Household Dust (Bunnies)

Many people will be celebrating Easter this coming weekend. Children will be searching for Easter eggs left by the Easter Bunny. A word of caution though: Not all bunnies are cute!

Household dust bunnies seem to magically reproduce and grow. They must be collected regularly either with a broom or a vacuum cleaner and disposed of. What exactly is household dust? A bit of internet searching reveals that household dust is composed largely of fibers from clothing, dead human skin and the bodies of dust mites - a very disgusting mixture indeed. The top trace in the figure below is the ¹³C CPMAS NMR spectrum of a sample of household dust. The middle trace is a similar spectrum of clothing fibers from a sample of dryer lint. The difference spectrum (in the bottom trace) is consistent with a complex mixture of proteins and largely represents the spectrum of dead human skin and the bodies of dust mites.

Free and Inexpensive NMR Processing Software for Students

Processing NMR data has just become more affordable for students. Our friends at Advanced Chemistry Development have recently decided to make their NMR processing software free of charge to academics. As a student, you can put this software on your personal laptop or PC and process your NMR data at home or anywhere your travels take you. You can register and download the software here. They have even started a BLOG dealing with use of the software. I think I speak for all students when I say "Thanks guys!!"

Also, our friends at Bruker Biospin have recently introduced an inexpensive personal student license for their TOPSPIN software which can be purchased online.
There are other excellent NMR processing software options available. I mention specifically those above as the University of Ottawa currently holds network licenses for them and our students are most familiar with them.

Watergate vs Presaturation

Biochemists and protein chemists are often interested in observing the NH protons in their samples. Since the NH protons usually undergo slow chemical exchange with water, it is desirable to run the samples in H₂O rather than D₂O so the NH protons will not exchange with the deuterium in the solvent which would make them invisible in the ¹H NMR spectrum. In practice, a mixture of 10% D₂O and 90% H₂O is used as a solvent so that a deuterium lock can be established and used while running the spectrum. The very high concentration of water compared to the very low concentration of solute necessitates the use of solvent suppression methods.

Both presaturation and WATERGATE are efficient techniques used to suppress strong water signals from proton NMR spectra, however, there are differences between the two methods of which the user must be aware. Presaturation employs a selective, long, low power pulse to saturate the water resonance. This pulse is usually several seconds in duration during which exchange can occur between the unsaturated NH protons and the saturated water protons. If this exchange occurs, the NH protons become partially saturated to an extent related to the rate of chemical exchange between the NH and the water. The intensity of the NH protons in the spectrum is non-quantitative. WATERGATE, on the other hand, uses a pair of gradients surrounding a composite pulse which in effect inverts all but the water signal. The duration of the composite pulse is about 4 orders of magnitude shorter than a presaturation pulse, so exchange between the NH protons and the solvent occurs to a much lesser extent during the WATERGATE sequence compared to presaturation. As a result, the NH region is much less attenuated and more quantitative in a spectrum collected using WATERGATE compared to a similar spectrum run with presaturation. This is illustrated in the figure below which shows the NH region of the ¹H spectrum of a small disaccharide substituted peptide.One can see that some of the NH's are greatly attenuated in the spectrum acquired with presaturation compared to the spectrum run using WATERGATE. The more attenuated the signal, the faster the chemical exchange for that particular NH with water.

The WATERGATE suppression sequence is very similar to the gradient spin echo sequences used to measure diffusion constants and DOSY spectra. As a result, when WATERGATE suppression is used, one expects diffusion losses for small molecules (which diffuse quickly) compared to large molecules (which diffuse slowly). This effect is demonstrated in the figure below which shows the NH/aromatic region of the ¹H spectrum of a partially degraded ¹⁵N labelled 10 kDa protein.Here, one can see the same exchange losses pointed out in the previous figure for the presaturation spectrum compared to the WATERGATE spectrum. In addition, one can see that the intensity of a very sharp peak marked in yellow (likely due to a CH proton from free histidine) is less intense in the WATERGATE spectrum compared to the presaturation spectrum. This loss is due to the fast diffusion of the small free amino acid compared to the very large protein. In conclusion, one must be aware of the differences between the two solvent suppression methods if quantitative results are being sought.

