The birth of X-ray crystallography is considered by many to be marked by the formulation of the law of constant angles by Nicolaus Steno in 1669 (Figure 1). Although Steno is well known for his numerous principles regarding all areas of life, this particular law dealing with geometric shapes and crystal lattices is familiar ground to all chemists. It simply states that the angles between corresponding faces on crystals are the same for all specimens of the same mineral. The significance of this for chemistry is that given this fact, crystalline solids will be easily identifiable once a database has been established. Much like solving a puzzle, crystal structures of heterogeneous compounds could be solved very methodically by comparison of chemical composition and their interactions.

Figure 1: Danish pioneer in both anatomy and geology Nicolas Steno (1638 – 1686).

Although Steno was given credit for the notion of crystallography, the man that provided the tools necessary to bring crystallography into the scientific arena was Wilhelm Roentgen (Figure 2), who in 1895 successfully pioneered a new form of photography, one that could allegedly penetrate through paper, wood, and human flesh; due to a lack of knowledge of the specific workings of this new discovery, the scientific community conveniently labeled the new particles X-rays. This event set off a chain reaction of experiments and studies, not all performed by physicists. Within one single month, medical doctors were using X-rays to pinpoint foreign objects such in the human body such as bullets and kidney stones (Figure 3).

Figure 2: German physicist Wilhelm Conrad Röentgen (1845 – 1923).

Figure 3: First public X-ray image ever produced. Pictured is the left hand of Anna Berthe Röentgen. The uncharacteristic bulge is her ring.

The credit for the actual discovery of X-ray diffraction goes to Max von Laue (Figure 4), to whom the Nobel Prize in physics in 1914 was awarded for the discovery of the diffraction of X-rays. Legend has it that the notion that eventually led to a Nobel prize was born in a garden in Munich, while von Laue was pondering the problem of passing waves of electromagnetic radiation through a specific crystalline arrangement of atoms. Because of the relatively large wavelength of visible light, von Laue was forced to turn his attention to another part of the electromagnetic spectrum, to where shorter wavelengths resided. Only a few decades earlier, Röentgen had publicly announced the discovery of X-rays, which supposedly had a wavelength shorter than that of visible light. Having this information, von Laue entrusted the task of performing the experimental work to two technicians, Walter Friedrich and Paul Knipping. The setup consisted of an X-ray source, which beamed radiation directly into a copper sulfate crystal housed in a lead box. Film was lined against the sides and back of the box, so as to capture the X-ray beam and its diffraction pattern. Development of the film showed a dark circle in the center of the film, surrounded by several extremely well defined circles, which had formed as a result of the diffraction of the X-ray beam by the ordered geometric arrangement of copper sulfate. Max von Laue then proceeded to work out the mathematical formulas involved in the observed diffraction pattern, for which he was awarded the Nobel Prize in physics in 1914.

Figure 4: German physicist Max Theodor Felix von Laue (1879 – 1960) won the Nobel Prize for discovery of the diffraction of X-rays by crystals.

The simplest definition of diffraction is the irregularities caused when waves encounter an object. Diffraction is a phenomenon that exists commonly in everyday activities, but is often disregarded and taken for granted. For example, when looking at the information side of a compact disc, a rainbow pattern will often appear when it catches light at a certain angle. This is caused by visible light striking the grooves of the disc, thus producing a rainbow effect (Figure 5), as interpreted by the observers' eyes. Another example is the formation of seemingly concentric rings around an astronomical object of significant luminosity when observed through clouds. The particles that make up the clouds diffract light from the astronomical object around its edges, causing the illusion of rings of light around the source. It is easy to forget that diffraction is a phenomenon that applies to all forms of waves, not just electromagnetic radiation. Due to the large variety of possible types of diffractions, many terms have been coined to differentiate between specific types. The most prevalent type of diffraction to X-ray crystallography is known as Bragg diffraction, which is defined as the scattering of waves from a crystalline structure.

Figure 5: The rainbow effects caused by visible light striking the grooves of a compact disc (CD).

Formulated by William Lawrence Bragg (Figure 6), the equation of Bragg's law relates wavelength to angle of incidence and lattice spacing, Equation 1, where n is a numeric constant known as the order of the diffracted beam, λ is the wavelength of the beam, d denotes the distance between lattice planes, and θ represents the angle of the diffracted wave. The conditions given by this equation must be fulfilled if diffraction is to occur.

(1)

Figure 6: Australian-born British physicist Sir William Lawrence Bragg (1890 – 1971).

Because of the nature of diffraction, waves will experience either constructive (Figure 7) or destructive (Figure 8) interference with other waves. In the same way, when an X-ray beam is diffracted off a crystal, the different parts of the diffracted beam will have seemingly stronger energy, while other parts will have seemed to lost energy. This is dependent mostly on the wavelength of the incident beam, and the spacing between crystal lattices of the sample. Information about the lattice structure is obtained by varying beam wavelengths, incident angles, and crystal orientation. Much like solving a puzzle, a three dimensional structure of the crystalline solid can be constructed by observing changes in data with variation of the aforementioned variables

Figure 7: Schematic representation of constructive interference.

Figure 8: Schematic representation of destructive interference.

At the heart of any XRD machine is the X-ray source. Modern day machines generally rely on copper metal as the element of choice for producing X-rays, although there are variations among different manufacturers. Because diffraction patterns are recorded over an extended period of time during sample analysis, it is very important that beam intensity remain constant throughout the entire analysis, or else faulty data will be procured. In light of this, even before an X-ray beam is generated, current must pass through a voltage regular, which will guarantee a steady stream of voltage to the X-ray source.

Another crucial component to the analysis of crystalline via X-rays is the detector. When XRD was first developed, film was the most commonly used method for recognizing diffraction patterns. The most obvious disadvantage to using film is the fact that it has to replaced every time a new specimen is introduced, making data collection a time consuming process. Furthermore, film can only be used once, leading to an increase in cost of operating diffraction analysis.

Since the origins of XRD, detection methods have progressed to the point where modern XRD machines are equipped with semiconductor detectors, which produce pulses proportional to the energy absorbed. With these modern detectors, there are two general ways in which a diffraction pattern may be obtained. The first is called continuous scan, and it is exactly what the name implies. The detector is set in a circular motion around the sample, while a beam of X-ray is constantly shot into the sample. Pulses of energy are plotted with respect to diffraction angle, which ensure all diffracted X-rays are recorded. The second and more widely used method is known as step scan. Step scanning bears similarity to continuous scan, except it is highly computerized and much more efficient. Instead of moving the detector in a circle around the entire sample, step scanning involves collecting data at one fixed angle at a time, thus the name. Within these detection parameters, the types of detectors can themselves be varied. A more common type of detector, known as the charge-coupled device (CCD) detector (Figure 9), can be found in many XRD machines, due to its fast data collection capability. A CCD detector is comprised of numerous radiation sensitive grids, each linked to sensors that measure changes in electromagnetic radiation. Another commonly seen type of detector is a simple scintillation counter (Figure 10), which counts the intensity of X-rays that it encounters as it moves along a rotation axis. A comparable analogy to the differences between the two detectors mentioned would be that the CCD detector is able to see in two dimensions, while scintillation counters are only able to see in one dimension.

Figure 9: Single crystal X-ray diffractometer with a CCD detector. The incident beam is generated and delivered through the silver apparatus on the right side of the sample, and the detector is the large black camera to the left of the sample.

Figure 10: Image of a powder X-ray diffractometer. The incident beam enters from the tube on the left, and the detector is housed in the black box on the right side of the machine. This particular XRD machine is capable of handling six samples at once, and is fully automated from sample to sample.

Aside from the above two components, there are many other variables involved in sample analysis by an XRD machine. As mentioned earlier, a steady incident beam is extremely important for good data collection. To further ensure this, there will often be what is known as a Soller slit found in many XRD machines. A Soller slit acts much like polarized sunglasses: it organizes random X-ray beams into a stack of neatly arranged waves parallel to the plane of rotation of the detector. Some machines have a Soller slit between the sample and the detector, which drastically reduces the amount of background noise, especially when analyzing iron samples with a copper X-ray source.

This single crystal XRD machine (Figure 9) features a cooling gas line, which allows the user to bring down the temperature of a sample considerably below room temperature. Doing so allows for the opportunities for studies performed where the sample is kept in a state of extremely low energy, negating a lot of vibrational motion that might interfere with consistent data collection of diffraction patterns. Furthermore, information can be collected on the effects of temperature on a crystal structure. Also seen in Figure 9 is the hook-shaped object located between the beam emitter and detector. It serves the purpose of blocking X-rays that were not diffracted from being seen by the detector, drastically reducing the amount of unnecessary noise that would otherwise obscure data analysis.

Over time, XRD analysis has evolved from a very narrow and specific field to something that encompasses a much wider branch of the scientific arena. In its early stages, XRD was (with the exception of the simplest structures) confined to single crystal analysis, as detection methods had not advanced to a point where more complicated procedures was able to be performed. After many years of discovery and refining, however, technology has progressed to where crystalline properties (structure) of solids can be gleaned directly from a powder sample, thus offering information for samples that cannot be obtained as a single crystal. One area in which this is particularly useful is pharmaceuticals, since many of the compounds studied are not available in single crystal form, only in a powder.

Even though single crystal diffraction and powder diffraction essentially generate the same data, due to the powdered nature of the latter sample, diffraction lines will often overlap and interfere with data collection. This is apparently especially when the diffraction angle 2θ is high; patterns that emerge will be almost to the point of unidentifiable, because of disruption of individual diffraction patterns. For this particular reason, a new approach to interpreting powder diffraction data has been created.

There are two main methods for interpreting diffraction data:

Another important aspect of being able to study compounds in powder form for the pharmaceutical researcher is the ability to identify structures in their natural state. A vast majority of drugs in this day and age are delivered through powdered form, either in the form of a pill or a capsule. Crystallization processes may often alter the chemical composition of the molecule (e.g., by the inclusion of solvent molecules), and thus marring the data if confined to single crystal analysis. Furthermore, when the sample is in powdered form, there are other variables that can be adjusted to see real-time effects on the molecule. Temperature, pressure, and humidity are all factors that can be changed in-situ to glean data on how a drug might respond to changes in those particular variables.