High Pressure Studies Using NMR Spectroscopy

760 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

with one of the major axes of a single crystal sample can be used to obtain information on lattice quality and the localization within the lattice of impurity atoms. A full discussion of channelling is beyond the scope of this article. PIXE (and to a certain extent RBS) are now more commonly used in the spatially-resolved mode. This is because their high yield contributes to the attainment of high spatial resolution.

List of symbols E = energy; Cz = concentration of element; m = particle mass; nT = number of target atoms; N = number of projectile atoms, number of photons; q = terminal charge; Q = total beam charge; V = voltage; Y = number of reaction products; Y(Z,E) = yield of interaction; z = charge; σ = crosssection; ε = detector efficiency; Ω = solid angle. See also: Atomic Absorption, Methods and Instrumentation; Atomic Absorption, Theory; Atomic

Emission, Methods and Instrumentation; Atomic Fluorescence, Methods and Instrumentation; Fluorescence and Emission Spectroscopy, Theory; Geology and Mineralogy, Applications of Atomic Spectroscopy; Inductively Coupled Plasma Mass Spectrometry, Methods; Proton Microprobe (Method and Background); X-Ray Emission Spectroscopy, Applications; X-Ray Emission Spectroscopy, Methods; X-Ray Fluorescence Spectrometers; X-Ray Spectroscopy, Theory.

Further reading Breese MBH, Jamieson DN and King PJC (1996) Materials Analysis Using a Nuclear Microprobe. New York: Wiley. Johansson SAE and Campbell JL (1988) PIXE, A Novel Technique for Elemental Analysis. Chichester: Wiley. Johansson SAE, Campbell JL and Malmqvist KG (1995) Particle-Induced X-ray Emission Spectrometry. New York: Wiley. Watt F and Grime GW (1987) Principles and Applications of High Energy Ion Microbeams. Bristol: Hilger.

High Pressure Studies Using NMR Spectroscopy Jiri Jonas, University of Illinois at Urbana-Champaign, IL, USA Copyright © 1999 Academic Press

Introduction The use of pressure as an experimental variable in NMR studies of chemical and biochemical systems leads to added complexity in instrumentation but the unique information gained from the combination of high pressure and NMR techniques justifies fully its use. There are several fundamental reasons for carrying out NMR experiments at high pressures. First, to separate the effects of density and temperature on various dynamic processes, one has to perform the measurements as a function of pressure. Second, for liquids the use of pressure enables one to extend the measurement range well above the normal boiling point and thus to study supercritical fluids. Third, as noncovalent interactions play a primary role in stabilization of biochemical systems, the use of pressure allows one to change, in a controlled way, the intermolecular interactions without the major perturbations produced by

MAGNETIC RESONANCE Applications

changes in temperature and/or chemical composition. Fourth, pressure affects chemical equilibria and reaction rates. The following standard equations define the reaction volume, ∆V and the activation volume, ∆V‡:

where K is the equilibrium constant, and k is the reaction rate. With the knowledge of ∆V‡ values one can draw conclusions about the nature of reaction and its mechanism. Fifth, the phase behaviour and dynamics of molecular solids and model membranes can be explored more completely by carrying out high pressure experiments. Sixth, the combination of advanced 2D, and 3D NMR techniques with high pressure capability represents a powerful new experimental tool in studies of protein folding.

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 761

The usual range of pressures used to investigate chemical and biochemical systems is from 0.1 MPa to 1 GPa (0.1 MPa = 1 bar; 1 GPa = 10 kbar); such pressures only change intermolecular distances and affect conformations but do not change covalent bond distances or bond angles.

Pressure effects on NMR spectra This heading is related to the fact that the effects of pressure and/or density on chemical shifts and spin– spin-coupling in molecular systems are relatively small and difficult to interpret. In contrast, most of the high pressure NMR studies of chemical or biochemical systems deal with pressure or density effects on dynamical behaviour of the readily compressible gases, molecular liquids and aqueous solution of biomolecules. Increasing pressure changes both the density and shear viscosity of the liquids which in turn changes the relaxation times and chemical exchange. The majority of NMR studies deal with liquids of low viscosity for which T1 = T2 and the condition of extreme motional narrowing applies. In the case of rotational motions of a spherical top molecule the following expression can be used to calculate the rotational correlation time, τR, from proton relaxation times