I would like to thank Roger Tam from Robert Ben's Laboratory and Allison Sherratt from Natalie Goto's Laboratory for kindly providing the samples of the peptide and protein, respectively.

Fast 90 Degree Pulse Determination

Almost all NMR measurements rely on the correct calibration of 90° pulses. This is traditionally done by collecting a series of spectra as a function of pulse duration, finding a null for the 180° or 360° pulse and calculating the 90° pulse by simple division by 2 or 4 in the case of the 180° and 360° nulls, respectively. This determination, although trivial, can be very time consuming. Wu and Otting* have presented a much faster method of determining a 90° pulse based on measuring the nutation of a magnetization vector directly. Continuous nutation is depicted in the figure below. Here, the sample is subjected to continuous irradiation about the x axis. While being irradiated, the magnetization vector rotates in the z-y plane at a nutation frequency proportional to the pulse power. The magnetization on the -y axis is defined by a sine function. Fourier transformation of this magnetization gives an antiphase doublet centered at zero whose splitting Δν is twice the nutation frequency. The reciprocal of the nutation frequency is the time it takes the magnetization vector to rotate one complete cycle in the z-y plane and therefore the time it takes to rotate by one quarter of a cycle (i.e. the 90° pulse duration) is defined as 1/(2 Δν). The problem with continuous irradiation is that the sample must be irradiated at the same time magnetization is being detected. To eliminate this problem, a scheme similar to homonuclear decoupling is used where the radiation is turned off long enough to sample a data point. This is depicted in the figure below.Here each dwell period is divided up into a period for irradiation and a period for detection. The duty cycle for the irradiation is the fraction of time for which the sample is being irradiated. The magnetization is sampled when the power is off. As in the case for continuous irradiation, the magnetization vector still rotates in the z-y plane however, the rotation is slower as it is scaled according to the duty cycle. The duration of the 90° pulse is d/(2 Δν), where d is the duty cycle for irradiation. An example of this is shown in the figure below. The nutation spectrum was measured for HDO using a duty cycle, d = 0.10 and a power level of 12 dB (Bruker). Since the response of the amplifiers is linear, the 90° pulses at higher power levels can be calculated. Each decrease by 6 dB cuts the duration of the 90° pulse in half. In this case the 90° pulse at 0 dB was calculated to be 10.93 µsec at 0 dB based on the measured 90° pulse of 43.71 µsec at 12 dB. This pulse agrees to within a couple of percent of that measured by the more traditional method however, the measurement took only a few seconds. You can use a program called "pulsecal" on newer Bruker spectrometers to do this in complete automation.

--

* Peter S.C. Wu and Gottfried Otting J. Mag. Res. 176, 115 (2005).

Faster Relaxation Time Measurements in Solids

T₁ relaxation times are typically measured with the inversion recovery technique. In this method the magnetization is inverted and its recovery is monitored as a function of time. For nuclei with long T₁'s, the measurements are very time consuming as a recycle delay of at least five times T₁ must be used between scans. Typical T₁'s for ¹³C in the solid state range from several seconds to tens of minutes, so their direct measurement via the inversion recovery method could be prohibitively long.

High resolution ¹³C solid state NMR spectra of solids are routinely measured with cross polarization and magic angle spinning (CPMAS) in order to take advantage of the signal enhancement due to magnetization transfer between the abundant protons and isotopically dilute ¹³C nuclei. Additionally, the recycle delay needed for this measurement depends on the T_1ρ of the protons rather than the T₁ of the ¹³C. Proton T_1ρ's are typically shorter than ¹³C T₁'s by at least an order of magnitude, so many more scans can be collected per unit data collection time compared to a direct one-pulse measurement.

