, in cancer you have anaerobic metabolism. So we inject a 13C labelled precursor (usually pyruvate) with long T1. By using dissolution DNP in a silicate medium injecting it in an animal, after 3-5 minutes the pyruvate would have been metabolized and we would see by NMR what it turned into (lactate). That way we can determine metabolism in cancer, infarcts, etc.
Cancer cells metabolise by glycolysis à lactate build up
Cancer cells have different signals due to diff metabolic pathways
Can be detected by C13 since lactate has an OH instead of C=O at C2
- Describe optical pumping and Imaging of Xe129 or He3 in the lungs.
Optical pumping transfers angular momentum from photons to e- in vapor in alkali metals for better polarization
E- spin of metal can be polarized, will polarize nuc spin of He/Xe by collisional spin exchange
Polarized laser at 795nm polarizes spin of valence e- of metal by population of 1 of 2 spin states for valence e-
After spin exchange, metal atom spins are polarized again
Spin-exchanges collision with He/Xe, noble gas polarization increases (better overall polarization)
Rb and Xe have v.v long T1.
What is fragment based drug discovery and what are its advantages?
- “Fragment-based drug discovery (FBDD)” takes a different approach. Rather than screening millions of compounds to find drug-sized starting points, FBDD begins with much smaller collections of smaller com- pounds. Fragments are usually defined as having less than 20 non- hydrogen (or ‘heavy’) atoms.
Pros:
1) Bigger chemical space & smaller library sizes (~1-5000)
2) Higher hit rates should enable more difficult targets
3) Fragments are small and typically soluble, they are likely to have beCer pharmaceuDcal properDes and thus have the potenDal to produce superior drugs
Typical FBDD by NMR uses libraries of ~20-50 molecules at a Dme which are known not to self interact and are well behaved in terms of solubility.
Thus FBDD can be quite high throughput
In several cases, FBDD has rescued failed HTS trials and idenDfied novel binding sites
Your company is inves4ga4ng a GPCR and the possibility of allosteric modula4on by addi4onal probes. The protein is very hard to prepare and quite expensive. Design an NMR experiment to screen small molecules for their binding propensity. Assume you know everything about the primary ligand binding site and assume you have a wide variety of agonists, inverse agonists, and par4al agonists, known to interact with the primary binding site. Lets also assume that ac4vity assays are quite laborious so you want to address this by NMR if possible. (1 paragraph)
We can use very little of protein )<1uM)
We can use STD or FAXS. Taking advantage of weak binders. Correlation is small so narrower lines. We can still pick up NOEs. In CPMG we have a slight decay and can therefore tell contribution from the protein.
A paramagne*c “street lamp”. This is the worlds first NMR amplifier which allows us to “see” drugs binding just to the extracellular pockets. Here, the N-terminus is modified by appending a Gd3+ bearing chelate, or a Eu3+ or Pr3+ chelate to study ligand binding. This would allow:
i) Direct ligand interac*on studies through
shiMs, CEST, CPMG
ii) FAXS studies (compe**on
assays)
FAXS:
- FAXS (Fluorine chemical shi5 Anisotropy and eXchange for Screening )
C. Dalvit / Progress in Nuclear Magne6c Resonance Spectroscopy 51 (2007) 243–271
(i) high sensi*vity rela*ve to 1H compe**on binding assays
(ii) No signal from protonated solvents, buffers, or detergents
(iii) Absence of overlap permits the screening of large chemical mixtures and automated analysis of the spectra.
The spy molecule is designed to have a weak affinity with the receptor in ques-on such that the exchange broadening amounts to 5-10 Hz. In this case, there is no bound and free chemical shiK signature. Rather we observe an average associated with the fast equilibrium between “bound” and “free”a CPMG experiment will sensi-vely exchange effects
GPCRs possess a domain referred to as the extracellular ves4bule. As is the case for many receptors, this region serves as a selec4vity filter and a desolva4on pocket prior to ligand binding to the orthosteric region. Of the experiments described above, which might be ideally suited to the detec4on of fragments bound top this solvated region?
