NMR Partial Alignment 2 - Preparing Liquid Crystal Samples

In this series on partially alignment samples for solution NMR, we explore the theory, methodology and analysis of residual dipolar couplings (RDCs) and residual anisotropic chemical shifts (RACSs) for the refinement of molecular structures and dynamics.

Liquid crystals for partial alignment may produce highly (macroscopically) viscous samples, which are a challenge to prepare homogeneously. In this post, we investigate general considerations in preparing liquid crystal samples.

Preparing Liquid Crystal Samples

Multiple alignment media are available, including Pf1 bacteriophage, d(GpG) dinucleotide, pinacyanol acetate (PNA), squalamine, bicelle liquid crystals and distorted acrylamide gels.

Detailed protocols for preparing samples in PNA and stretch acrylamide gels are available. For many liquid crystals, sample preparation typically involves dissolving the crystalline powder in solution, mixing thoroughly and measuring the sample in the NMR tube.

A few general considerations for preparing samples:

  1. Bubbles. Bubbles in samples present regions with a different dielectric constant, which are difficult to shim. For the best line shapes and shimming, bubbles are removed by spinning NMR samples in a hand centrifuge. A hand centrifuge is recommended since many liquid crystalline samples are macroscopically viscous and may have a viscosity comparable to glycerol.

  2. Macroscopic viscosity and microscopic viscosity. Liquid crystal samples may be macroscopically viscous–yet remain microscopically non-viscous such that molecules in solution tumble rapidly and produce high-resolution spectra. Viscous solutions may be difficult to pipet and can be transferred to an NMR tube by cutting a funnel from a pipet tip and centrifuging the solution to the NMR tube.

  3. Inhomogeneity. The viscosity of samples may produce regions of the sample with different concentrations of liquid crystal. Samples should be thoroughly mixed, possibly by vortexing. The homogeneity of the liquid crystal can be monitored from the ²H splitting of ²H₂O in the sample.

  4. Changes in the degree of alignment. The degree of alignment and the ²H₂O RQC may change once the sample is placed in the magnet. It is worth waiting 30 minutes, once the sample is placed in the magnet, to see if the RQC has changed. A ²H 1D spectrum should be collected before every J-coupled experiment as well. In favorable cases, the RDCs can be scaled by changes in the RQC of the liquid crystal sample.

In the following post, we discuss the collection of ²H spectra and shimming to assess the homogeneity of the liquid crystalline sample.

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