Autoclave reactor synthesis of upconversion nanoparticles, unreported variables, and safety considerations

Introduction

In the context of chemical synthesis, autoclave reactors (sometimes simply referred to as “autoclaves”) are extremely strong enclosed metal vessels designed to contain reaction mixtures at elevated temperatures and pressures. Autoclave reactors serve as the “pot” in which many hydrothermal and solvothermal reactions occur. Autoclave reactors have the advantage that they can be heated with common laboratory equipment such as hotplates or ovens. There are a variety of autoclave reactors available on the market, which can accommodate reactions of different scales and single or multiple reaction vessels. Autoclave reactors are used in many aspects of chemistry and materials science, including the synthesis of many types of nano- and micromaterials^1,2, metal-organic frameworks¹, catalysis³, hydrogenation⁴, polymerisation⁵, materials testing⁶, digestion⁷, single-crystal casting⁸, and corrosion testing⁹. Autoclave reactors have been widely used as a method of synthesis for upconversion nanoparticles (UCNPs) and are, therefore, the focus of this perspective.

UCNPs are inorganic crystalline nanostructures consisting of a low-photon energy host lattice doped with photonically active trivalent lanthanide ions. There are many possible host lattices for UCNPs^10,11,12,13, with NaYF₄ being the most common¹⁴. The long-lived excited states of the lanthanide ions (typically ~100 µs to ~10 ms)¹⁵ enable multi-photon absorption and subsequent upconversion process, where multiple low-energy photons are absorbed and converted into a higher energy photon¹⁶. UCNPs can absorb and emit light at various wavelengths, primarily dependent on the choice of photoactive dopants hosted within the UCNP lattice structure. Sensitisers include Yb³⁺ ( ~ 976 nm), Nd³⁺ ( ~ 808 nm), and Er³⁺ ( ~ 1532 nm)^17,18. Er³⁺ and Tm³⁺ are common emissive activator ions, enabling emission from UV to visible at discrete “line-like” wavebands^19,20,21. UCNP properties can be further enhanced via incorporating core/shell architectures. Carefully designed and synthesised photonically active core/shell architecture can enable advanced multi-wavelength excitation, e.g., for display technologies, and enhance quantum yield^18,22,23. Core/shell architectures (e.g., layers of silica or the host lattice material) can also shield photoactive ions from solvent quenching that otherwise reduce UCNP emission²⁴. Typical quantum yields for UCNPs in non-polar solvents are ~0.01% to 0.1%^25,26. However, the greatest UCNP quantum yield to-date was 10.3% for core/shell hexagonal-phase NaYF₄:18%Yb³⁺,2%Er³⁺ for UCNPs in a dry form reported by Homann et al.²⁷. For comparison, the quantum yield of rhodamine 6 G is 95% in EtOH; whereas 25–75% is typical for quantum dots^28,29. Nevertheless, there are reports of bright luminescence from well-optimised UCNPs being visible to the naked eye^30,31. UCNPs offer several key advantages over other optically active materials, such as fluorescent dyes and quantum dots. Prominently, UCNPs do not photodegrade, photobleach, or blink^{32,33,34,35,36}. This makes UCNPs well suited to applications such as nanoscale temperature sensing³⁷, pressure sensing^38,39, transparent multicolour volumetric display technologies^18,40, as well as nanopatterned security inks^41,42,43. In biological environments, the diffuse NIR UCNP excitation can travel through several millimetres of blood¹⁷, and several centimetres of ex vivo tissue due to minimal absorption and scattering at NIR wave ranges^44,45,46. Further, NIR excitation does not induce visible autofluorescence, and has low phototoxicity⁴⁷. The combination of these optical properties makes UCNPs uniquely suited to all-optical reporting in life sciences applications. Beyond purely optical properties, dopants also enable multi-modal responses, for example, incorporation of Gd³⁺ and Dy³⁺ within or onto UCNPs induces a parametric response for magnetic resonance imaging (MRI) contrast⁴⁸ complimentary to x-ray computed tomography (CT)⁴⁹, and incorporation of isotopes such as Fluorine-18 within UCNPs enables positron electron tomography (PET) response⁵⁰.