where γ is the gyromagnetic ratio for hydrogen and rij is the distance between protons. However, for proton-containing molecules one has to separate the intramolecular and intermolecular contributions to the observed proton relaxation times. In the case of nuclei with spin I > the relaxation studies provide direct information about rotational motions because the electric quadrupole interactions provide the dominant relaxation mechanisms. For nuclei with spin I = 1 (e.g. 2H, 14N) the relaxation rate due to the quadrupolar interaction is given by the well-known expression

where χ is the asymmetry parameter, e2qQ/ is 2π times the quadrupole coupling constant in hertz, and τR is the rotational correlation time. It is well established that the rotational correlation time, τR, for the isotopic rotation of a spherical top

molecule can be expressed in terms of the macroscopic viscosity, η, using the Debye equation

where η is the viscosity, a is the radius of the molecule and the other symbols have their usual meaning. In the case of the intermolecular contribution to relaxation times of proton-containing molecules and for the effect of pressure on self-diffusion one can use the Stokes–Einstein expression, which relates the shear viscosity, η, to the self-diffusion coefficient D:

where all symbols have their usual meaning. For non-hydrogen bonded liquids the increase of pressure slows down both rotational and translational motions with the result that for low viscosity liquids one shortens the spin–lattice relaxation times and decreases the diffusion. The slowing down of exchange owing to increased pressure in chemically exchanging systems also leads to changes in the NMR spectra. As discussed elsewhere in this encyclopedia, high resolution NMR spectroscopy is widely used to study chemical exchange processes. As an example of the effect of pressure on chemical exchange Figure 1 shows the pressure dependence of hindered rotation about the C–N amide bond in N,N-dimethyltrichloracetamide (DMTCA). From the observed line shapes it is straightforward to calculate the observed reaction rate and from its pressure dependence one can calculate the volume of activation ∆V‡. In the case of DMTCA the experimental rotation rate, k, decreases with increasing pressure and the correlation of the rates with measured viscosity (shear viscosity, η) shows that the hindered rotation in DMTCA falls in the strongly coupled diffusive regime. There is yet another important origin of observed pressure changes in the NMR spectra of biomolecules in aqueous solutions. NMR studies of protein denaturation using temperature or chemical means are well established but pressure also leads to reversible denaturation as discussed in more detail in the section on applications. In the case of model membranes the use of pressure leads to very rich barotropic behaviour and high pressure NMR techniques can establish pressure–temperature phase diagrams for the membrane system studied.

762 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

High pressure NMR instrumentation

Figure 1 Pressure effects on the line shape of the N-methyl proton NMR spectrum of DMTCA in pentane at 282.3 K. The dotted lines denote the experimental line shapes, and the full lines denote the calculated line shapes.

In addition to gases and liquids the NMR spectra of molecular solids exhibit sensitivity to pressure. The relaxation behaviour is changed and, in addition, pressure also leads to changes in phase-transition temperatures. The effect of pressure on the phase-transition temperature can be evaluated using the Clausius– Clapeyron equation:

Since high resolution NMR spectroscopy represents a spectroscopic technique of major importance for chemistry and biochemistry the overview of high pressure NMR instrumentation focuses on high resolution, high pressure NMR techniques. Thanks to advances in superconducting magnet technology one can achieve a high homogeneity of the magnetic field over the sample volume even without sample spinning and, therefore, it is possible to construct high resolution NMR probes for work at high pressures. The high resolution, high pressure NMR equipment consists of three main parts: (a) pressure generating and pressure measuring systems; (b) nonmagnetic high pressure vessel; and (c) a NMR probe that includes a sample cell. All components necessary for building a high pressure setup, such as hand pumps, high pressure tubing, and valve intensifiers are currently available from commercial sources. Figure 2 shows a schematic drawing of a high pressure generating system capable of producing a hydrostatic pressure up to 1 GPa. In addition to high pressure NMR instrumentation based on a high pressure vessel made from non-magnetic metallic alloy, one can also obtain high resolution NMR spectra using glass capillaries or special sample cells made from sapphire. Table 1, which gives characteristic performance features of high pressure NMR probes, also includes information on the diamond anvil cell (DAV) which is suitable for broad-line work up to pressures of 8 GPa. However, the extremely small sample size makes DAV probes difficult to operate and relatively insensitive. A schematic drawing of a high pressure NMR vessel, and a sample cell used for high resolution NMR experiments at pressures up to 950 MPa, is shown in Figure 3. It is important to note that even at pressures of 1 GPa and sample diameter of 8 mm one can achieve a resolution of 3 × 10–9. The natural abundance 13C NMR spectrum of 2-ethylhexylcyclohexanecarboxylate recorded at 80°C and 500 MPa as shown in Figure 4 serves as an illustrative example of the high quality NMR spectra which can be routinely obtained at high pressures.

