32.18: X-ray Diffraction of Biological Samples

X-ray diffraction or XRD is an analytical tool that utilizes X-rays to study ordered structures such as crystalline organic and inorganic samples, polycrystalline materials, proteins, carbohydrates, and drugs.

According to Bragg's law, when X-rays strike the sample positioned on a stage, the rays are scattered by the electron clouds around the sample atoms. The X-ray diffraction or scattering is caused by constructive interference of the X-ray waves that reflect off the internal crystal planes. The scattering angle of the rays in specific directions by the atoms within the sample provides a diffraction pattern representing the electron density due to the atoms and bonds within the crystal. This information about the crystal lattice arrangement helps identify the material from its diffraction pattern, which resembles a fingerprint. The material can be identified by comparing this fingerprint with a database, such as the International Center for Diffraction Database (ICDD). XRD can look at the size, shape, and internal structure and help in the basic structural determination of a sample or unknown material.

For a single crystal, a typical diffraction pattern comprises spots that are 2D slices of 3-dimensional spheres. A computer program can integrate the resulting spots to determine the shape and intensity of the diffracted X-rays. Whereas, in a powder sample, the X-rays interact with many tiny crystals in random orientations. Hence, instead of spots, a circular diffraction pattern is observed. The intensities of the diffracted circles are plotted against the angles between the ring of the beam axis, giving a 2D plot known as a powder pattern. Amorphous or non-crystalline powders give a broad peak due to scattering from different directions compared to ordered scattering in crystalline powders, giving sharp concentric rings.

Certain precautions should be taken while studying biological samples using this technique. First of all, well-ordered crystals are a prerequisite for deciphering the protein structure through single-crystal XRD. Generally, single small crystals with well-defined faces, if they contain heavier atoms, will suffice and give a good diffraction pattern. For organic compounds, however, the crystals need to be larger. Without well-defined, viable crystals, this technique is not feasible. Some molecules are inherently more crystalline than others; thus, the difficulty of obtaining X-ray quality crystals can vary between compounds and is the main limiting factor of this technique. Further, the powder samples should be fine powder and have no clumps.

X-ray diffraction or XRD is a non-destructive analytical technique used to study the structural and spatial arrangement of atoms. It is commonly used to study biological samples such as protein crystals, nucleic acids, and ribosomes. It is also used to study crystalline powders of pharmaceutical formulations.

The XRD analyzer consists of a source of highly focused X-ray beams that strike the sample, like a crystalline solid.

These X-rays diffract, producing an interference pattern that is recorded by an electronic detector.

Early insights into the DNA double-helical structure were obtained using XRD, which shows the X-shaped diffraction pattern and other dimensional details of the DNA helix.

Single-crystal XRD is used in protein crystallography to determine its three-dimensional structure.

In powder XRD, a polycrystalline sample is seen as concentric rings. The phase and purity of the material can be determined by comparison with the database.