Annual Reports on NMR Spectroscopy: 51

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This would mean that the first point of sine-modulated data should be zero, and in this way the weighting error would be negligible. The level of the noise is proportional to the intensity of the cross peak. This t1 noise is caused by general hardware instabilities like inconsistencies in RF pulse synthesis and pulse sequence timing. The level of t1 noise can not be reduced in the spectrum through processing of the acquired data. Baseline correction can be used to shift the middle point of the noise, but this can also affect the volume of cross peaks that appear on this t1 noise ridge.

Such cross peaks should be cautiously used for quantification. The number of acquired points should be sufficient to give a good digital resolution to the spectrum.

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Therefore the acquired data will need zero filling to provide the aimed digital resolution. The same rules apply to the 2D NMR spectra as to 1D NMR spectra; if the digital resolution is too small, the line shapes will be too coarse to accurately describe the resonance, and the peak volume will be biased. Apodization or weighting of the acquired 2D data has a major effect to the appearance of the spectrum.

Apodization is used either for enhanced signal-tonoise ratio or enhanced resolution. It can be also used to smooth a truncated acquisition data to avoid distortions that appear on the base of peaks. In case of HSQC data, apodization that mimics the natural decay of the signal i. Exponential windowing may not always be the best option if good resolution is aspired, and then a good compromise is to use apodization with a small resolution enhancement, like squared cosine windowing.

Effects of apodization and linear prediction on the quantitativity of the spectrum have been discussed in literature by several authors. Integration of the cross peak can be performed in two ways: There are several third-party NMR processing software packages available Quantitative 2D NMR Studies 21 featuring some advanced NMR data processing tools— which offer more flexibility in processing of the spectra. When quantitative analysis is conducted for a large set of samples, the recorded spectra must be processed in identical manner, preferably by the same operator.

This ensures that the processing deviations between the spectra are minimized and quantitative comparison between the samples should be straightforward. Reference in quantification In quantitative NMR spectroscopy the concentration of the analyte is typically determined by comparing the analyte resonance integral to an inert, internal reference with known concentration. It is then quite straightforward to calculate the analyte concentration in the sample. In some cases there are no inert references available that could be applied internally to the sample.

In these cases a reference chemical in a coaxial glass insert within the NMR sample tube can be used, so that no contact will take place between the sample and the reference chemical. Other possibility is to use external standard in a separate NMR tube. The electronic reference to access in vivo concentrations also known as the ERETIC method provides a reference signal, synthesized by an electronic device, which can be used for the determination of absolute concentrations.

This approach shows potential as a universal reference in quantification. The selection of the used experiments was also quite diverse; all typical 2D homo- and heteronuclear correlation experiments were among them. In overall, 2D NMR quantification offered a better linearity and accuracy in these comparisons due to better resolved peaks. Additionally, the approach often facilitated analysis of minor components that were not distinguishable from 1D NMR spectra.

The main part of the papers that demonstrated 2D NMR quantification focused on the analysis of natural products like animal and plant metabolites. Some articles were also found where quantitative 2D NMR was applied to quality control in food industry, and on characterization of the products of oil industry. The following sections give a short summary of the quantitative 2D NMR on the aforementioned topics. Metabolic profiling Study of natural products, biological systems and processes taking place in living organisms has become a major research.

NMR spectroscopy has been an important technique in characterization and quantification of single natural products in plant extracts, dietary materials, and materials representing different metabolic stages of organisms. In comparison to conventional 1H NMR quantification the author stated that the likelihood that a peak in the projection spectrum contains more than one resonance is significantly smaller, thus providing additional accuracy for summarizing and interpreting the metabolomic data.

Their tests with dog urine, fish liver extract and leukaemia cell extract samples indicated that combined use of sine-bell and exponential apodization resulted in a better resolution and reproducibility, which should be beneficial in quantitative studies. There are several reports dating from the s where metabolic analyses were performed with COSY.

The paper also demonstrated the quantification of the lactate levels on the basis of lactate cross peak volumes. The authors reported a good linear correlation R2 0. Based on that finding the authors were able to follow the changes of lactate level in gastrochemius frog muscles during the 18 h observation time.

They were able to identify, and also quantify, the presence of maltodextrinphosphate in this rumen bacterium. The magnitude calculations with the DQFCOSY caused broad lines, making the integration more difficult; other implications were also discussed. It was also noted that the heteronuclear scalar couplings were averaged to zero with TOCSY during the isotropic mixing period, and the effects related to long-range heteronuclear couplings were avoided.

They reported that the improved resolution facilitated detection of minor metabolic components in urine giving more accurate and statistically relevant analysis of changes in the metabolic fluxes. In the study by Dumas et al.

They applied linear discriminant analysis to extract quantitative variables from the spectra in order to reveal metabolism involved in endocrine disruption processes. The authors discussed the implications to the quantitativity of the integrals with respect to the differences between polarization transfer optimization and the true 1JCH couplings. They stated that when variation among true 1JCH values was in range 5—8 Hz, as obtained for uronic acid residues, it did not affect to a significant degree to the calculated content in glycosaminoglycan individual residues.

However, there were cases where observed couplings of certain groups differed 25 Hz, and differences of these magnitudes, according to the authors, can generate significant errors in the compositional analysis. HSQC was also applied by Lewis et al. They reported identification and quantification of ca. Lignin chemistry While a large part of natural product studies is focused on plant extracts containing small molecules, a significant amount of research is also going on with polymers of the flora.

Pulp industry that uses cellulose of wood in paper making has also carried out research on the other major polymer of the plant kingdom known as lignin. Zang and Gellersted presented an extensive study on the influence of T2 relaxation in quantification with HSQC experiment. They proposed an approach where cross peaks with the same T2 relaxation profiles were analyzed as individual groups, so that the T2 errors in quantification could be minimized.

Their results in quantification of milled wood lignin of Picea abies agree well with the literature values,,, acquired with chemical analysis or 1D NMR methods. Natural degradation of lignin has also been under interest. Lignin degradation products form a large part of humic substances in soil as well as in river and lake waters. NMR spectroscopy has been used in several studies to characterize these highly complex samples. Information was used to interpret the processes of lignin degradation.

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Food analysis and quality control Food production for human and animal consumption is an important branch of industry. The production, processing, as well as storage, is controlled by national and international legislation. This legislation is aimed to assure that the food is safe for human or animal consumption by regulating, e. There is also need to verify the origin of the foodstuff e. Many of these regulations need verification by methods of analytical chemistry.

NMR spectroscopy has also been applied to food analysis, and there are some excellent reviews on this topic. With application of principal component analysis they distinguished six lagers from each other based on their amino acid contents. They also discussed how the amount of compounds that have an important effect to the taste, like tyrosol, can be estimated with the method. A relaxation reagent was used in the milk samples to speed up the acquisition.

Calibration curves for sodium citrate, acetylglucosamine, tributyrin, triolein, trilinolein and lecithin measured against an external reference 1,1,2,2-tetrachloroethane in a coaxial glass insert showed a good linearity; R2 was above 0. The amounts of the chemicals in milk were in accordance with the reference values determined by gas chromatography.

A possible reason can be that experimental set-up, acquisition and processing of 2D NMR experiments are considered to be too difficult or timeconsuming compared to 1D NMR experiments. Also, restrictions and regulations from the laboratory environment quality systems, standards can hinder application of new methodology in quantification. Considering the challenging sample matrices the 2D NMR methods would be excellent tools in quantification.

Product properties of oil fractions Oil industry has a long history of application of NMR spectroscopy for characterization of crude oils, products and oil fractions. The methodology has been mainly 1D proton- or carbon-detected experiments. Due to environmental concerns oil companies are nowadays more interested in development of lubricant base oils that have low aromatic and olefin contents.

Hydrogenation of unsaturated components also improves the stability of the base oils, which is an important property for the end-product. Quantitative analysis of a saturated oil fraction with NMR is a major challenge. When the oil fraction contains only aliphatic compounds, the spectrum width that contains the resonances narrows to ca. This causes excessive resonance overlap in the 1D NMR spectra, thus rendering their usefulness in characterization of oil fractions quite limited.

Therefore, elevated interest in 2D NMR spectroscopy for characterization and quantification of oil fraction components has risen. In addition, there have been attempts to make statistical models how the quantitative information derived from 2D NMR spectra can be used to predict oil product properties. The part of the spectra containing methyl cross peaks, i. The analysis predicted viscosity indexes of the oil fractions with correlation coefficient 0.

The analysis also indicated what kind of branching in the hydrocarbons most likely promoted the desired high viscosity index. While there is a limited number of publications about application of quantitative 2D NMR in oil fractions, they still demonstrate how much more details 2D NMR can offer. The reason for the low number of papers can be also explained by the nature of the business; oil companies might feel to lose an advantage in the markets if they publish their latest analytical methodologies.

The industry itself is now on the verge of changes due to depleting oil resources and general concerns of environmental pollution. Motor oils and other oil products will be needed also in the future, but the oil consumption need to be controlled and reduced. Development of new oil products fulfilling these new requirements needs tools of analytical chemistry, and by that, NMR spectroscopy. Compared to quantitative 1D NMR, quantitative 2D NMR is more demanding with respect to experimental set-up, processing and cost of measurement time, but with the invested effort, 2D NMR offers significant improvement in resolution, and therefore challenges 1D NMR techniques in quantification of complex samples.

Combination of sophisticated statistical analysis and the quantitative 2D NMR is an approach that has found a fertile ground in, e. Ando, Ultraviolet and Visible Spectroscopy: Analytical Chemistry by Open Learning. Second Edition, Wiley, Chichester, De Hoffmann and V. Third Edition, Wiley, Chichester, Heyden, London, , Chapter 9.

A Quilliam and J. Heyden, London, , Chapter 4. Bailleul, Fuel, , 65, B, , , Akoka, Talanta, , 71, Nishizawa, Fuel, , 71, Sebald, Solid State Nucl. Ernst, Chimia, , 29, Bacher, Phytochemistry, , 68, Wiley, New York, Density Matrix and Product Operator Treatment. Prentice Hall, New Jersey, Basic Principles and Experimental Methods. A, , , Encyclopedia of NMR, D. Clarendon Press, Oxford, , Chapter 8.

Clarendon Press, Oxford, , Chapter 4. Basics of Nuclear Magnetic Resonance. Wiley, Chichester, , Chapter 2. Canet, Nuclear Magnetic Resonance: Wiley, Chichester, , Chapter 4. A Guide for Chemists, Second Edition. Oxford University Press, New York, Acta, , , NMR, , 6, NMR, , 8, Holmes, Xenobiotica, , 29, Ather, Advances in Phytomedicine, , 2, Methods and Protocols, Humana Press Inc. Tjeerdema, Metabolomics, , 1, Viant, Metabolomics, , 3, Van der Meijden, A. Wright, Biochemistry, , 23, Portais, Phytochemistry, , 68, Fundamentals and Applications, Second Edition.

Academic Press, San Diego, Brauns, The Chemistry of Lignin. Academic Press, New York, Occurrence, Formation, Structure and Reactions, K. Wood and Cellulose Chemistry, D. Wood Structure and Composition, M. Advances in Lignocellulosics Characterization, D. Methods in Lignin Chemistry, S. Linn, Fuel, , 60, Smith, Fuel, , 62, Atkins, Fuel, , 63, Smith, Energy Fuels, , 1, Fuchs, Fuel, , 70, Bhatnagar, Energy Fuels, , 12, Singh, Fuel, , 87, Beloeil, Fuel, , 74, A Practical Reference Ryan T.

McKay 34 34 35 37 38 38 45 49 52 53 55 57 59 60 61 64 67 67 69 72 73 73 74 Contents 1. Problems with ignoring solvent signals 2. How to suppress solvent? Suppression Pulse Sequences 3. Selective excitation family 3. Comparisons, Conclusions, and Advice 4. Comparison of suppression 4.

Bandwidth, artefacts, and baseline 4. McKay provided including recommendations on sample preparation and spectrometer optimization. This publication attempts to provide practical background and general knowledge for both novice and experienced users seeking to select and optimize the most appropriate tools for their NMR challenges. Nuclear magnetic resonance spectroscopy, Solvent, Water suppression, Radiation damping, Demagnetization field, Bulk susceptibility effect, Presaturation, Watergate, Purge, Metabonomics. Objectives There are a fantastic number of exploratory manuscripts and reviews thoroughly covering aspects of biomolecular NMR and the relationship with solvent suppression over the last 30 years.

Biochemicallya focused NMR spectroscopy has been developing for well over a half century. The first was the overwhelming volume of material to cover in order to perform a comprehensive review. The second and perhaps more important reason was the quality of reviews completed by other authors and at best the redundancy that any comprehensive work on our part would necessarily encounter.

Interestingly, larger reviews on solvent suppression seem to cover approximately years of the literature, and the timing of this review is no exception. For example, a review by Hore2 was published in , while Coron et al. Other excellent reviews have emphasized integral techniques such as the incorporation of pulsed field gradients. During the past few years there have been three developments necessitating an updated review.

The first is a recent resurgence of active development in the field of solvent suppression. The second is the rapid incorporation of cryogenically cooled probes into biomolecular NMR laboratories and the related challenges of increased signal to noise. This is an interesting point as most spectroscopists would never think of increased signal to noise ratios as a challenge. Do not forget that the solvent signal gets more intense as do artefacts.

In addition, as the noise floor drops any previously hidden problem tends to stand out. Even researchers experienced with NMR may not be familiar with all the subtleties of optimizing solvent suppression. Therefore we will attempt to reference the excellent previous works wherever possible and only re-address similar topics in the cases where a review will facilitate a better understanding of a new development or where we can yield directed assistance for less experienced users. This publication will attempt to tackle the limitations and robustness of techniques on identical, biologically relevant samples to facilitate direct comparison and evaluation for the potential user.

Best of luck and let us begin. There are many reasons behind the need for solvent suppression. We also always add 2,2-dimethylsilapentanesulfonic acid DSS 13 for internal referencing of biological samples. Just like trying to see something in the sky close to the sun, small peaks next to, or on top of the solvent peak are obscured and distorted. Common spectral errors obtained by not suppressing the solvent were shown in Figure 1B and C. None of the spectra were particularly useful. While it may be easy to suggest and visualize the physical removal of 1H solvent resonances from the sample by the full substitution of a maximally deuterated solvent e.

The concentration of 1H resonances in fully protonated water samples is on the order of M, which vastly exceeds even the most optimistic biological samples. Even if the solubility of a subject protein were such that solute signal could be on par with the solvent, any subsequent work would be hindered by concerns of nonspecific binding, dimerization or higher order polymerization , and functional relevance in any of these cases.