Applications of high pressure NMR spectroscopy where T is the transition temperature, P is the pressure, ∆V is the molar volume change and ∆H is the change in molar enthalpy.

Dynamic structure of liquids

Theoretical and experimental evidence indicate that many physical properties of liquids may be

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 763

Figure 2

Table 1

a b c

Schematic diagram of the high pressure generating equipment.

Characteristic performance features of high pressure NMR probes

Probe type

Pressure range (MPa)

Temperature range (K)

Sample diameter (mm)

Resolution (× 10 –9)

Capillary cell

0.1–250

1–1.5

3

Sapphire cell

0.1–100

3.4

1.5

HP vessel

0.1–950

8

DAVb

0.1–8000

a a 253–373 200–300

3 c

<<1

1

H Frequency (MHz)

a a 60–500 1–100

Temperature and frequency range is determined by the commercial probe used. Diamond anvil cell. Broad line.

determined, to a large extent, by the size and shape of the constituent atoms or molecules. Owing to the close packing of molecules in liquids, even a small change in density can produce a considerable change in the molecular dynamics of the liquid; therefore, to test rigorously a theoretical model of a liquid, or a model of a specific dynamic process in a liquid, one must perform isochoric, isothermal, and isobaric experiments. Figure 5, which shows the temperature dependence of the self-diffusion coefficient, D, in liquid tetramethylsilane at constant density and constant pressure, illustrates the importance of separating the effects of density and temperature on molecular motions. Four main research directions can be identified in the high pressure NMR studies of liquids: (a) studies of self-diffusion; (b) investigations of reorientational motions; (c) studies of angular momentum behaviour; and (d) tests of

applicability of hydrodynamic equations at the molecular level. The effect of pressure on the anisotropic reorientation of acetonitrile-d3 in the liquid state can serve as in illustrative example of high pressure NMR studies of molecular reorientation in liquids. The deuteron and nitrogen spin–lattice relaxation times of acetonitrile-d3 have been measured as a function of pressure up to 200 MPa at 23°C, using the NMR T1 relaxation technique. Since the quadrupole coupling constants for nitrogen and deuterons in acetonitriled3 are known, the experimental T1 data are interpretable in terms of the rotational diffusion constants for motion perpendicular D⊥ and parallel D
to the symmetry axis. From Figure 6, which plots the pressure dependence of D⊥ and D
, it is readily apparent that ∆V‡(D⊥) >> ∆V‡(D
). This experimental finding

764 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

Figure 3

Schematic drawing of a high pressure NMR probe and sample cell.

reflects the difference in frictional torques connected with the reorientation about the individual molecular axis, or more generally stated, reflects the symmetry of intermolecular potentials. The low ∆V‡(D
) value indicates that the intermolecular potential energy is largely independent of the angle of orientation about the main symmetry axis and thus the rotational frictional coefficient is very small.

Supercritical fluids

Figure 4 High resolution natural abundance 13C NMR spectrum of liquid 2-ethylhexylcyclohexanecarboxylate at 80ºC and 500 MPa.

NMR techniques can be used to study supercritical fluids as the employed pressures range from 0.1 to 80 MPa. The reason for renewed interest in the properties of supercritical fluids can be traced to the great promise of supercritical fluid extraction techniques. High pressure NMR spectroscopy can not only be used to investigate transport and intermolecular interactions in compressed supercritical

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 765

Figure 5 Temperature dependence of self-diffusion in liquid tetramethylsilane (TMS) at (䊊) constant pressure and (∆) constant density.

fluids but an in situ NMR technique has also been developed for the determination of the solubility of solids in supercritical fluids. The basic idea behind the determination of the solubility of solids in supercritical fluids is based on the radically different spin–spin relaxation rate between T2, dissolved and solid material (T2, solid dissolved). Using the 90º–t–180º spin echo sequence with a pulse separation of t << 1 ms ensures that no contribution to the NMR echo signal can result from the quickly relaxing protons in the solid material. Figure 7 gives the experimental solubilities for solid naphthalene in supercritical carbon dioxide as determined by the spin echo NMR technique. The three isotherms measured were selected near the upper supercritical end point (UCEP) for naphthalene in CO2. In Figure 7 the 58.5°C isotherm shows a large increase in solubility at about 23.5 Mpa; the slope of the isotherm is near zero. This indicates that one operates in close proximity to UCEP. This NMR technique can yield both solubility data and phase information when studying equilibria in supercritical fluid mixtures. Phase transitions in molecular solids

Because molecular solids exhibit significant compressibility even in the pressure range 0.1 to 900 MPa one can use high pressure NMR relaxation techniques to investigate phase transitions. The high degree of molecular rational freedom in plastic crystals (orientationally disordered crystals)

Figure 6 Pressure efffects on the rotational diffusion constants D⊥ and D
of acetonitrile-d3 at 23 oC.