Summarize water LOGSY
taregts highly hydrated and therefore bound waters. Ligands see water therefore there is NOEY between ligands and free water compared to ligands and bound water. Bound water have the sample tumbling rates as the protein unlike free. We want to selectively invert bound water. We rely on gradient pulses (guassian). More it moves the less the signal is picked up
Assuming you used a paramagnetic beacon FAXS experiment how do you an4cipate differences resul4ng from using Gd, Eu, or Ln as the “beacon”?
FAXS requires a spy molecule that is fluorinated and has high affinity for the protein target. Gd and Eu are paramagentic due to unpaired electrons and have a large magnetic moment. This means v.v. large T1 and therefore 1/T1 ~500MHz which is almost exactly the protn larmour frquency! Very efficinet T1 relaxation. Relaxation reagent.
Amplify exchange broadening through dipole interactions. For Eu it is so fast it doesnt effectively give T1 relaxation due to dipolar shift and this is what we call a shift reagent.
Ln is diamagnetic and therefore the control
Summarize (in 1-3 sentences each) the principle behind SAR by NMR, Studying binding by relaxa4on effects, the STD experiment and binding by satura4on, Waterlogsy and FAXS.
Saturation transfer difference (STD) spectroscopy allows to detect transient binding of small molecule ligands to macromolecular receptors. Receptor species include proteins - free in solution or immobilized, whole virus particles, etc.
Range of applicable dissociation constants is approximately 10-3-10-8 M [1].
STD method can be used to determine which part of the ligand molecule is responsible for binding, since most strongly interacting groups of ligand will show stronger STD effect.
Method of saturation transfer difference relies on the possibility to selectively saturate protons of macromolecular receptor by irradiating the spectral region containing “wings” of broad resonances of the macromolecule which is also free of any smaller molecule signals. Due to effective spin diffusion saturation quickly propagates across the entire receptor. If the smaller molecule ligand binds the receptor, saturation will also spread onto the ligand. The result will be that intensity of the ligand signal will be attenuated. Substraction of resulting spectrum from the reference spectrum without saturation yields the STD spectrum containing only signals of the binding ligands.
FAXS: The FAXS method is an NMR-based ligand binding-competition approach used to explore new binders of target proteins by employing 19F-containing “spy” molecules with weak affinity
Water LOGSY:
WaterLOGSY is a widely applied 1D ligand-observation technique for the detection of protein–ligand interactions. As the STD approach, WaterLOGSY is based on the NOESY experiment, and implies transfer of magnetization via a intermolecular NOE and spin diffusion. The originality of WaterLOGSY comes from the intervention of water molecules in the transfer pathway. The bulk water magnetization is excited and transferred during the NOESY mixing time to the bound ligand via different mechanisms. The WaterLOGSY spectrum, which is recorded for the free ligand, contains the bound-state perturbed magnetization as long as the relaxation time T1 of the ligand is greater than the dissociation rate constant koff. The inverted water magnetization can be transferred via different pathways to the bound ligand:
(1) direct transfer from water molecules immobilized in the protein binding site (water residence times greater than nanoseconds)
(2) chemical exchange between excited water and protein labile protons (amide, hydroxyl, amino, etc.) and propagation of the inverted magnetization to the ligand by intermolecular dipole–dipole crossrelaxation as well as spin diffusion via the protein– ligand complex
(3) transfer from the water molecules found in the protein surface via the protein–ligand complex. In the three mechanisms, the ligands interact with water via water–ligand–protein or protein–ligand complexes, whose rotational correlation times yield negative cross-relaxation rates and exhibit a negative NOE with water. By contrast, small molecules that only interact with bulk water (non-binders) will experience much faster tumbling, which translates into a positive NOE. Therefore, opposite signs for signals from free versus protein-bound ligands are observed in a WaterLOGSY spectrum, which enables one to easily discriminate binders and non-binders.
Suppose you wish to perform an STD ligand binding experiment on a 50 kDa protein. The protein, which facilitates rapid cogni4ve func4on, must be extracted from the spinal fluid of the finest students from St Georges campus and is thus in short supply. You have iden4fied a series of ligands with 1 μM, 7 μM, and 50 μM dissocia4on constants. If you prepare three 300 μL samples, each containing 1 mg of protein, calculate the frac4on of ligand bound and the frac4on of protein bound in each sample if you then add 100 μL of 1 mM stock solu4ons.
[PL]=0.5*(kd+[Po]+[Lo]-sqrt(.25(kd+[Po]+[Lo])^2)-[Lo][Po})
fLR=[PL]/[Lo]. fpb=[PL]/[Po]
Take a cell with 13C glucose and enrich at alpha and beta carbons or a mix and then look at unique spectra through scalar couplings. Measure in HECCOR (like HSQC) to directly detect 13C and indirect 1H
- What is SABRE-SHEATH hyperpolarization, how does it work, and how could it be implemented in metabolomics? What are the limitations?
SABRE-SHEATH hyperpolarization technique method is a low-cost, efficient catalytic polarization transfer method for hyperpolarizing nuclear spins at room temperature. SABRE (signal
amplification by reversible exchange) works via the transient binding of parahydrogen to a transition-metal complex to form a network that permits the transfer of spin order from the para-hydrogen to the nuclei on the substrate molecule or molecules of interest. Set-up and equipment costs are typically less than a few hundred dollars, yet enhancements on the order of 10,000x are possible and the hyperpolarization state lasts for over an hour. This has huge implications for low field (1-10 Tesla) metabolite NMR and MRI via (1H, 13C, and 19F)
13C-optimized 1.5-mm cryoprobe that uses high temperature superconductors has enabled 13C NMR studies of small quantities of natural products to be done at natural 13C abundance
SOFAST HMQC and NUS 2D NMR are key to reducing 2D NMR times by ~ 10x
- What is the difference between an oncometabolite and a metabolic marker and what are the possible medical consequences?
indicative of positive treatment. ONco not most downstream
- What are:
i) arthritogens,
ii) atherotoxins,
iii) cardiotoxins,
iv) dementogens,
v) diabetogens,
vi) hepatotoxins,
vii) immunotoxins,
viii) nephrotoxins,
ix) neurotoxins,
x) obesogens,
xi) oncometabolites,
xii) osteotoxins, and
xiii) teratogens?
i) arthritogens: Altered area acts as binding site for arthritogens. An initial infection causes malaise, fatigue, and pain in muscles and small joints in hands
ii) atherotoxins: blood vessles
iii) cardiotoxins: Cardiotoxins are a confusing group of toxins, because the name is often used for certain snake venom components, even though in humans, they generally do not affect the heart significantly. These snake venom toxins are based on phospholipases, as are many other venom components.
iv) dementogens, A dementogen is a chemical that increases your risk for cognitive decline
v) diabetogens: Adjective. diabetogenic (comparative more diabetogenic, superlative most diabetogenic) (pathology) That produces diabetes.
vi) hepatotoxins: A hepatotoxin (Gr., hepato = liver) is a toxic chemical substance that damages the liver. It can be a side-effect of medication, or found naturally, as microcystins, or in laboratory environments.
vii) immunotoxins: An immunotoxin is an artificial protein consisting of a targeting portion linked to a toxin. When the protein binds to that cell, it is taken in through endocytosis, and the toxin kills the cell. They are used for the treatment of some kinds of cancer and a few viral infections.
viii) nephrotoxins: A nephrotoxin is a toxic agent or substance that inhibits, damages or destroys the cells and/or tissues of the kidneys. This is an example of a cytotoxin. Inflammation of the kidneys is called nephritis. Study of the kidneys is called nephrology.
ix) neurotoxins: Neurotoxins are toxins that are destructive to nerve tissue. Neurotoxins are an extensive class of exogenous chemical neurological insults that can adversely affect function in both developing and mature nervous tissue.
x) obesogens: Obesogens are foreign chemical compounds that disrupt normal development and balance of lipid metabolism, which in some cases, can lead to obesity
xi) oncometabolites: oncometabolite (plural oncometabolites) (biochemistry, pathology) Any metabolite that is associated with a cancer quotations
xii) osteotoxins: A chemical substance that causes damage to bones and/or joints
xiii) teratogens an agent or factor which causes malformation of an embryo.