Using autoclave reactors

Advantages and disadvantages of autoclave reactors in comparison to other UCNP synthesis approaches

Given the utility of UCNPs and the expense of purchasing UCNPs from commercial vendors (see Supplementary Section B for the cost of purchasing UCNPs), it is often necessary for researchers to synthesise UCNPs for their research projects. A variety of UCNP synthesis approaches have been developed, the major ones being microwave reactions, hot-injection reactions, polymer-assisted reactions, and autoclave reactor approaches. Each method has advantages and disadvantages when it comes to (a) equipment and expertise required, (b) range of materials that can be produced, and (c) scalability. For brevity, these methods are discussed in depth in supplementary material (Sections D-G). A comparative summary of equipment and skill requirements, costs, and capabilities for each UCNP synthesis method is provided in Table 1.

Table 1 Comparative summary of equipment needed for each UCNP synthesis method

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Use of an autoclave reactor enables hydrothermal and solvothermal synthesis of UCNPs at moderate costs in comparison to other approaches (see Table 1), and can give rise to a wide range of UCNP morphologies based upon various lattice materials (see Supplementary Figs. S4 and S5 for examples). In such reactions, the autoclave reactor acts to safely contain any pressure build-up arising from the gas pressure of the reaction mixture at elevated temperatures (typically 180 °C for standard autoclave reactors). For contextual information regarding the hydrothermal and solvothermal synthesis of oleic-acid capped UCNPs (OA-UCNPs) and polyethyleneimine capped UCNPs (PEI-UCNPs), the reader is referred to the case studies provided in Supplementary Sections H and I. However, there are some disadvantages to autoclave reactor synthesis. For example, control of the heating rate and cooling rate is generally limited, and many autoclave reactors do not accommodate internal temperature monitoring. Further, scaling up autoclave reactions requires adopting either a parallel synthesis approach (e.g., multiple small-capacity autoclave reactors) or a much larger autoclave reactor; both approaches involve considerable expense.

Given the temperature and pressure hazards, autoclave reactor synthesis requires (1) careful attention to safety (see sections “Autoclave design, safety, and operation”), (2) an appreciation of the related legal regulations (section “Legal considerations of autoclaves”), and (3) an understanding of the experimental variables which are often unreported in the literature (section “The unreported variables in autoclave synthesis”).

Autoclave design, safety, and operation

Safety is a major concern with pressurised autoclave reactors because an autoclave failure or erroneous opening under pressure can result in the release of large amounts of energy (i.e., an explosion). The hazards associated with autoclave reactor synthesis are compounded by: (a) the design of the autoclave reactor and (b) the type of reaction occurring and the resultant pressure build-up. In hydrothermal UCNP synthesis reactions approaching 100 °C, water will, of course, form steam, which generates an elevated pressure (we typically record 10 to 25 bars of pressure in such hydrothermal syntheses). In solvothermal reactions, UCNP synthesis will only generate excess pressure if heated to temperatures in excess of the boiling point of the solvents used, e.g., ethylene glycol [197 °C], oleic-acid [286 °C], and 1-octadecene (ODE) [178–179 °C]^51,52,53. Therefore at typical autoclave reactor synthesis temperatures (i.e., ~180–200 °C), solvothermal synthesis (assuming dry starting products) will not generate significant elevated pressures.

Autoclave reactor designs range from simple screw-thread systems to more sophisticated high-safety systems incorporating multiple redundant safety features. In general, autoclave reactors will include a liner (e.g., PTFE or suitable glass), to hold the reaction mixture. The simplest varieties of autoclaves may feature an over-pressure disk. However, if the autoclave reactor does not have a pressure gauge or internal temperature reading system, then one has to rely on the good judgement of the operator. Additionally, care must be taken with screw-thread systems to preserve the integrity of screw threads and to ensure there is no excess pressure within the autoclave reactor before it is opened. Therefore, simple autoclave reactors have higher risks of accidental failure and more advanced high-safety autoclave reactors are preferable.

Advanced high-safety autoclave reactor systems will feature engineering controls to help ensure safety. As a minimal example, the Berghoff DAB pressure vessels (see Fig. 1a) include a rupture disk to release pressure build-ups beyond design tolerances⁵⁴. Both the Berghoff DB series (not shown) and Asynt PressureSyn series (see Fig. 1b) offer high-safety reactors that use clamps instead of threads, pressure release valves, and over-pressure bursting disks, temperature probe ports, and pressure gauges^55,56. These features allow operators to identify and control potential pressure hazards during operation. Therefore, there is a lower risk of misuse.

