Chemical crystallization

Single crystal X-ray diffraction (SCXRD) is an essential tool for analyzing 3D structures of small organic molecules, yielding direct atomic-level information on both the molecular and extended structure of a crystalline material e.g., absolute stereochemical assignment.

Diffraction methods are heavily reliant on the ability to grow high-quality crystals of the target molecules, which often becomes a bottleneck in the process and consumes large amounts of time and sample. As a result, the desire to screen more crystallization space with significantly less sample is driving new methods to deliver ever more data from both SCXRD and electron diffraction. 

Methods for crystallizing small organic molecules

Crystallisation Image 1

In the field of chemistry, classical methods of producing high-quality single crystals have largely evolved around time-consuming manual techniques. These methods involve slow growth solution phase crystallization based on evaporation, thermal control, or diffusion (either liquid-liquid or vapor). Such techniques suffer from inherent limitations, mostly centred around the relatively large amount of material consumed during sample preparation coupled to the significant amount of time required to produce a quality crystal. Additionally, these methods cannot give information on samples that are liquids at room temperatures. 

In recent years, a number of new methods have emerged to overcome these classical limitations and facilitate crystal production for SCXRD. These can be broadly arranged into two categories, namely “host-aided” and under-oil based approaches.  

Host-aided techniques include crystalline sponges, a porous metal organic framework (MOF) which encapsulates (guest) small molecules and promotes long range ordering and crystal formation. Advantages of this approach are its ability to work with both liquids and non-crystalline solids, coupled to the fact that it does not require crystallization of the analyte itself. Drawbacks are the lack of solid-state packing information and restrictions on the size of analyte that can be accommodated in the pores, making the selection of an appropriate host challenging. A second host-aided method uses a family of organic molecules with tetrahedral-like symmetry, known as Tetraaryladamantane (TAA), which co-crystalize with the guest analyte to form a chaperone. While this approach does not provide solid state packing information, it is compatible with liquid analytes and can be tailored to match the TAA to the guest target. 

Under-oil techniques can be divided into two types: a traditional micro-batch approach for water soluble organic salts, and an encapsulated nanodroplet method for organic soluble molecules. In the micro-batch method, exposure of the analyte to a wide variety of counter ions is set against control of evaporative loss using an oil layer. Crystalline forms of organic salts are of particular interest to the pharmaceutical industry for formulating of active pharmaceutical ingredients (APIs). Not only does this method require only a small amount of material (a few hundred milligrams), it also allows for screening a wide range of conditions providing not only structural information but crucial packing information not obtainable in sponge type experiments. However, as this approach is limited to water-soluble polar organic molecules, it leaves approximately 50% of possible APIs that are not water soluble. To address this concern, an encapsulated nanodroplet crystallization technique has been developed – ENaCt.

What is ENaCt?
Encapsulated Nanodroplet Crystallization of organic-soluble small molecules is a technique developed to allow automated high-throughput, low volume extensive screening of solution phase crystal growth conditions using a comparatively tiny amount (only a few milligrams) of substrate (analyte).

Dr Mike Probert gave a talk highlighting the impacts of technologies operating at the nano-scale as part of the Pint of Science festival. Specifically he breaks down the benefits of ENaCt within the realms of pharmaceutical development. Watch the full broadcast here.

Where can I access this technique as a service?

If you have a sample or project that you think might benefit from this crystallization technique you can access it as a service from the following providers:


Commercial Entities

Indicatrix >


Academic Users in the UK

UK National Crystallography Service >

Frequently asked questions

A very small volume (50 nL) of near saturated analyte, dissolved in an organic solvent is placed into a droplet of inert oil (typically 200 nL) which regulates the rate at which further sample concentration can occur by evaporation. Experiments are set up using a mosquito Xtal3 liquid handler in 96-well glass sandwich plates using a range of hydrocarbon, fluorinated and silicon based oils.

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  • Automated

  • Rapid

  • Screen hundreds of parallel experiments for fractions of the usual sample requirements

  • Reduce costs - up to 50,000 times less material per experiment than traditional methods

  • Exert a degree of control over the crystallization itself

  • Obtain crucial sold state packing information

  • Enhanced success rate allows more samples to be analyse
  • Perfect for a wide range of analytes which are solids under ambient condition
  • Glass SBS format plates make downstream optical screening (manual or automated) for crystals easy

Whilst ENaCt is still a fairly new technique, it has been successfully applied to an ever- broadening range of projects. These include but are not limited to: 

  • Screening for new polymorphs of APIs to understand the impact of physical properties on bioavailability1
  • Crystallizing organic salts soluble in organic solvents where traditional methods have failed e.g. bicyclic triazonium salts4
  • Elucidating structural information for isolated natural products, especially in cases where sample quantity is very limited e.g. hypocrellins5
  • Absolute stereochemical determination in medicinal chemistry e.g. alpha-ketoamide inhibitors to block viral replication in coronavirus main protease3

ENaCt Method (patent pending) 

1. Tyler AR, Ragbirsingh R, McMonagle CJ, Waddell PG, Heaps SE, Steed JW, Thaw P, Hall MJ, Probert MR. Encapsulated Nanodroplet Crystallization of Organic-Soluble Small Molecules.Chem. 2020, 6(7), 1755-1765.

Overview of Crystallization Techniques 

2. Metherall JP, Carroll RC, Coles SJ, Hall MJ, Probert MR. Advanced crystallisation methods for small organic molecules. Chem. Soc. Rev. 2023, 52(6), 1995-2010.

Applications of ENaCt 

3. Cooper MS, Zhang L, Ibrahim M, Zhang K, Sun X, Röske J, Göhl M, Brönstrup M, Cowell JK, Sauerhering L, Becker S, Vangeel L, Jochmans D, Neyts J, Rox K, Marsh GP, Maple HJ, Hilgenfeld R. Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease. J. Med. Chem. 2022, 65, 19, 13328–13342 

4. Zhu J, Moreno I,  Quinn P, Yufit DS, Song L, Young CM, Duan Z, Tyler AR, Waddell PG, Hall MJ, Probert MR, Smith AD, O’Donoghue AC. The Role of the Fused Ring in Bicyclic Triazolium Organocatalysts: Kinetic, X-ray and DFT Insights. J. Org. Chem. 2022, 87(6), 4241–4253 

5. Al Subeh ZY, Waldbusser AL, Raja HA, Pearce CJ, Ho KL, Hall MJ,  Probert MR, Oberlies NH, Hematian S. Structural Diversity of Perylenequinones is Driven by their Redox Behavior.J. Org. Chem. 2022, 87(5), 2697–2710 

6. Straker H, McMillian L, Mardiana L, Hebberd G, Watson E, Waddell PG, Probert MR, Hall MJ. Polymorph prediction through structural isomorphism and discovery of a new crystalline form of cannabidiol. Cryst. Eng. Comm. 2023, 25(16), 2479-2484.

High Pressure Crystallography 

7. Taylor CR, Mulvee MT, Perenyi DS, Probert MR, Day GM, Steed JW. Minimizing Polymorphic Risk through Cooperative Computational and Experimental Exploration.J. Am. Chem. Soc. 2020, 142(39), 16668–16680

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