One might think that ¹³C T₁'s can simply be measured with cross polarization using a simple inversion recovery scheme by applying a 90° pulse to the ¹³C spins immediately following the contact pulse and then following their recovery over time. This method would have both the advantages of signal enhancement due to CP and more scans per unit time. The problem however, is that the enhanced magnetization of the inverted spins relaxes back to its unenhanced Boltzmann value and not its enhanced value. So, in order to measure the T₁, the direct ¹³C magnetization would have to be measured first (without CP) which would be very time consuming. This difficulty can be eliminated with the pulse sequence introduced by Torchia in 1978^* shown in the figure below.
This sequence uses a simple two step phase cycle to subtract out the effect of the direct ¹³C Boltzmann magnetization. The first part of the sequence uses a (90°_-y) pulse to return the CP enhanced magnetization to the z axis. The decay of the enhanced magnetization down to its Boltzmann value is followed using a (90°_x) pulse with detection of signals on the -y axis. The second part of the sequence uses a (90°_y) pulse to put the CP enhanced magnetization on the -z axis. The recovery of the enhanced inverted magnetization back to its equilibrium Boltzmann value is followed using a (90°_x) pulse with detection of signals on the y axis. The addition of the first and second parts of the experiment by way of the phase cycle allows for a simple calculation of the ¹³C T₁ with both the advantages of CP enhancement and the ability to collect more scans per unit time. An illustration of this method is shown in the figure below where the ¹³C T₁'s of glycine were measured. (The small peak in the spectrum is a spinning sideband of the carbonyl carbon)

* D.A. Torchia, J. Mag. Res. 30, 613, (1978).

NMR WIKI

An excellent resource for NMR users has been gaining more and more popularity on the web over the last few years. The brainchild of Evgeny Fadeev (Director of the Biomolecular Spectroscopy Facility at the University of California Irvine), NMR Wiki is an information sharing site offering a question and answer forum, job postings, a pulse sequence library, course material, history, information on meetings etc.... I think this is a fantastic place to learn more about NMR and Evgeny is to be commended for his efforts.

The Scale on an NMR Spectrum

Some people new to NMR spectroscopy have trouble with the meaning on the scales of their NMR spectra. Usually the data are plotted with a chemical shift scale (δ). This scale increases to the left and is usually reported in units of parts per million (ppm). The chemical shift of a resonance in a sample, δ_sample , in ppm is defined as follows:where ν_sample is the absolute frequency of the sample resonance and ν_reference is the absolute frequency of an agreed upon reference compound. For ¹H, ¹³C and ²⁹Si NMR, tetramethylsilane (TMS) is the agreed upon reference standard. When the scale is plotted in this manner, the peak positions are relative to that of the standard compound. This scale is particularly useful, as it independent of a single absolute frequency and therefore does not depend on the magnetic field strength, which varies from laboratory to laboratory. Spectra recorded using magnets of unequal field strength can be compared more easily.

Scales are also plotted in frequency units (Hz), usually with the reference compound assigned a value of 0 Hz. This scale also increases to the left and is usually of use when coupling constants (which are independent of field) are being measured.

Although absolute electronic shielding values are inconvenient to measure, one may hear people refer to one resonance being "more shielded" or "less shielded" than another, The shielding constant, σ, for a particular nucleus in a particular environment can be expressed by rearranging the Larmor equation.The shielding scale increases to the right.

In the age of continuous wave NMR spectrometers, NMR spectra were measured by irradiating the sample with continuous wave radiation and sweeping the magnetic field from a low value (on the left) to a high value (on the right). The scales were sometimes plotted in magnetic field units and one still hears about one peak being described as "upfield" or "downfield" from another. These terms have little relevance in FT NMR and should be avoided.

Some of the older literature reported ¹H NMR peak positions on a τ scale. Here, τ was equal to (10 ppm - δ ) and the scale increased to the right. This is no longer in use and has caused considerable confusion. It should be avoided.