- What functions are primarily observed in metabolites originating from: i) urine, ii) cerebrospinal fluid, iii) sweat, iv) saliva, v) fecal water, and vi) blood/serum
i) urine,
ii) cerebrospinal fluid,
iii) sweat: Skin microflora
iv) saliva,
v) fecal water:
vi) blood/serum
- How can the PSA test be improved from the perspective of prostate serum antigen biomarkers? What type of metabomolic study might be combined with this protein biomarker study?
Normally limit of detection is terrible with false positives and negatives. Other glycoforms and antigens would be better bio markers.
PSA: Prostate-specific antigen
. How is the metabolome potentially advantageous over proteomic or genomic analyses?
Non-invasive
In expensive
downstream - lots of intro from metabalome amplification from genetic facotrs
personalized medicine
What is the difference between an oncometabolite and a metabolic marker and what are the possible medical consequences?
oncometabolites initiate or sustain tumor growth. Thus, their elimination from say the blood could contribute to treatment. A metabolic marker is simply a diagnostic of a condition.
Perform a search for 2-hydroxyglutarate on HMDB.ca and identify how it is an oncometabolite and how it can be detected?
blood, CSF, feces, saliva, urine
part of butanoate metabolic pathway, produced by phosphoglycerate dehydrogenase (PHGDH). More importantly, 2-hydroxyglutarate mimics 2-oxogluratate (2OG) and thus inhibits a range of 2OG-dependent dioxygenases, including histone lysine demethylases (KDMs) and members of the ten-eleven translocation (TET) family of 5-methylcytosine (5mC) hydroxylases. This inhibitory effect leads to alterations in the hypoxia induced factor (HIF)-mediated hypoxic response and alterations in gene expression through global epigenetic remodeling. The net effect is that 2-hydroxyglutarate causes a cascading effect that leads genetic perturbations and malignant transformation.
- ~1 uM levels in blood in normal patients. Can be up to 1000x higher depending on the condition
- In one study in urine (normal patients) 2-hydroxyglutaric acid was measured by NMR at 33.0 (13.3–77.9) (uM/mM creatinine), 100% occurrence. Thus likely NMR or MS could detect this compound
1.Recall S/N equation
What savings is gained by a 13C,1H HSQC over a direct detect 13C NMR experiment?
ye and yd both in S/N. For inadequate and DEPT both different, for homo joined
- What is an INADEQUATE Experiment? What is observed in the indirect and direct dimensions?
double quantum-some of 2 coupled spectra
direct-antiphase chemical shift of other species
Most of proton spectrum never excited, effective T1. Can look at proteins and small molecules
What are optical detection technologies?
NMR spectrometers rely on the inductive detection of oscillating magnetic fields generated by precessing nuclear spins
Sensitivity & detection volumes are suboptimal for metabolic analysis of single mammalian cells or use in microfluidic assays
Quantum sensors based on nitrogen-vacancy (NV) centers in diamond have emerged as alternative NMR detection modalities
NV centers consist of 2 stable unpaired electrons which adopt a triplet state
Strong permanent magnets (1.5 T) are used to generate nuclear spin polarization (See Fig 1B next slide)
Detection is performed at 13 mT using Helmholtz coils (Fig 1C), simplifying the task of stabilizing NMR linewidths to sub-hertz
levels while enabling the use of high-sensitivity diamond quantum sensing protocols at low microwave frequencies
What is DNP?
Dissolution DNP uses microwave radiation to transfer the high polarization of electron from stable radicals to low abundance NMR active nuclei like 13C, 15N in molecules of interest thereby increasing the number of polarized nuclear spins tremendously and enhancing the signal to noise ratio by up to three orders of magnitude. The signal enhancement makes it possible to study biochemical phenomena and diseases in real time in vivo by 13C NMR/MRI.
