The technologically advanced AFA is often confused with more general sonication technologies because both utilize acoustic energy. In contrast to AFA, sonication is an older and simpler technology using low frequency and unfocused acoustic energy which is in the audible range and is administered with either a “probe” or a “bath” sonicator. The unfocused acoustic energy dissipates rapidly and in the process is converted to heat energy (1st law of thermodynamics). The rapid dissipation of the low frequency acoustic energy results in decreased efficiency, and therefore, the sonication process requires a relatively large amount of acoustic energy to yield a desired effect. The heat generated from the excess energy heats the sample resulting in thermal molecular damage and non-uniform processing of samples.
A probe sonicator is in direct contact with the sample and thereby can impart more concentrated energy to the sample than bath sonicators. However, the direct contact is problematic for small volumes, as well as leading to sample cross-contamination and contamination by erosion of the probe tip. Bath sonicators isolate the sample from the energy source but require significantly more energy input than probe sonicators because the entire water bath is energized. Both probe and bath sonicators are notoriously unpredictable and often over-heat samples when attempting to achieve a desired biological or chemical event.
A fundamental difference between Covaris AFA and probe or bath sonication is the operating frequency (see figure below). Fundamentally, higher frequency sound waves have shorter wavelengths, whereas low-frequency sound waves have longer wavelengths. The short wavelength acoustic energy developed by AFA can be precisely focused on the sample, enabling highly controlled sample preparation.
The operating frequency used in sonication is 10 to 30 kHz. At this frequency, the wavelength is approximately 100 mm which is too long to focus on a biological sample. The operating frequency of AFA is in the 500 kHz to 1MHz range depending upon the application. The resultant wavelength is only a few millimeters (see figure below). In a laboratory environment, the size of a typical sample processing tube is anywhere from 10 to 100 mm long, with the sample occupying typically less than 20 mm in height. The wavelength of Covaris AFA (approximately 3mm in water) is intentionally scaled to match the sample vessel to optimize the focused acoustic process. The focused wave allows Covaris AFA to deliver acoustic energy precisely and reproducibly into a localized area in the sample tube.
Using high frequency focused acoustic wave propagation, Covaris AFA forms a localized high pressure region precisely located in the target vessel. This creates a controlled pressure flux/acoustic field, which under high intensity “doses” will generate cavitation bubbles. The cavitation bubbles are created when the peak pressure reaches 1 Mpa. Focused-ultrasonication requires only 0.8 W power to create 1MPa in the sample, while bath sonicators apply approximately 130W and probe sonicators apply 4.6W to reach 1 MPa. The figure below demonstrates that Covaris AFA uses approximately 1% of the input energy of a bath sonicator and less than 20% input energy of a probe sonicator to generate similar pressure amplitudes.
With relatively low energy input, Covaris AFA generates a high pressure focal region, comparable to the sample vessel size Computer modeling of finite element analysis of acoustic systems.
With the low frequency unfocused acoustic waves used in bath sonication a significant amount of energy is dissipated into the water bath surrounding the sample vessel and never reaches the targeted sample. The energy dissipation makes it difficult to create the high pressure required to generate cavitation bubbles in the target vessel. To raise the pressure in the vessel to the point of cavitation, the transducer in a bath sonicator applies substantial amounts of energy to the bath. This excessive input energy inevitably leads to significant increase in the sample temperature, causing side effects, such as thermal damage (see isothermal process section for details).
Probe sonicators use a solid probe to guide waves from a transducer into the target sample. However, the differing physical properties of the probe material and the sample create acoustic impedance, reducing the transfer of mechanical energy from probe into the sample. In addition, the strong vibration of the probe generates heat in the probe; as the probe is in direct contact with the sample, the heat is directly transferred to the sample leading to a significant increase in sample temperature, resulting in thermal damage.
The Effect of Standing Waves
In Bath sonicators the samples are processed in a water bath with variable water levels, depending on the sample and application. Both the air above the water bath and the walls of the water tank are good reflectors of acoustic waves. Interference patterns are created by incident waves from the transducer and reflected waves from water and air boundaries. These become patterns of “hot spots” (high pressure locations) and “cold spots” (low pressure locations) in the water bath. These wave patterns, referred to as standing waves or stationary waves, do not move in space but do change amplitude over time. As the acoustic energy is not focused, the pattern of the standing waves is difficult to predict. During sonication, these standing waves introduce uncertainty and a lack of uniformity to sample processing. The pattern of the standing waves becomes even more complicated when sample vessels are introduced. This problem is amplified if tubes are moved within the bath during processing. The effect of standing waves is minimized when using the focused ultrasonic process developed by the Covaris AFA system because the sample vessel is positioned precisely in the focal region of the acoustic energy, and the majority of the input acoustic energy is directly projected and transmitted into the sample vessel.
A Probe sonicator transmits the mechanical energy directly into the sample by immersing the probe in the sample. The hot spots of standing waves are built between the probe tip and the vessel and cause an extreme temperature rise in the sample. Immersion of the probe into the sample may cause damage to the probe tip by cavitation and consequential contamination of the sample.
A heat-map computer modeling of temperatures after 30 seconds processing with either Covaris AFA, a bath or Probe sonicator. Green regions represent 20 C air, and blue regions represent water bath with initial temperature of 5 C before sample processing. A: Covaris AFA maintains the temperature by the end of the process; B: in bath sonicator, moderate temperature increase is inside of the plastic vessel and above the transducer surface; C: in probe sonicator, temperature of the sample is increased dramatically.
Finite element analysis software modeling of the different acoustic technologies
Controlling the temperature of the sample during the process is critical to obtaining reproducible and predictable results without causing thermal damage to the sample.
The temperature rise in the sample is mainly caused by the absorbed acoustic energy and heat flux from the acoustic device. The majority of the input energy is eventually converted to heat caused by molecular friction. It is technically challenging to simultaneously remove the heat in the system and maintain the precise delivery of acoustic energy.
Using AFA the Covaris instruments focus high frequency acoustic energy directly into the liquid phase of the sample with little scatter and high efficiency.
Probe and Bath sonicators using unfocused low frequency acoustic energy apply significant amounts of energy to the sample vessel or bath in order to generate high pressure to initiate cavitation. Various methods have been used in attempts to reduce the thermal effect. Probe sonicators require the samples sit in ice or cold water during processing, because the vibration in the probe generates substantial heat. As shown in the figure above, material near the probe surface experiences a significant temperature rise after thirty seconds and is liable to thermal damage. The cooling effect of the surrounding ice or water is low as the sample is not mixed.
There are several challenges to keeping samples cool in a Bath sonicator. