DOSY processing

DOSY experiments can resolve signals from the individual components of a mixture of compounds without physically separating the compounds. This is achieved by using the different rates that molecules diffuse through solution to separate the signals. DOSY data are typically presented as a multi-dimensional NMR spectrum where one of the dimensions corresponds to the diffusion rate. Unlike the frequency dimensions, the diffusion data is not processed with a fourier transform. Instead, non-linear curve fitting is used to extract rates from which a pseudo spectrum is created.

The chemical shift of chloroform

Chloroform is one of the most commonly used solvents in the NMR Facility and many users use the residual solvent peak to reference their spectra. Unfortunately, I have seen a range of values for the chemical shift of chloroform. Its possible that chloroform's chemical shift is temperature dependent and the different values were obtained at different temperatures. To test this, and to determine the correct chemical shift to use, I decided to measure the temperature dependence of the residual chloroform peak in deuterochloroform.

COSY experiments

The COSY experiment was one of the very first 2D NMR experiments developed and still remains an essential tool. The original experiment has several shortcomings, however, and numerous modifications have been proposed. Three of the most useful are the gCOSY, the DQF-COSY, and the CLIP-COSY. This post discusses the problems of the original experiment and how the new versions attempt to solve them.

Probe sensitivity specifications

Despite my best attempts to convince users otherwise, many still want to run 1D ¹³C spectra. Because of the poor sensitivity of ¹³C this is a time consuming, and thus costly, experiment. Sensitivity is mainly determined by the strength of the magnet and the type of probe being used. Since we are currently considering acquiring new equipment, I was curious to see how different probes and fields would impact the time taken to record ¹³C spectra and so collected sensitivity specs for a variety of probes.

T1 noise

T₁ noise is streaks found in the indirect dimension(s) of multidimensional spectra. It is normally found around the most intense peaks in a spectrum and can obscure crosspeaks. How t₁ noise arises, and what can be done to reduce it, are discussed below.

Protein structure determination

Many people are aware that NMR can be used to determine peptide or protein structures, but they are not so clear on what the process involves. Protein NMR structure determination relies on the measurement of nuclear Overhauser enhancements, or NOEs, whose intensity depends on the distance between two nuclei. With a large number of NOEs, a computational model of the protein structure can be twisted and pulled until the distances between the atoms are consistent with the intensity of the NOEs. Sounds straightforward right? Read on for all the details and complications.

Binding: Metal ions

In addition to monitoring small molecule binding and protein-protein interactions, NMR has a long history in being used to study the binding of biologically important metal ions. Nature uses metals structurally and to provide chemical functionality and NMR can be used not only to demonstrate that metals are bound, but also to identify the binding site and define its geometry.

Binding: Saturation Transfer Difference

One of the strengths of NMR is the variety of methods that have been developed to understand different aspects of molecular interactions. The previous post discussed the waterLOGSY experiment, which identifies small molecules that bind to larger molecules. The saturation transfer difference experiment is another method that identifies molecules that bind another, but it relies on a different mechanism than the waterLOGSY experiment and so is often useful in different situations.

Binding: the waterLOGSY experiment

NMR is one of the best methods for studying molecular interactions. To identify small molecules that bind to macromolecules many different experiments can be used, one of which is the waterLOGSY experiment. This is a 1D, ¹H-detected experiment that can be run in five minutes and is best suited for weakly interacting complexes (mM K_Ds), a binding regime that is difficult to probe by other techniques. To demonstrate how it works I will show spectra of a mixture of L-tryptophan and D-sucrose in the presence and absence of bovine serum albumin.

Indirect referencing

Most chemists use the residual protonated solvent peak to reference their spectra, but in two dimensional heteronuclear spectra, like HMBCs, the residual solvent peak is often distorted or not present at all. The ¹H dimension of such experiments can usually be referenced by aligning it to a 1D ¹H spectrum. The heteronuclear dimension, however, is more difficult to reference correctly. The best way to reference such spectra is to use indirect referencing to calculate the chemical shifts based on the frequency ratio of the nuclei involved.

Temperature calibration

Temperature is an often overlooked parameter when recording NMR spectra. Temperature directly affects molecular dynamics so changing the temperature can change the appearance of an NMR spectrum. Peaks, particularly those of exchangeable protons, move as the temperature is altered. Interconversion of conformers can be slowed or increased with temperature so that peaks can separate or coalesce. This allows determination of thermodynamic parameters, as long as the temperature can be accurately measured. All modern NMR spectrometers provide temperature control, but the systems need to be calibrated.