Of course the more obvious problem would be the difficulty in obtaining and purifying enough sample see Appendix , often labelled via expression in expensive 13C, 15N, and possibly even 2H-enriched media, to create such a hypothetical sample. As the solubility of almost any sample can never come close to these concentrations, suppression of the solvent becomes inescapable. Problems with ignoring solvent signals 1.

Receiver and analogue to digital converter errors We have seen what happens if we simply ignore suppression concerns for the moment and acquire a spectrum Figure 1. A receiver overflow occurs when the intensity of the receiver voltage exceeds the maximum that can be accurately read. Otherwise the start of the FID that should show precession of a sinusoid contains only maximum values until enough signal decay has occurred to bring the intensity back into scale Figure 2.

Until then, no accurate amplitudes can be measured. One solution is to turn down the amplification of that signal and any other weaker signals that might be of interest. This means that the maximum water signal amplitude fits inside the largest number possible for the digital converter, and the receiver can record the voltage levels in the coil.

Annual Reports On Nmr Spectroscopy Vol. 51 (Hardcover)

However this does not mean the solute is being measured correctly, only that the largest signal is within the scale range. This may seem like a lot, but if the water signal is times larger than the signals of interest i. Some newer consoles now contain bit ADCs but this does not solve receiver overflows, or other distortions occurring due to the massive solvent signal. Digitization noise Digitization noise can most easily be described as mistakes in the measured voltage when translated to a digital number.

Essentially there are not enough points to accurately describe the voltage, and the nearest digital number is selected somewhat similar to simple rounding errors. Effective digitization of a signal intensity less than the smallest value has been discussed,21 although the reasoning that noise levels should theoretically increase solute signals into a relatively detectable scale is certainly not the most palatable argument.

Digital noise errors should be most common when extremely weak signals are sought after in the presence of very strong ones and everything has to fit in a limited range of values.

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While receiver overloads occur when intensities are too Figure 1 Effect of solvent. All were run with a simple one pulse-read sequence containing no pre- or post-acquisition solvent suppression. Spectra were recorded on an Oxford 2. The short pulse was necessary to reduce the water signal intensity sufficiently to avoid receiver overflow. Even with minimal gain the water signal was sufficient to overload the receiver.

Two simple carton FIDs are shown. The left FID amplitude A is within scale and shows accurate measurements for each recorded point of acquisition. The right FID B shows a signal recorded with the gain set too high and the first series of points off scale. These points can only be incorrectly recorded by the hardware as maximum values. They are not meant to imply hard and fast rules or requirements to be imposed on all samples and spectroscopists though some are certainly required at our facility , but instead simply methods we have found to effectively acquire consistent data in the most reasonable amount of time.

Crap in equals Crap out censored version for our younger readers , 2. Cleanliness is next to Godliness, 3. Use the simplest experiment to answer the question. We recommend a uL sample i. While spectrometer vendors recommend volumes as high as uL, we have found that uL is the practical minimum with uL giving excellent results via a reasonable amount of shimming see Section 2.

The sample length is important because modern biomolecularoriented NMR probes i. A uL sample can be carefully centred in even 18 mm long cryogenically cooled receiver coils and yield good results. We have seen that uL samples can be run, but the sacrifices in terms of final shim quality begin to outweigh the advantages gained by not diluting the solute concentration. For example, it is better to dilute a sample up to at least uL or even better to uL than it is to run it at uL.

The uL sample suffers from overall inhomogeneity lineshape and solvent suppression problems that remove any expected advantages of higher concentration. It is easy to imagine that the best overall magnetic field homogeneity should be achievable on a consistent cylinder of infinite length passing through perfect rotations of coil wiring. While we certainly cannot achieve infinite length samples, it is our experience that 10—11 mm of sample on either end of the receiver coil is sufficient in order to properly shim a sample.

When the amount of available sample is the limiting factor not solubility , we have turned quite successfully to susceptibility-matched plugs such as those supplied by Shigemi NMR Tubes Inc. Shigemi manufactures tubes with solvent-matched plugs in the bottom of the NMR tube and a top plug that can be lowered to the surface of the sample. Using these tubes we have found that — uL fills the receiver coils to 20 mm of length and maintains enough material in the top reservoir to avoid refluxing of the solvent into the upper tube areas.

Refluxing and subsequent capillary action can result in micro-bubbles forming in the receiver area. Bubbles are very disruptive to any resulting spectra as they form directly inside the receiver coils causing a drop of the lock level indicative of a disruption to the homogeneity of the magnetic field over several hours. For example, samples that are shipped for studies from sea level to higher altitudes often degas during or shortly after transportation. A simple way to check for bubbles or any other non-soluble contaminant without removing the sample from the magnet is by the observation of a distortion in the gradient profile Figure 3.

This can be repeated if necessary until all bubbles are removed, though some patience and care may be essential. When clear of bubbles, the plunger position can be readjusted and secured in place. Once 40 Ryan T. A sample yielding the gradient profile seen in B would be very difficult to shim to satisfactory levels and water suppression efforts would certainly suffer. The gradient profile of Figure 3A should confirm the result once temperature equilibrium has been re-established.

Tubes Many researchers are seduced into saving money by buying cheap NMR tubes. These tubes are not only inexpensive but also poorly manufactured with relatively high error tolerances that deliver inconsistent results. In the past, when liquid samples were routinely spun at 15—20 Hz, the quality and precision of the tube manufacturing was critical to reduce artefacts such as spinning side bands. Today most spectrometer users do not spin their samples as the use of pulsed field gradients for coherence selection prohibits the physical rotation of the sample.

This does not mean that consistent, high accuracy tube manufacturing is not important or that simple 1D-1H experiments that do not commonly use coherence transfer selection via gradients would not benefit from spinning. On the contrary, for reproducible results and reducing the need for shimming adjustments from sample to sample, high quality precision tubes are ideal. Research groups focusing on high throughput e.

Unfortunately these same projects are faced with high initial costs associated with large or massive numbers of NMR tubes that are subsequently either reused i. Our experience has been that cheaper tubes disrupt routine operation more often. Admittedly, we have used Wilmad P or equivalent for facility standard samples e.

The frequency of the internal deuterated solvent is continuously followed by the spectrometer hardware as a reference point, and a small magnetic field is applied to bring the reference frequency back to a preset static position. If the main magnetic field drifts far enough away from the set point, then the lock field generator will no longer be able to compensate, and the set point must be re-established in the new range. If external influences, such as large metal bodies, cryogen Dewars, temperature changes, vibrations, pressure changes, and so on, affect the magnet, then the lock circuitry must quickly compensate to maintain the position of the deuterium resonance.

Like any signal, the phase of the observed resonance can be critical in the ability to evaluate the actual frequency. Conversely, searching for the lock frequency when far off resonance would be difficult and time consuming regardless of the accuracy of the phase of measurement. We have found that it is very important to set the lock position and phase prior to acquiring high field and high-resolution data. Incorrect settings of the lock phase by even a few degrees can cause distortions originating from intense peaks, especially when gradients are utilized on cold probes.

The answer and solvent suppression method depends on the application. Initial thermal equilibrium, often based on the lock level stability, can be reached quickly taking as little as a few minutes e. Increasing the airflow to the variable temperature controller can speed this equilibrium process but unfortunately can easily introduce microphonics in the resulting spectra due to minute vibrations i.

This is of course deleterious, and a compromise between temperature stability and positional stability must be reached for each spectrometer. For cryogenically cooled probes this becomes 42 Ryan T. Increased airflows are often required to minimize temperature gradients throughout X-, Y-axis or along Z-axis the sample. For samples requiring runs of seconds or minutes, thermal equilibrium may be all that is required. This can be determined by the stability of the lineshape during shimming of the sample.

For long-term experiments, equilibrium of the refluxing solvent must also be reached. As the solvent refluxes and condenses higher in the tube the volume of the sample in the receiver coil is reduced. The sample is no longer centred in the receiver coils and the lineshape degrades. We have found that aqueous solvents take about 18 hours to reach equilibrium depending on the cleanliness of the tube. To accommodate this finding we often insert a sample in the magnet the night before experiments are due to begin. This assists with overall shim stability and solvent suppression. We have found that each spectrometer behaves differently: Each magnet, and perhaps even each probe, can have a distinct response to different shims.

While counterintuitive, the overall homogeneity and lineshape improves despite the reduction in the relative lock level more on this below. The adjustment is not nearly so pronounced on the 5 mm cold probe, but is still present and appropriate. Now whether this is a compensation for a lengthwise temperature gradient through the sample or an idiosyncrasy of our particular probe coil design is uncertain, but it is merely a fact and is performed almost without thought by experienced users on that particular instrument and always results in better lineshape.

None of the other magnets that we manage has this peculiarity or at least that has the same magnitude of lock drop. We hypothesize that the lock drop is due to deuterated lock solvent at the edges of the coil moving out of resonance as the field inside the receiver coil is improved. The overall amount of lock solvent on resonance therefore decreases even though the lineshape is improving for the solute. So why is it so important to learn the requirements for your system? The long and short of shimming is that the better the overall quality of shimming the narrower and more symmetric all resonances will be in the spectra.

This is always a desired aspect for any form of solvent suppression no matter how good the pulse sequence may be. There are of course many methods to shim based on iterative manual shimming, shimming to obtain the highest lock level, automatic shimming on the lock via a pre-programmed algorithm, Z-gradient-based autoshimming, full three-dimensional shimming using Z-gradients and the X and Y Recent Advances in Solvent Suppression for Solution NMR 43 shim coils capable of properly shimming a magnet from installation , or any combination of the previously mentioned techniques.

To minimize the time taken to shim, we have adopted a weekly routine on an ideal standard sample, and users are supplied an updated optimal shim set upon which to start their experiments. Users are strongly encouraged to use moderate to high quality NMR tubes with exactly uL of sample volume reproducing the volume used in the standard samples and to centre the NMR tube in the spinner carefully in relation to the receiver coils.

Users are often able to interactively shim on the resulting lineshape requiring only slight corrections to the Z1, Z2, X1, and Y1 shims. Spectroscopists can also utilize Z-gradient shimming that typically yields reasonable results. It is our experience that short samples of uL or less go vastly astray in the iterative portion of Z-gradient shimming with the Z5 and Z6 shims quickly moving out of range, seemingly overcompensating for one another. On normal or long samples, gradient shimming and subsequent interactive manual shimming of the spectra in an interactive manner usually requiring less than 5 min will result in acceptable lineshape and solvent suppression characteristics Figure 4.

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Conversely if a user ignores the facility recommendations e. For any type of high-throughput experimentation we cannot emphasize enough the utility and efficiency of having a set length of sample with established content and supplied routine shims , consistently centred via the sample spinner for delivery into a well-maintained magnet. Mo vector model considered in the rotating frame is one of the easiest and most dramatic optimizations that can be applied to an NMR sample.

The paper is therefore highly recommended. Presaturation 91 Hz B1 field was applied for 2. A total of four steady state pulses were used prior to the 32 acquired transients. This was the sample used for sequence comparisons. This requires controlled gating of the 1H amplifier while the receiver circuitry is open otherwise harm may befall the instrumentation.

The frequency of nutation in this case the magnitude of the observed splitting of the observed resonance is the B1 field strength and therefore one can easily determine the correct pulse width for the sample. Varian Inova require a pulse sequence utilizing explicit acquisition periods. Also note that the authors used a fold oversampling that appeared in their calculations, and users need to replace this with their own value if applicable.

Carrier position The last easily optimized parameter is the carrier position of the spectrometer. The carrier is often set using the lock frequency based on solvent tables , or after a single pulse read experiment. We have found that a simultaneous array of both the high power carrier position and lower power presaturation frequency if applicable on the actual sample works best.

As will be discussed later see Section 2. Summary Each high quality sample or group of similar samples needs to be carefully shimmed and locked. As a minimum set of optimizations the pulse width and carrier position should be accurately established. These simple parameters should provide the background for at least manageable suppression. More complicated sequences may require further optimizations that will be discussed in greater detail in the following sections.

Understanding water In biological systems the interaction of aqueous solvents with amino acids and polypeptide chains has been examined27 and even exploited28 for decades. Two brilliant papers were written by John Edsall and Hugh McKenzie, regarding how proteins might fold and thereby interact with water. Solvent in exchange While most researchers first experience NMR incorporating organic solvents such as deuterochloroform CDCl3 or deuterated-benzene C6D6 , relevant biomolecular NMR often requires an electrostatic solvent such as water.

Besides the hydrophobicity difference of organic solvents, one of the most obvious differences is the exchangeable nature of water. Since protons in H2O can easily exchange in a pH dependent manner,31,32 the complexity of residual solvent signal suppression vastly increases. Also the desire to avoid inadvertently suppressing exchangeable solute signals is evident. When the protons in water are actively suppressed e.

McKay solute, not only are the exchange sites themselves removed but also the immediate neighbours of the solute can show bleaching effects. The water peak is not only broad due to exchange but also depends on the stability and homogeneity of the main magnetic field Bo. Again we see the need for careful shimming of the sample once equilibrium has been reached Section 2.

Solvent in this portion of the sample experiences different homogeneity of the main field as well as reduced induced fields B1 from hard and soft pulses. All of this results in resonances skewed away from the central resonance frequency and experiencing little or no suppression more on this later. Radiation damping The entire utility of NMR is the ability to record the magnitude and phase information of precessing nuclei in a large hopefully static external field. Cumulative precession is digitized from a carefully nurtured voltage in the receiver circuitry. However, any oscillating signal will generate an electromagnetic field.

Radiation damping occurs when large coherent precessing resonances like the massive solvent signal produce a current in the hardware of the receiver coil such that an effective field BRD counter to the induced field B1 is generated. Water can relax on the order of seconds in the absence of radiation damping, but will only require — ms when radiation damping is in effect Equation 1.

One cannot rely on a single optimized calculation, based on off-resonance peaks e. The solvent relaxes not with the expected T1 time constant i. Room temperature probes typically have a Q of perhaps a few hundred units but cryogenically cooled probes can push this value to f or even upwards of 40 is possible though not applied in biomolecular probes. The increased rate of relaxation due to radiation dampening is immediately evident. We usually think of T2 relaxation leading to a reduction of the resulting vector magnitude in the XY-plane, followed by the longer T1 period with the zero net magnetization slowly climbing up the Z-axis to equilibrium.

Radiation damping results from an induced field BRD caused by the solvent spins. Errors in pulse lengths become common when one remembers that RD fights the initial pulse B1 e. Demagnetization field effects 2. Identifying the problem For over a decade several groups have identified small chemical shift changes during particular 2D experiments. The assumption of all NMR experiments is that the primary field of the magnet is stable during the course of the experiment, or at least that any changes are compensated for via the lock circuitry and associated field s applied to bring the lock signal back to a set point.

This assumption allows all calculations to be done on a non-time dependent Bo field. Malcolm Levitt published an excellent full review on this subject in The effect has arguably been attributed to other phenomena e. Bloch—Siegert shifts43 and intermolecular multiple-quantum cross peaks For example, it was originally hypothesized that multiple-quantum peaks could be generated between water and solute accounting for artefacts in multidimensional spectra.

We now know that demagnetization can be thought of as a field created by the presence of a large number of spins such as an aqueous solvent. The perturbation caused by such a large number of spins affects the magnetic field experienced by the remaining nuclei in solution. Every other resonance including residual solvent resonances proceeds under a slightly different magnetic field and their frequencies of 48 Ryan T.

McKay precession are correspondingly altered. This begs the question: A technique fully optimized on an organic solvent may fail on an aqueous sample for precisely this reason. Even presaturation, one of the simplest solvent suppression techniques depends strongly on the saturation pulse position. If water starts out resonating at one frequency but moves once suppression is initiated, then either the frequency of saturation must be changed during the saturation pulse to compensate, or a compromise intermediate position must be used that can be established by arraying the carrier frequency of the saturating pulse.

We believe that demagnetization has a much larger effect than has been previously considered or reported. While investigating what we thought to be hardware lock instability on our MHz spectrometer we discovered what we now suggest is a large demagnetization effect on cold probes. What we found was a noticeable change in lock levels during presaturation periods i. The change was occurring despite the sample being locked perfectly on resonance and the lock resonance carefully phased, shown in Figure 5A and B region iii.

Turning the lock compensation off Figure 5 iv resulted in large changes when the same presaturation pulses were used. The lock circuitry was not able to completely compensate for the field disturbance in the H2O sample as shown in Figure 5A. To confirm the demagnetization field hypothesis, we repeated the same experiments with presaturation pulses both on and off resonance for both samples off-resonance pulses had no effect , and then proceeded to repeat all tests on a MHz spectrometer equipped with a room temperature probe.

Finally we moved to a different MHz spectrometer located in a different building equipped with a 5 mm cold probe and repeated the experiments data not shown. The tests on the MHz spectrometers confirmed that the lock perturbations were proportional to the spectrometer magnetic field strength, the presence of a high Q cooledprobe, and the concentration of 1H in the sample. The marked intervals correlate to: There are only so many ways to distinguish one NMR signal from another e. Water is especially irritating due to exchange characteristics when compared with highly deuterated common organic solvents like dichloromethane CD2Cl2 or benzene C6D6.

McKay The spectroscopist requires a rapid technique that can isolate the solvent resonance s while causing little or no perturbation to the solvent or the quantum mechanical manipulation applied to the solute during the course of the experiment. We shall now briefly review a few of the more common methods for more detailed reviews, see References 2, 4—7. Methods in this category require multiple careful optimizations, and compromises must commonly be made in terms of overall suppression amplitude and bandwidth of effect. Refocusing of the solvent coherence must be avoided but is quite often overlooked.

The coherence pathway selection aspect applies most to heteronuclear systems.

The coherence order is selected without the need for phase cycling. Solvent resonances cannot meet the heteronuclear selection criteria and are drastically suppressed though not entirely removed. Coherence selection against solvent can be improved with moderate solvent suppression techniques e. Selective pulses Selective pulses, also called shaped or soft pulses, can be used to isolate the response of solvent resonances exploiting chemical shift differences.

Shaped pulses utilize altered phase, frequency, or amplitude or any combination thereof to mould the frequency response. Presaturation response is shaped by the extreme length several seconds of the pulse at relatively low power. Shaped pulses attempt to also achieve efficient selection, but in a far shorter period of time typically hundreds of microseconds to a few milliseconds.

Modern spectrometers and the accompanying software usually supply the user with a graphic interface for the design and selection of a wide range of shaped pulses optimized for excitation, decoupling, inversion, and refocusing Recent Advances in Solvent Suppression for Solution NMR 51 to name but a few. Combinations of shaped pulses resulting in the simultaneous selectivity of multiple resonances are possible and becoming common. The basic principle is to use a relatively long selective pulse to manipulate water.

Evaluation criteria for suppression It has always been desirable to have an analytical process for the evaluation of solvent suppression methods. One of the first attempts at quantitation was done by Peter Hore in his review of solvent suppression. Each solvent behaves differently in terms of relaxation, coupling, and chemical shift among other properties. In addition, each spectrometer has different characteristics and each user individually evaluates the quality of shimming to be used and the time available for optimizations.

This makes establishing a standard sample to work on and how any pulse sequence will be evaluated challenging to say the least. Residual solvent amplitude Residual solvent amplitude is perhaps the most obvious parameter when judging the effectiveness of a suppression sequence. The magnitude of the remaining solvent resonance s is easily identified in the spectra and can be judged in comparison to the solute present in the sample. While certainly not the only criterion, it is indeed important and most sequences realize the need to suppress solvents such as water by a factor of or to bring samples into dynamic range.