Figure 7 Experimental solubilities for solid naphthalene in supercritical CO2 expressed in moles of napthalene dissolved per litre of solution.

766 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

provides a good illustrative example. Adamantane, C10H16, is a saturated hydrocarbon with an fcc crystal structure in its high temperature plastic phase (α-phase) whereas at lower temperatures it undergoes a phase transition to a tetragonal structure (βphase) in which the rotation is quenched. Taking advantage of the abrupt change in T1 or T2 at the phase transition provides the means of detecting the precise phase-transition pressure. At the phase-transition pressure, the T1 of adamantane decreases abruptly by a factor of 40 in passing from the orientationally disordered plastic α-phase to the βphase. Figure 8 shows the pressure dependence of the proton spin–lattice relaxation times, T1, in solid adamantane in the α- and β-phases at different temperatures. The relaxation time can be expressed in terms of the rotational correlation time τ, and the

frequency units. For the measured T1 one can assume ω0τ >> 1 for the β-phase rotation. Then the expressions for the relaxation times are as follows:

where Cαω0 = 8.91 × 109 radian2 s–2 and 2Cβ/ω0 = 2.07 × 10–7. Assuming that rotational motion is a thermally activated process and that the second moments are independent of temperature and pressure within our experimental range, one can obtain the activation volume ∆V‡(T) at constant temperature from the pressure dependence of ln(T1):

and the activation enthaply, ∆H‡(P), at constant pressure: second moments (M2) where ω0 is the Larmor frequency (Eqn [1]). The constant C is proportional to the proton second moment expressed in angular

Figure 8 Pressure dependence of the proton spin–lattice relaxation times, T1, of solid adamantane in its two phases at different temperatures. The open symbols denote values obtained in the compression run whereas the filled symbols give values for the decompression run.

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 767

Figure 9 Normalized spin-echo amplitude as a function of time in polyethylene at 400 MPa and temperatures of 217 to 223°C.

where Equations [3] and [4] have a plus sign when ω0τ >> 1, and a minus sign when ω0τ << 1. The interesting feature of the experimental results given in Figure 8 is the hysteresis of the observed transition pressure for the compression α → β run and the decompression β → α run. Analysis of this hysteresis can provide valuable information about the nature of order–disorder transitions. Pressure effects on crystallization kinetics of polymers

NMR relaxation or line width measurements are sensitive to the degree of crystallinity in solid polymers, because the protons in the crystalline lattice experience strong dipole–dipole interactions which cause fast spin–spin relaxation and line broadening. As a result, line width and relaxation studies have been used to measure crystallinity in a broad range of polymers. The use of high pressure as an experimental variable in NMR studies of polymer crystallinity offers new details about polymer crystallization. The study of pressure and temperature effects on the

Table 2

Mechanistic studies of chemical reactions in solution

High pressure NMR studies of various chemical reactions provide information about the reaction mechanism. Almost all reactions exhibit a characteristic pressure dependence of the reaction rate which can be used to calculate the volume of activation, ∆V‡. The ∆V‡, extrapolated to ambient pressure, together with the partial molar volume for the reactants and products, or with the reaction volume

High pressure kinetic studies of solvent exchange in inorganic systems

System [Cu(DMF)6]2+, [Cu(H2O)6]2+ [Ln(PDTA)(H2O)2] (Ln = Tb, Dy, Er, Tm, Yb) [Er(EDTA)(H2O)2] –

