Microwave Evolution

Microwave irradiation, as a known form of energy, has been around since the late 1940s, but it was not until 1986 with publications from Professor Richard Gedye and Professors Raymond J. Giguere and George Majetich that microwaves began to make their ways into the organic laboratory in large numbers. Gedye and Giguere/Majetich found the application of microwave irradiation to their reactions, as opposed to “conventional” heating techniques, such as heating a reaction on a hotplate, enabled more rapid product formation. Reactions that typically were complete only after 8 hours or more took mere minutes in the microwave.

Throughout the 1980s and during the early 1990s, scattered references exist as to the use of microwave irradiation, but it was largely an underutilized technology (more widespread acceptance and utilization of the technology would come later). The concept of performing reactions in short periods of time with this advanced energy source was starting to take hold, but was not yet fully mature, due in a large part to issues with instrumentation. Most of the microwave ovens used in these first papers were kitchen-type, or domestic, ovens, without the controls commonly associated with organic laboratory equipment: stirring was not incorporated, the temperature was not monitored during the reaction, and no real control existed over the amount of power applied. As a result, for as many benefits associated with microwave irradiation, there were equally as many drawbacks, including lack of reproducibility, vessels that spilled their contents throughout the cavity, and even some that exploded. Microwave ovens needed to further evolve before they could fully take hold.

Most of the reactions performed initially in domestic microwaves were done in sealed or round bottom flasks with polar solvents or by using an additional agent to help absorb microwave energy. This additional component, such as vermiculite, could be packed around the sample to assist in rapid heating if sample was very small or low in polarity and the microwave itself was not able to heat it (the vermiculite could also serve as an absorbing agent, should the vessel rupture). This energy-absorbing medium could also be incorporated into the reaction itself by absorbing the reagents onto a solid support such as alumina or clay. The solid support was able to absorb microwave energy very rapidly and easily transfer this energy to the reaction components. Many microwave reactions also utilized a water line or beaker of water in the cavity to help eliminate any microwave energy not absorbed by the reaction. All of these techniques enabled the microwave energy more easily “find,” and therefore, heat the sample, preventing the instrument from suffering major damage.

The drawbacks associated with early microwave reactions were somewhat alleviated with the use of industrial-type microwave ovens. Temperature monitoring could be utilized, stirring could be incorporated, and an apparatus, known as a mode stirrer, could be used to reduce the number of “hot spots,” or pockets of high and low energy generated, thereby helping with reproducibility issues. The use of industrial microwaves with pressure monitoring systems also enabled the safe use of pressurized vessels in the microwave.

There was, however, a need to explore reactions on a smaller scale. A single reaction of a few milliliters proved challenging for the industrial type multiple mode microwave systems to effectively heat without modifications similar to those in the early domestic papers (such as a microwave absorbing medium around the sample). In order to address the need to more easily test single reactions on the milligram scale, single mode microwave systems were designed in the late 1990s.

The main difference between the new single mode and previously existing multimode design is the generation of a single mode (as opposed to multiple modes) of energy during the irradiation cycle. The microwave is a wave, constantly moving forward and transitioning between positive and negative values. As the wave moves, it generates pockets of high energy and low energy as the moving wave either reinforces or cancels. This leads to the presence of high-energy fields, low energy fields, and a point where the amount of energy is equal to zero, called the node (Figure 1).

Figure 1: Modes And Nodes

Instead of allowing the presence of multiple modes and nodes of microwave energy, the single mode cavity is designed for the length of only one wave, therefore generating only one mode of microwave energy. The wave generates a center of high electromagnetic (em) field intensity with homogenous energy distribution in the cavity where the synthesis takes place (Figure 2). In a multimode system, there are many centers of high em intensity, called “hot spots,” but there are also several low energy spots, creating a “cold spot.” Mode stirrers cause the microwaves to move around in the cavity, reducing most of the differences in hot spots and cold spots. When performing reactions in parallel, or with larger sample sizes, the presence of these multiple areas of energy is not as significant; the samples move around and are large enough to effectively absorb the existing microwave energy. With a single smaller sample, however, the chance of the one sample of a few milliliters in volume within the cavity effectively absorbing the microwave energy moving around a 50 liter cavity is very low, resulting in slow or non-existent heating profiles and a significant amount of energy being wasted.

Figure 2: Single Energy Distribution in Single Mode vs. Multiple Nodes Generated in Multiple Mode Microwave

The other major difference between single mode and multiple mode cavities is the energy density. Although most single mode cavities have a maximum power output of only 300 Watts, the amount of energy in a given amount of space within the cavity is actually much greater. Only one area of microwave energy exists in the small space in a single mode microwave, as opposed to multiple areas of high em energy in the larger multiple mode cavity, giving rise to a greater energy per unit area. As a comparison, a typical single mode cavity, with a volume of 0.5 L, can generate 900 W/L with 300 W of power applied to the reaction. A multimode cavity, on the other hand, will generate 20-25 W/L with 1200 W of energy applied.

How Microwave Irradiation Accelerates Chemical Transformations

Microwave radiation is a form of energy, relatively low in the electromagnetic spectrum. This form of irradiation falls below x-rays, UV, visible, and infrared in the energy spectrum (Figure 3), with a frequency of 300 to 300,000 megahertz (MHz). Because microwave irradiation is at such a low frequency, below that of x-rays, UV, and even infrared, it will only cause bonds to rotate, not break.

Figure 3: The Electromagnetic Spectrum

A microwave travels very rapidly, at the speed of light. However, it varies in length, based on frequency. There are four frequencies that are allowed by the government for commercial microwave use: 915 MHz, 2450 MHz, 5800 MHz, and 22,125 MHz. The frequency 2450 MHz has a length of 12.2 cm, which has an appropriate penetration depth (the distance a microwave can travel into a standard sample) for use with small samples.

But how does that energy get into the sample? Heating mantles transfer thermal energy into a reaction system by using energy to warm the heating mantle, the heating mantle in turn warms the vessel, the vessel then warms the reaction mixture until everything is one, relatively, homogenous temperature (some thermal gradients generally exist). This thermal energy will supply the necessary energy to the reaction mixture to cause the formation of products.

The mechanism of energy transfer in a microwave is significantly different from a hotplate or heating mantle. Microwave energy heats the sample through direct activation. Instead of heating the microwave, then the vessel, energy is transferred to the reaction components within the solution, providing two different benefits: 1) more efficient energy transfers to the reaction mixture, instead of the vessel and 2) reaction components at the center of the reaction are heated at the same rate as reactants near the walls of the vessel.

Figure 4: A Microwave

Figure 5: Rotation Of Molecules With Microwave

Figure 6: Solvent Chart

As mentioned above, each of the energy transfers generated by microwave irradiation occurs very rapidly—every nanosecond (10-9 seconds). The almost constant energy input is achieved at a rate greater than the molecular relaxation rate, which is on the order to 10-5 seconds. Because the energy is added at a rate faster than the molecules are able of fully relaxing, all of the molecules in solution will be in a constant state of disequilibria. This disequilibria situation will provide more than enough energy to overcome the activation energy barrier (Ea) and drive the reaction to completion (Figure 7).

Figure 7: Reaction Coordinate

In fact, microwave energy will sometimes allow the formation of otherwise low-yielding products, as is the case with thermodynamic products. Conventional synthesis does not always provide enough energy to overcome the greater Ea of the thermodynamic product. Although the thermodynamic product, Product 2, is energetically more stable than the kinetic product, the thermodynamic product has a higher energy barrier to surmount. The reaction is forced to take the lower energy path to the kinetic product (Product 1), meaning the majority of the products will be from the kinetic reaction instead of the thermodynamic reaction. The addition of energy directly to the reactants, via microwave irradiation, allows the thermodynamic energy barrier to be overcome in a microwave, forming the desired lower energy product, Product 2 (Figure 8).

Figure 8: Thermodynamic vs. Kinetic Reaction

Microwave irradiation can also serve to activate a resonance-stabilized intermediate, thereby helping to drive the reaction in down the thermodynamic path. Although most intermediates are too short-lived to be activated (on the order of 10-13 seconds), a resonance-stabilized intermediate will linger long enough to interact with the microwave and will be able to gather energy directly from the microwave, in a similar fashion as one of the starting materials, to assist in overcoming the thermodynamic Ea.

The Impact of Microwave Energy on Peptide Synthesis

The development of solid phase peptide synthesis (SPPS) overcame many of the problems of classical solution phase synthesis and has become the standard method for research scale synthesis of peptides up to 50 amino acids. This method employs use of a solid resin bead to assemble a peptide chain from C to N terminus (Figure 9). The carboxy group of the C-terminal amino acid is attached to a linker group, thereby attaching it to the resin. A temporary protecting group on the a-amino group is first removed. The second amino acid, with N-terminus temporary protection, is then added to the reaction in excess with its carboxy group activated to generate an activated ester capable of amide bond formation. Excess reagents are then removed from the reaction, usually by filtration, and the resin washed repeatedly with solvent. The N-terminus protecting group of the dipeptide attached to the resin is then removed and the cycle continues until the desired sequence is made. The last step involves deprotection of the final N-terminus protecting group and removal of the peptide from the resin. This step also removes any side chain protecting groups used during the stepwise synthesis yielding an unprotected peptide in solution. The two main approaches of SPPS used are the Fmoc and Boc methods. The Boc method is the older of the two and has largely been replaced by the Fmoc method.