Typically, a sample containing the metabolite of interest (e.g. pyruvate, lactate) and stable radical is frozen, and then irradiated by microwave radiation in the DNP apparatus at ≤1.2 K for approximately 1 hour. The sample is then rapidly melted and transferred to an automatic injection device using a heated buffer solution, where it can be introduced into the tissue, organ, or cell culture to be studied. The hyperpolarized signal can then be observed for 3 - 5 minutes after the dissolution process has been initiated.
What is STD NMR?
Solve
Solve
Solve
What are the absorption and what are the dispersion modes?
How do we describe I1x and I2x ?
What does I1z, I2Z represent?
I1x, I2x, etc are described as (in-phase) single quantum coherence since they give rise to doublets where both peaks are of the same sign.
I1z, I2Z represent longitudinal magnetization
What do these operators represent?
2I1xI2x and 2I1xI2y
epresent multiple quantum coherences (these are not directly detectable but they are genuine coherences with characteristic frequencies
Product operators with at least one longitudinal component (e.g. 2I1xI2z ) are termed?
What about 2I1zI2z?
Product operators with at least one longitudinal component (e.g. 2I1xI2z ) are referred to as anti-phase magnetization on spin one, leading to detectable signal on spin 1 in the form of an an symmetric doublet.
Similarly, anti-phase terms like 2I1xI2z evolve under J12 into I1y
2I1zI2z represents a non-equilibrium populaton
Solve
Solve:
Solve for I1x
Do this for I1x, I1y,
2I1xI2z
2I1yI2z
Show how I and S are either refocused or not in a spin echo:
What is coherance transfer?
What is this technique also called?
Show and example of the spin evolution
Coherence Transfer
Via the scalar coupling, it is possible to transfer the transverse component of an an6-phase spin state from one spin to another.
ie 2I1yI2z can be converted to -2I1zI2y through two 90 degree pulses
How do selective COSY experiments work?
What are multiple quantum coherances?
Multiple quantum coherences are easily generated from anti-phase states
Solve:
Solve
Solve HMBC
Solve HMBC 2.0
Solve HECTOR
Two 23Na NMR resonances are observed to be 0.5 ppm apart on a 600 MHz (1H NMR) NMR
spectrometer.
Calculate the separation in Hz and rads/s.
Two spins differ in frequency by 75 Hz. Calculate how long it takes for them to dephase by π radians.
2.1 In an NMR spectrum, the peak from TMS is found to occur at 500.134271 MHz. Two other peaks in the spectrum are found at 500.135021 and 500.137921; compute the chemical shifts of these two peaks in ppm. Given that the receiver frequency is 500.135271 MHz, recompute the chemical shifts of the two peaks. What would the frequency separation in Hz and rad s-1 be between these two peaks if the spectrum were recorded using a different spectrometer operating at 400 MHz for protons? The receiver reference frequency of the spectrometer is 400.130000 MHz.
Predict a tree diagram to predict the form of the multiplet expected for spin A when coupled to B and C using coupling constants of JAB=10 Hz and JAC= 2 Hz. Calculate each line and label it with the spin states of the coupled spins; assume the multiplet is centred at 0 Hz. Repeat the process for the cases (a) JAB= 10 Hz, JAC = 12 Hz and JAB= 10 Hz and JAC=10 Hz. What special feature arises in the latter case?
2.4 Make sketch graphs of x- and y- components of a rotating particle as a function of time for the case where the starting phase is (a) 0 degrees, (b) 135 degrees, (c) 2 radians and (d) 3pi/2 radians. In each case comment on the form of your graphs noting whether they are sine or cosine functions
Two peaks in a proton spectrum found at 1.54 and 5.34 ppm. Spectrometer frequency = 400.13 MHz, what is the separation in Hz and rad s-1?
Calculate the Boltzmann ratio for a 1H nuclei in a 900 MHz magnet