[Ln(DTPA-BMA)(H2O)] (Ln = Nd, Eu, Tb, Dy, Ho)

a

kinetics of crystallization of linear polyethylene can be used as an illustrative example of this specific application of high pressure NMR techniques. One can determine the amount of amorphous polyethylene present from the initial maximum by spin-echo measurements 90°–τ–180°–τ–echo with τ equal to 300 µs. Figure 9, which plots the amplitude of the echo signal against time, shows the crystallization data at 400 MPa for polyethylene. Before each measurement the sample was equilibrated at the desired temperature and a pressure 75 MPa below the crystallization pressure. The very fast relaxation of the crystalline material prevented it from contributing to the echo intensity. The isotherms as depicted in Figure 9 show the characteristic crystallization behaviour: an induction time before crystallization, a primary crystallization, and a gradual decrease in crystallization rate as the final crystallinity is approached and secondary crystallization and perfection pressures occur. The quantitative analysis of the high pressure crystallization kinetics yields information about the formation of extended crystals, the value of the Avrami coefficient, the mechanism of extended chain crystallization, and the effect of pressure on the surface energies of the crystal nuclei. High pressure NMR experiments are interesting because of their direct sensitivity to polymer chain dynamics on the microscopic scale. In this way NMR complements techniques such as dilatometry which are sensitive to bulk properties.

–

Solvent

Pressure (bar)

Temperature (K)

Nuclei

DMF, H2O

2000

217–300

17

H2O

2000

272–292

H2O

2000

na

17

2000

278–332

14

2000

288–333

17

[M(en)3](CF3SO3)2 (M = Co, Fe, Mn)

en

[Cp*M(H2O)3]2+ (M = Rh, Ir)

H2O

Ethylenediamine.

O

17

a

O O N O

768 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

of the overall reaction, allows one to create a volume profile that describes the volume changes along the reaction coordinate. The location of the transition state with respect to reactants and products determines the mechanism of the reaction investigated. Both organic and inorganic reactions can be studied. Specifically, solvent and ligand exchange, and ligand substitution reactions in inorganic systems have extensively been studied by high pressure NMR techniques. Table 2 gives the activa-

Table 3

DPPCb DPPC-d62 DPPC-d62-TTC

a

c d

Biochemical applications Model membranes

Phospholipid bilayers and monolayers have important roles in nature as components of cell membranes

Examples of NMR studies of the pressure effects on model membranesa

System

b

tion parameters of solvent exchange studied in inorganic systems.

c

Experiment

Result

Natural abundance 13C,T1,T2

Phase transitions

2

Phase diagram; order parameter; pressure reversal of the anaesthetic effect of tetracaine

H line shapes

DPPC-TTCe

31

DPPC-d2 (2,2); (9,9); (13,13)

2

Order parameters; chain motions

DPPC-d62-cholesterol

2

Phase diagram

DPPC POPCd

1

Lateral diffusion

P line shapes, T1

H line shapes, T1, T2 H line shapes HT1 and T1ρ

Pressure range from 0.1 to 500 MPa. DPPC = dipalmitoylphosphatidylcholine. TTC = tetracaine. POPC = palmitoyloleylphosphatidylcholine.

Figure 10

Pressure–temperature phase diagram of DPPC-d62.

Structure and dynamics of the head group; phase diagram

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 769

and lipoproteins. In cell membranes, phospholipid bilayers constitute the permeability barriers between aqueous compartments. Aside from their structural interfacial roles, aggregated phospholipids modulate the functions of associated proteins and enzymes by

direct binding and by physical effects. Therefore, synthetic phospholipid bilayers, in multilamellar or small bilayer vesicle forms, have become models for the study of the structural and dynamic properties of natural phospholipid aggregates.

Figure 11 Histidine region of the 1H NMR spectra of RNase A in D2O at various pressures. The standard in the insets is sodium 3(trimethylsilyl) tetradeuteriopropionate (TSP).

770 HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY

Figure 12

Phase diagram of RNase A.

Since the 1970s, NMR methods, in particular 2H NMR, have been applied very effectively to investigate the biophysical properties of phospholipid bilayers. NMR experiments with high pressure and variable temperature measurements allow one to access diverse gel phases in phospholipid bilayers and to study the order and dynamics of phospholipids in bilayers as a function of volume changes. Table 3 gives illustrative examples of high pressure NMR studies of model membranes and indicates the type of information that can be obtained from these studies. Figure 10 shows the pressure–temperature phase diagram of deuterated dipalmitoylphosphatidylcholine, DPPC-d62, as determined by high pressure NMR techniques using direct measurement of the deuteron quadrupole splitting of the methyl group and first moment analysis. Pressure denaturation of proteins

The relationship between the amino acid sequence and the structure of the native conformation of proteins represents the key unsolved problem of biochemistry and biology. Temperature and chemical perturbation represent the usual approach used in studies of protein stability and the folding problem. However, it is advantageous to use pressure to study protein solutions as pressure perturbs the protein environment in a controlled way by changing