**Fig. 1: Examples of autoclave reactor systems.**

Autoclaves are typically single-chamber apparatus, but it can be desirable to scale up synthesis via parallel batch production or simply larger autoclaves (e.g., autoclaves as large as 500 L are currently commercially available from manufacturers such as Büchiglasuster). Some manufacturers provide inserts to turn a single-chamber autoclave into a multi-chamber system, whilst others offer purpose-designed multi-reactor systems. Such multi-chamber reactors can speed up iterative synthesis.

Autoclave reactors can be heated by numerous means, including simple ovens, heating blocks, and advanced heating systems. Indeed, advanced heating systems may provide advantages with regard to space efficiency, temperature control, and sensing of the reaction parameters. The most advanced autoclave reactor systems also enable automated temperature data logging and feature active cooling to recover products faster than passive cooling will allow, thereby increasing potential synthesis rate, and enabling safety features such as automatic shut-down if nearing maximum limits. Advanced systems may also enable magnetic stirring, which typically cannot be accommodated in an oven. Notably, the most advanced systems available remove the need for manual handling, thereby preventing burn hazards and allowing maximum accessibility to all users—after all, autoclaves are made from solid metal (e.g., stainless steel) and are, therefore, cumbersome to handle. These features can help ensure safety and to enable reproducible synthesis.

To summarise: contemporary high-safety autoclave reactor systems offer benefits in terms of (1) safety, (2) accessibility, and (3) reaction monitoring. However, they are more expensive than (arguably unsafe) simple screw-thread autoclave reactors. Further, more sophisticated autoclave reactor systems with larger or multiple reaction chambers may also help reduce the time required to iterate synthesis towards optimisation or simply enable the generation of a greater quantity of desired product.

Some additional “last line of defence” measures above standard laboratory procedures may be considered for best safety practices when using autoclave reactors. (1) A form of secondary shielding around the autoclave reactor system (e.g., polycarbonate shields) to protect partially against accidental discharge or over-pressure. (2) Provision of hearing protection for operators in case of rupture of an over-pressure safety release valve.

The maximum heat and pressure a given autoclave reactor can safely sustain is primarily determined by the materials it is manufactured from; this is often stainless steel, with other options available on the market including alloys, glass, nickel, titanium, and zirconium options available for various temperature ranges, including temperature in excess of >200 °C. However, it is worth noting that PTFE liners are known to soften and deform if exposed to sufficiently high temperatures and pressurises arising from erroneous usage (see Fig. 1c). Instead, liners made of borosilicate glass may be better suited to high-temperature reactions.

Legal considerations of autoclaves

There are various regulations worldwide regarding the use of pressurised equipment. In the UK, employers have a legal duty to comply with the “Pressure Systems Safety Regulations 2000 (PSSR)” as part of the broader legal duties specified in the “Health and Safety at Work Act” (1974)^57,58,59. The PSSR regulation aims “to prevent serious injury from the hazard of stored energy as a result of the failure of a pressure system or one of it’s component parts”, and applies to any “compressed or liquefied gas, including air, at a pressure than greater than 0.5 above atmospheric pressure” and “pressurised hot water above 110 °C” ⁵⁷. There are some exceptions to the PSSR, which include (but are not limited to) pressure systems to be used for “weapons systems”, “vehicle tyres”, and “experimental research”. However, we note that most research is conducted at universities that have a duty of care to their students and staff, so we suggest that it would be good practice to abide by the PSSR when using autoclaves for nanomaterial synthesis research. In the European Union, the directive 2014/68/EU⁶⁰ governs the certification and testing of equipment pressurised to >0.5 bar⁶⁰. Guidance in other countries varies and cannot be comprehensively covered here. We encourage all users of autoclave reactors to (a) familiarise themselves with the legal requirements and guidance regarding autoclave reactors for their specific country and (b) to source autoclaves that are compliant with the highest international standards.

Autoclave reactors should only ever be purchased from reputable scientific suppliers who pre-test and certify their autoclave reactors to governmental standards. Examples of reputable manufacturers include (but may not be limited to): Berghof GmBH (Germany), Büchiglasuster (Switzerland), Asynt Ltd (United Kingdom), LBBC Baskerville Ltd (United Kingdom), Mettler Toledo (USA/global), and Parr Instrument Company (USA). It is also worth noting that, in our experience, some manufacturers will provide certification of testing but may not provide a user manual or equivalent example operating procedures. This can result in some unexpected issues for inexperienced users. For example, any ferrules that are used to introduce thermocouple probes to autoclave reactor ports will likely become permanently conjoined with a thermocouple probe after autoclave usage. Further, appropriate high-temperature, high-pressure grease (e.g., CRC Lithium Grease 30570 for temporary operation up to 200 °C) will aid smooth situation of clamp components, and non-flammable leak detector fluid (e.g., SNOOP®) can be beneficial to check that all fittings are secure and pressure is contained.