Figure 9: Solid Phase Peptide Synthesis Cycle

While certain peptide sequences could be synthesized relatively easily it was noticed early on that other sequences were much more difficult. During chain assembly sudden decreases in reaction rates were observed. In some cases repeated or prolonged reaction time showed no improvement in chain assembly. Optimal reaction conditions require a fully solvated peptide-polymer matrix that allows for efficient reagent penetration. It has been observed that during the synthesis of a difficult peptide the reaction matrix becomes partially inaccessible, typically during the 6-12th residue of the chain. This is thought to occur due to secondary structures of aggregates that result in poor solvation of the peptide-polymer matrix. As a peptide chain is built stepwise on a resin bead it can form aggregates either with itself or neighboring chains. This involves inter or intramolecular hydrogen bonding of the peptide backbone that leads to b-sheet formation.

In peptides, the N-terminus amine group and peptide backbone are polar which makes them constantly try to align with the alternating electric field of the microwave. During peptide synthesis this can help break up chain aggregation due to intra and interchain association and allow for easier access to the growing end of the chain (Figure 10). One must also consider the solvent used for SPPS. Both DMF and NMP are medium absorbers of microwave energy, while DCM is a lower absorber. The dielectric loss for NMP, DMF, and DCM are 8.855, 6.070, and 0.382 respectively. Conventional heating relies on energy to migrate in from the outside of the vessel and is a slow and non-specific transfer. Whereas with microwave, energy transfer occurs in 10-9 sec with each cycle of electromagnetic energy. The kinetic molecular relaxation from this energy is approximately 10-5 sec. Thus a large amount of energy can be applied directly to the molecules involved in the reaction in a very efficient manner.

Figure 10: Effect of Microwave on Aggregation during Peptide Chain Assembly

Microwave applications have expanded from the original use of the industrial-type multiple mode microwaves in drying, and food processing to more analytical type applications, including digestion and extraction of samples. Expansion into other areas of chemistry includes synthetic organic work such as peptide synthesis, inorganic work, pharmaceutical research, polymer chemistry, even nanoparticle synthesis. The range of applications that have been impacted by microwave use within the chemistry arena is almost endless. Work in the last 4-5 years in the organic field has moved from a focus on high temperature transition metal-type coupling reactions, easily thought a target due to the high microwave absorbance level of the catalyst and high temperature of the reaction, to all types of chemical transformations, including radical-based reactions, substitution and elimination reactions, peptide synthesis, enzymatic digests, even low temperature microwave synthesis. As microwave irradiation expands its horizons and proves its utility as both a timesaving tool and a novel means to perform challenging transformations, it is becoming an increasingly important in the industrial community.

As they have over the past 60 years, microwave systems will continue to evolve to meet the changing needs presented by chemists. Microwave irradiation has already proven its value as a tool within the chemistry community, pushing reactions to completion more rapidly than previously possible and opening doors to perform novel transformations. Microwave acceleration has proven to be a valuable tool for any synthetic chemist and will only continue to become more prevalent in the future.

Since the successful synthesis of a peptide necessitates near 100 percent completion of two reactions per cycle, microwave energy represents an efficient way to drive these reactions to completion. Common problems in SPPS, typically from aggregation, necessitate a great deal of time and reagent cost to drive to completion. Microwave energy represents a fast and efficient alternative that is useful for providing efficient energy to accelerate both the deprotection and coupling steps.

Hayes, B.L. Microwave Synthesis: Chemistry at the Speed of Light; CEM Publishing: Matthews, NC, 2002.

Microwaves in Organic Synthesis; Loupy, A. Ed.; Wiley-VCH Publishing: Weinheim, 2002.

Microwave Assisted Organic Synthesis; Tierney, J.P. Lindström, P., Eds.; CRC Press: Boca-Raton, FL, 2005.

Microwaves in Organic and Medicinal Chemistry; Kappe, C.O, Stadler , A., Eds.; Wiley-VCH Publishing: Weinheim, 2005.

Fmoc Solid Phase Peptide Synthesis – A Practical Approach; Chan WC, White PD, Eds.; Oxford University Press: Oxford, 2000.