Figure 13 2D 1H Correlation spectroscopy spectrum with presaturation pulse for solvent suppression of 2 mM sucrose at 25ºC and 475 MPa pressure recorded at 500 MHz.

only intermolecular interactions. Moreover, by taking advantage of the phase behaviour of water, high pressure can lower the freezing point of an aqueous solution and allow the study of cold denaturation of proteins. The reversible pressure denaturation of bovine ribonuclease A (RNase A) can be used as an illustrative example for pressure denaturation. In this specific protein the ε1 hydrogen signals of the four histidine residues are well resolved from other proton peaks in the 1D 1H NMR spectrum of the native RNase A in D2O. These peaks have been assigned and used to investigate the folding and unfolding of the protein. Figure 11 shows the pressure effects on the proton NMR spectrum in the histidine region of RNase A at pH 2.0 and 10ºC. As expected with increasing pressure, the intensity of the native histidine peaks decreases and at about 400 MPa their disappearance indicates that the protein is denatured. The appearance of peaks D and D′ with increasing pressure signals the denaturation. It is important to point out that during the pressure-assisted cold denaturation many proteins show significant residual secondary structure which parallels the structure of folding intermediates. It appears that pressure denaturation and pressureassisted cold denaturation of proteins studied by high resolution NMR techniques provide novel information about the folding of proteins. In addition, these experiments allow the determination of phase diagrams for proteins. Figure 12 shows the

HIGH PRESSURE STUDIES USING NMR SPECTROSCOPY 771

pressure–temperature phase diagram for RNase. Above the curve the protein is in the denaturated state. The most promising direction of the high pressure studies of proteins is the application of advanced 2D and 3D NMR techniques. Figure 13 shows the high quality of 2D NMR spectra which can be routinely obtained at high pressures. Very often the 2D spectra obtained at high pressure offer new insights into the pressure-induced unfolding and pressure-induced dissociation of proteins when compared with results obtained by other high pressure spectroscopic techniques such as fluorescence methods.

List of symbols a = molecular radius; D = Self-diffusion coefficient; I = nuclear spin quantum number; k = rate constant; K = equilibrium constant; P = pressure; rij = distance between protons; R = gas constant; t = pulse separation time; T = absolute temperature; T1, T2 = relaxation times; ∆V = reaction volume; ∆V‡ = activation volume; γ = gyromagnetic ratio; η = shear viscosity; χ = asymmetry parameter; ω0 = Larmor frequency. See also: Biofluids Studied By NMR; Diffusion Studied Using NMR Spectroscopy; High Pressure Studies Using NMR Spectroscopy; Labelling Studies in Biochemistry Using NMR; Membranes Studied By NMR Spectroscopy; NMR Relaxation Rates; Proteins Studied Using NMR Spectroscopy; Relaxometers; Two-Dimensional NMR, Methods.

Further reading Benedek GB (1963) Magnetic Resonance at High Pressure. New York: Interscience Publishers. Jonas J (1973) NMR studies in liquids at high pressure. Advances in Magnetic Resonance 6: 73–139. Jonas J (1975) Nuclear magnetic resonance at high pressure. Annual Reviews in Physical Chemistry 26: 167– 190. Jonas J (1978) Magnetic resonance spectroscopy at high pressure. In: Kelm H (ed) Proceedings NATO ASI, Series C, High Pressure Chemistry, pp 65–110. Dordrecht: Reidel. Jonas J (1982) Nuclear magnetic resonance at high pressure. Science 216: 1179–1184. Jonas J (1991) High pressure NMR studies of the dynamics in liquids and complex systems In: Jonas J (ed) High Pressure NMR, pp 85–128. Vol 24 in Monographs NMR Basic Principles and Progress. Heidelberg: Springer-Verlag. Jonas J (1993) High pressure NMR studies of chemical and biochemical systems. Proceedings NATO ASI, Series C, High Pressure Chemistry, Biochemistry and Materials Science, 401: 393–441. Dordrecht: Reidel. Jonas J and Jonas A (1994) High pressure NMR spectroscopy of proteins and membranes. Annual Reviews in Biophysical and Biomolecular Structure 23: 287–318. Jonas J and Lamb DM (1987) Transport and intermolecular interactions in compressed supercritical fluids. In: Squires TE and Paulaitis ME (eds) Supercritical Fluids Chemical and Engineering Principles and Applications, pp 15–28. Vol 329 in ACS Symposium Series. Washington DC: American Chemical Society. van Eldik R (1987) High pressure studies of inorganic reaction mechanisms. In: Proceedings NATO ASI 197: 333–356.