It should be noted that there are some dubious low-cost autoclave reactors readily available via non-reputable manufacturers. These autoclave reactors will likely not be compliant with government standards at these low-price points. This could introduce a high risk of spurious failure and potentially lethal hazards. Therefore such low-cost autoclaves should not be used. We strongly recommend that research teams purchase autoclaves from certified reputable suppliers and ensure that their autoclave equipment meets appropriate governmental certification requirements.

The unreported variables in autoclave synthesis

Many studies report autoclave UCNP synthesis, yet most of these studies do not report which autoclaves were used and how they were operated. For example, much of the literature simply states that reaction mixtures were “added to a Teflon-lined autoclave” and “heated at” a temperature for some time. This raises many questions, such as:

What volume is the autoclave reactor?
How much internal space does the liner occupy?
What is it made of?
How is it heated?
Was it pre-heated?
Is the quoted temperature the internal reactor temperature or the externally applied heating temperature? These are often different—see Supplementary Fig. S3.
What timepoint counts as reaction onset?
Was the reaction stirred? What pressure did the reaction occur at?

This is a huge parameter space of unreported variables. In our experience, this combinatorial pitfall of variables can make it difficult for researchers to reproduce autoclave synthesis—particularly hydrothermal autoclave UCNP synthesis (see Supplementary Section H)—even when using the same autoclave equipment in the same laboratory environment. If it is challenging for researchers using the same equipment and reagents to reproduce, then how can we possibly reproduce UCNP synthesis studies where the equipment used is fundamentally unreported? The absence of comprehensive reporting has the potential to waste significant time and effort within our research community. Therefore, in the interest of robust, reproducible, and open science, we strongly recommend that a number of key autoclave parameters and variables be reported; these are summarised in Table 2.

Table 2 Key parameters and variables to report in autoclave reactor synthesis in order to maximise reproducibility

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For context, we have detailed two case studies of autoclave synthesis UCNPs. The first is hydrothermal synthesis of oleic-acid (OA) coated NaYF₄:Yb,Er,Mn UCNPs (976 nm excitation) (see Supplementary Section G). The second example is the synthesis of PEI-coated NaYF₄:Yb,Er@NaYF₄:Yb, Nd core/shell UCNPs featuring dual band 808 nm and 976 nm excitation in Supplementary Section H. These case studies are intended as illustrative examples of the importance of the aforementioned variables in autoclave synthesis, rather than comprehensive scientific reports.

Future directions for autoclave reactor synthesis of UCNPs

In the longer term, there may also be scope to improve the wider standards of autoclave reactor synthesis of UCNPs. For instance, the field may consider the adoption of more transparent standardised operating protocols and/or standardised apparatus. This could also facilitate inter-group comparator experiments, where for example, a standardised output/reference material is synthesised to demonstrate verified high-quality UCNP synthesis and/or to compare measurements between research groups¹³. For example, in the wider field of nanomaterials research, standardised approaches have been developed and proposed for nanomaterial synthesis⁶¹, physiochemical properties⁶¹, optical properties (e.g., quantum yield)⁶², toxicity^61,63,64, dynamic light scattering (hydrodynamic diameter)⁶⁵. Whilst full discussion relating to standardised UCNP reference nanomaterials is beyond the scope of this paper, it should be noted that standardisation is a complex problem in the wider field of nanomaterials which requires input from the relevant global scientific communities^64,66.

Outlook

A large number of publications have reported the use of autoclave reactors for the synthesis of nanomaterials, including UCNPs. However, to date, many important experimental and operational parameters associated with autoclave reactor equipment have not been reported. The lack of these crucial experimental details reduces experimental reproducibility across the nanomaterial literature. Herein, we have provided suggestions for experimental parameters that can and should be reported in detail of autoclave reactor synthesis procedures (see Table 2). We recommend that these details be provided wherever practicable in order to enhance future experimental reproducibility. We have also summarised some regulations and legalities covering the use of autoclaves for research and work purposes and have noted that these legalities should be carefully considered by all researchers involved in autoclave reactor usage. We recommend that research should only use autoclaves made by reputable manufacturers and which are certified/tested to comply with appropriate international regulatory standards. Further, we note that currently available high-safety autoclave reactor systems offer advantages in terms of safety, experimental control, and reproducibility.

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