RRC Reporter

The Newsletter of the Research Resources Center

This is the first in a series of articles about analytical techniques available within the Research Resources Center at the University of Illinois at Chicago. This issue deals with nuclear magnetic resonance spectroscopy. At the end of the newsletter we include announcements and some issues of concern to a broader readership.

Nuclear Magnetic Resonance Spectroscopy

All types of spectroscopy require the interaction of matter with electromagnetic radiation. The highest energy (shortest wavelength) radiation is cosmic radiation. Energy then decreases through X-ray, ultraviolet, visible, infrared, microwave and radio frequency (RF) radiation. This article is about Nuclear Magnetic Resonance (NMR) spectroscopy, which operates in the lowest energy of these frequencies. Subsequent articles will discuss other analytical techniques that use higher energy irradiation.

Both NMR Spectroscopy and Magnetic Resonance Imaging are based on the same underlying measurement, as shall be discussed below. It is ironic that the word "nuclear" was dropped from Nuclear Magnetic Resonance Imaging (now called MRI) due to fears this word would frighten the general public that the technique involved ionizing radiation. Nuclei that can be detected by this technique tend to be the isotopes that are NOT radioactive, and the technique's sensitivity is limited by the low energy of the transition it measures. In fact, NMR spectrometers have to be designed to protect them from local radio stations, which use the same RF frequencies.

NMR detects atomic nuclei by placing them inside a strong magnetic field and exciting them with Radio Frequency (RF) waves. The nuclei then are observed as they "relax" from this excited state. Since these nuclei are part of atoms that make up molecules, NMR can provide specific information about molecular structure and interactions within or between molecules.

Whether the nucleus of an atomic isotope has a nuclear "spin" depends on the number of protons and neutrons it contains. Nuclei that have nuclear spin (also referred to as a magnetic moment) will align themselves when placed into a magnetic field. In the simplest case (which turns out to be the most important for most organic and biologically important compounds), the nuclei can align themselves in only two energy levels corresponding to nuclei pointing 'toward' or 'against' the direction of the magnetic field. The energy difference between the two magnetic alignments of the nucleus is so small that thermodynamic redistribution causes the two energy levels to be almost equally populated. The lower energy state has only about one-in-a-million more nuclei than the higher energy state. Since NMR detects only the nuclei that switch between these two energy levels during the measurement, it tends to be the least sensitive of the spectroscopic techniques.

Quantum mechanics indicates that if a particle is moving with a periodic motion (oscillating), then the particle can absorb electromagnetic radiation if the frequency of that motion exactly matches the frequency of the radiation. This is called resonance.

The frequency at which an atomic nucleus with a nuclear spin will interact with RF irradiation is given by the Larmor equation:

in which 'w' is the frequency of resonance of the nucleus (known as the Larmor frequency). The magnetogyric ratio, 'y', is a constant that is characteristic of each atomic nucleus (eg., H-1, C-13, P-31, etc.). 'B' is the strength of the magnetic field into which the sample is placed to make the measurement. The frequency and magnetic field are measured in millions-of-Hertz (MHz) and Gauss, respectively.

The Larmor equation provides a wealth of information for both NMR and MRI. For example, if one observes only one kind of nucleus (i.e., such as the protons in water), then there is only one value of 'y' and the frequency of resonance, 'w' depends only on the value of 'B'  If one varies the magnetic field strength over the volume of the sample in a known manner, then the frequency of resonance becomes an indicator of the location within the sample. This is the underlying principle upon which MRI is based. At steeper magnetic field gradients, the spatial resolution becomes greater and permits micro-MRI (i.e., NMR Microscopy). On the other hand, if the magnetic field is made to be very uniform (i.e., the value of 'B' is kept constant), then the frequency of resonance depends on the value of 'y' and provides spectroscopic information about which elements the sample contains. Furthermore, since the motion of electrons in molecular orbitals will alter the magnetic field actually felt by nuclei in the various functional groups of a molecule, a spectrum of frequencies will be observed in nuclear magnetic resonance of a compound or mixture of compounds. With modern NMR instrumentation, it is possible to do both spectroscopy and imaging. The Bruker 500 MHz spectrometer in the RRC, for example, could accept auxiliary imaging equipment that would enable it to image small samples.

The simplest NMR experiment consists of placing the sample in the magnet and then turning on a high powered RF transmitter for a burst of energy lasting microseconds. This burst causes a rapid exchange of the nuclei's state such that the levels now have equal populations. A short time after the transmitter shuts off, an extremely sensitive RF receiver turns on. After the burst, the nuclei return to their former equilibrium populations having slightly different numbers. The instrument records the frequencies that are picked up from the sample, converts them from an analog signal into a digital signal and stores a plot of amplitude vs time. This plot of the signal amplitude decreasing with time is called a Free Induction Decay (FID). Via a Fourier transform, the FID is mathematically converted into a plot of signal amplitude versus frequency. The amplitude versus frequency plot is the NMR spectrum which is then analyzed.