Suppression bandwidth This is another important criterion that is easily overlooked. When judging the pulse sequence simply by the value of remaining solvent signal, one forgets that there is often a price to be paid via the suppression range. Peaks that are close to or under the solvent resonance may be fully or partially suppressed by any selective technique.

The goal of all solvent suppression techniques of course is to be as effective as possible, in a region as narrow as possible, and not require too much time to execute. Pulse sequence length This leads us to the third criterion of sequence performance. HN backbone or side chain atoms , or can add an unacceptable amount of time to the acquisition. This can occur from individual pulse sequence lengths, or from the requirement of multiple acquisitions via extensive phase cycling and subsequent cancellation, to delays required for full relaxation before the next FID can be acquired.

An ideal suppression technique will therefore need to be powerful, selective, and quick, but are there additional characteristics? Artefacts Any technique can inadvertently introduce changes to the rest of the spectra. For example, baseline distortions are timing errors in the first few acquisition points. Another common problem involves phase changes linear and non-linear introduced throughout the spectrum both 1D and higher dimensionality that easily occur depending on the method used.

Homonuclear couplings develop during spin-echo sequences and are not easily suppressed interfering with quantitative analysis. A good suppression sequence must not create these problems or should attempt to compensate in some other way. Calibrations and tolerance Now we add one more level of complication. Not only will the sequence need the characteristics described above i. All NMR users, regardless of experience, would undoubtedly appreciate a pulse sequence that is easy to conceptualize, requires the least number of experimental parameters to be optimized, and yields simple reproducible results for efficient optimization.

If a pulse sequence takes hours to optimize e. Alternatively, a pulse sequence that gives only average suppression but requires little or no optimization will likely receive enthusiastic usage even though the overall performance may not meet that of other suppression choices. While neither extreme is likely we are often faced with several comparable choices and must evaluate the experimental needs and the robustness of the chosen suppression method s. Suppression techniques prior to or during acquisition are considered.

Suppression in the solid state67 and post-acquisition mathematical suppression techniques e. Selectivity is achieved by elongating the pulse so that an extremely narrow effective bandwidth ensues. Even the most modern spectrometers, with ever increasing RF pulse and field performance, cannot deliver identical signals for an indefinite period of time, and the inhomogeneity causes an eventual loss of coherence in the water signal. Since the selectivity is proportional to the pulse duration, one naturally considers increasing the pulse length hopefully resulting in perfectly selective solvent suppression.

While a wonderful idea, this can never be the result. Thinking about establishing equilibrium in the real spectral world, one can easily envision a situation where longer and longer pulses have an ever-decreasing improvement on the quality of suppression i. The efficacy of presaturation depends on the power delivered, the extent of pulse length, and the position at which this power is delivered to the sample.

A broad asymmetric peak makes the selection of the carrier position difficult since a compromise frequency must be selected to immediately saturate the greatest number of spins and then again during exchange with the other resonances. As the symmetry of the resonances in the sample improves most likely through shimming and the peaks become taller and narrower, the ideal presaturation position becomes evident.

Once the shims have been optimized yielding the narrowest peak possible and the best carrier position has been selected see Section 2. Radiation damping fights the induced saturation field requiring more power than initially surmised by simple extrapolation from higher power levels even on non-compressing amplifiers. Increasing the power yields better water suppression but a larger effective suppression band that reduces the intensity of neighbouring peaks.

Outlined Gaussian shapes indicate selective pulses while gradients are represented on their own horizontal line by filled shapes. For full phase cycle discussions please refer to the referenced manuscripts. However at higher fields equipped with a cold probe we did not find this modification to be entirely distinct from the original presat see Figure The first increment of the 2D is acquired while the delay increments, phase cycles, and quadrature detection related to the indirectly detected dimension are not used Exorcyle77 and other phase cycling is still performedg.

This sequence also seems to be the present method of choice for metabonomics studies. The solute would be observed at full intensity while the solvent would have little or no signal to contribute. This method is one of the few sequences that allows resonances to be observed in proteins close to or even under the water peak. Water then relaxes at a normal slow rate allowing the solute to reach equilibrium before the solvent. NMR crystallography , John A. Ripmeester and Roderick E. CrystEngComm 15 , Basic Principles and Practice , David C.

Ashbrook, and Stephen Wimperis Eds. Vol 89, Nb 7 ; Part 2: Principles and Applications , Vladimir I. Bakhmutov, CRC Press McDermott and Tatyana Polenova Eds. Wasylishen, and Melinda J. Physics and Biophysics Springer, Dordrecht Walstedt, Springer, Berlin Pascal, with animations by Jennie M. Google book Spin Dynamics: Levitt, Wiley, Chichester Jacobsen, Wiley, New Jersey Freitas, Elsevier, Amsterdam Applications in Medical and Pharmaceutical Sciences, Part 3: Bakhmutov, Wiley, Chichester Duer, Blackwell Publishing, Oxford Elliott Burnell and Cornelis A. Ernst Springer, Wien Smith, Pergamon, Amsterdam Pergamon Materials Series, Series editor:

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