The spectrum usually contains many signals occurring at different frequencies. Each peak has an area that can represent the relative amount of that type of signal and a frequency that is related to the electronic environment around each nucleus. The electronic environment is indicative of the chemical structure surrounding each nucleus. Knowing the area of the signals and the frequency at which they occur provides quantitative and direct structural information about the compound of interest.

The Larmor equation points out a difficulty in reporting the positions of peaks in an NMR spectrum. The frequency of resonance depends on the strength of the magnetic field, B. For example, with commercial NMR instruments hydrogen (H-1) resonates over the range from 64 MHz with UIC's MRIs to 750 MHz for very high field NMR spectrometers. In order to be able to compare spectra from different NMR spectrometers, a dimensionless unit that normalizes the spectral frequency range to a 'unit' magnetic field is needed. The Parts Per Million unit (ppm) was defined as the frequency range of a spectrum from a known reference compound divided by the MHz operating frequency of the spectrometer (times one million to make the numbers ppm). A resonance from a given sample will occur at the same ppm value in the spectrum regardless of the strength of the magnet used. For example, a proton signal at 1.4 ppm would resonate 420 Hz from the reference in a 300 MHz spectrometer, but 700 Hz from the same reference compound in a 500 MHz instrument. The ppm scale in NMR does not refer to units of concentration.

In general, compounds that are liquid or fully dissolved in solution yield much better spectral resolution and signal-to-noise than solid or tissue samples. The most common NMR experiments have the compound of interest dissolved in deuterated solvents such as chloroform, water, pyridine or benzene. The deuterated solvent provides a reference signal that "locks" the spectrometer to this NMR signal during the experiment while also diminishing the signal from hydrogen nuclei in the solvent.

However, it is not obligatory to have fully dissolved samples in deuterated solvents. Perfused organs and tissue culture samples are commonly studied. These studies try to optimize the physiological aspect while maintaining the essential NMR requirements. Probes designed for perfused organs and large NMR tubes with superfusion lines, associated pumps and aeration chambers have been used in the RRC's NMRL magnets since 1976. The UIC was among the first to utilize NMR for in situ metabolic studies. NMR spectra also can be recorded with solid samples, but this will be the topic of a future report.

Numerous nuclei such as Hydrogen, H-1, and Carbon isotope 13, C-13, have magnetic moments and thus can come into magnetic resonance. Some of the other commonly studied nuclides (i.e., nuclei with magnetic moments) are: P-31, F-19, N-15, N-14, O-17, Si-29, B-10, B-11, H-2, Na-23, Cl-35 and Pt-195. Note that the isotopes most studied with NMR are NOT radioactive.

The sensitivity of an NMR experiment depends on:

1. Nuclide observed, with H-1  >  F-19  >  P-31  >  C-13
2. Natural abundance of that nuclide
3. Volume of the sample
4. Solubility of the compounds of interest.
5. Purity of compounds of interest.

Sometimes the low natural abundance of isotopes like C-13 or N-15 can be used advantageously by specifically labeling compounds with these nuclei as non-radioactive tracers in synthetic and metabolic studies. Since the natural occurrence of these nuclei is low, the fate of the tracer compound can be easily followed.

The volume of a sample depends on which instrument is used and ranges from 8 mL down to less than 0.2 mL. The following table provides a crude guideline for sample requirements in 1 mL solutions:

Hydrogen (H-1)	100 nanomoles	(0.1 mM )
Carbon (C-13)	1 micromole	(1 mM)
Phosphorus (P-31)	1 micromole	(1 mM)
Fluorine (F-19)	100 nanomoles	(0.1 mM)

Small molecules having a molecular weight of less than 1000 are the meat and potatoes of most NMR laboratories. Simple H-1, then C-13 information is acquired. Later, more complex pulse sequences can determine which of several conceivable structures is the actual one. 2D studies correlating the hydrogen and carbon NMR spectra are the major refinement in the past decade. From these experiments the 3-dimensional structure of isolated or synthesized compounds can be determined. A representative reference for biological small molecule experiments is:

E.J. Kennelly et al. "Abrusoside E, a Further Sweet-tasting Cycloartane Glycoside from the Leaves of Abrus Precatorius," Phytochemistry, 1996, 41, 1381.

Biological polymer experiments with proteins, polysaccharides or polynucleotides are usually limited to observing H-1 because only concentrations less than 1 millimolar are possible. The H-1 signals can be separated into those that are bonded to specific types of carbons, nitrogens or phosphorus groups. Although these studies typically involve days of acquisition time, the results can provide specific 3D structures.

S. Samarasinghe, A. Balasubramaniam and M. Johnson, "Proton Nuclear Magnetic Resonance Studies of the Structure of Neuropeptide Y and its Analogs," Current Medicinal Chemistry, 1997, 4, 151.

Membrane binding of ions
N. Hamasaki, et. al "Inhibition of Chloride Binding to the Anion Transport Site by Diethylpyrocarbonate Modification of Band 3," J. Membrane Biology, 1990, 116, 87.

pH measurement by P-31 NMR
R.J. Labotka and R.A. Kleps, "Phosphate-Analogue Probe of Red Cell pH Using Phosphorus-31 Nuclear Magnetic Resonance," Biochemistry, 1983, 22, 6089.

Energy metabolism changes in perfused rat hearts.
A. Omachi, et. al. "Inhibition of the calcium paradox in isolated rat hearts by high perfusate sucrose concentrations," American Physiological Society, 1994, H1729.

C-13 metabolic studies in perfused rat liver.
K.J.M. Liu et.al. "Alanine utilization for gluconeogenesis in Cancer: a C-13 NMR study," Surgical Forum, XL, 1989, 11.

The NMR phenomenon is on the same time scale as room temperature molecular motion, thus NMR can capture information about molecular conformations and chemical exchanges.

A molecule can be studied through many different nuclei. Thus, signals from different functional groups can be isolated and molecular linkages determined. The functional groups found to exist from H-1 spectra can be combined with the functional groups found through C-13, P-31, N-15 or F-19 NMR experiments to suggest a small number of possible structures, or often a unique molecular structure.

Often the linkages between functional groups can be determined, providing the researcher with unambiguous sections of the structure. The most likely attachment points between these sections can often provide the exact structure including its optical isomer.

Most other types of spectroscopy have their signals produced by all or most of the molecules in the observation volume. As indicated above, NMR is capable of detecting only about one in every million atomic nuclei and, thus, exhibits significantly lower sensitivity than other spectroscopic techniques. For small molecules in organic solvents, solubility is usually not a problem, but with large biological polymers it can be the limiting factor for experimentation.

NMR equipment is expensive compared to that of many other spectroscopic techniques. The spectral resolution is directly related to the field strength of the magnet, and this is related to the amount of money the investigator has to spend. To maintain the strong magnetic field, superconducting magnets require that the solenoid be immersed in liquid helium (4 degree K) and be further isolated from room temperature by a dewar of liquid nitrogen. This requires routine handling of cryogens. Pace makers, surgical implants, credit cards and mechanical watches should not be brought within 2 meters of the magnet. Pace makers could change their settings, implants could move, credit cards will lose their magnetic coding and mechanical watches may stop.

The RRC-West maintains 3 NMR spectrometers: 200, 360 and 500 MHz. All have been used to study live, biological samples. All spectrometers can transfer data from the instrument console via various networks to servers or local PCs for off-line NMR processing. The 500 MHz instrument was recently upgraded. This corrected problems with the magnet's stability and included the installation of a new console with modern NMR experiments and processing capabilities. In addition, new 400 and 500 MHz spectrometers are in the process of being installed in RRC-East. An announcement will be made in a future Reporter when this new laboratory is in operation.

The NMR Laboratory in RRC-West is available 24 hours a day and 365 days per year. Both user training and service are available for each instrument. For additional information contact: Rob Kleps at (312) 996-8550 or (312) 996-7600. To learn more about services available from the RRC visit our web site at: http://www.rrc.uic.edu/

Announcements

New Arrival
Dr. Mei Ling Chen joined the RRC EMF microscopy staff on Aug. 1 to operate and develop its Confocal Microscope services. She is a pathologist and was director of the confocal microscope facility at the U. of Nebraska Medical Center in Omaha. (x6-0543)

Additional Services
As of July 1, the staff and operations of the Protein Research Laboratory (PRL) were transferred to the RRC from the Department of Biochemistry. Its staff includes facility head Dr. Bao-Shiang "Bob" Lee, Dr. Shalini Gupta, and part time workers Tsu-Hua Chen and Dr. Ming-Yuan Zhou. (x6-1411)

Also, as of July 15, operations of the DNA Core Facility were transferred to the RRC from the Hematology/Oncology section in the Dept. of Medicine. Dr. Amittha Wickrema is the part time faculty manager and Sonia Lottinville does oligonucleotide sequencing and other services. (x3-9284)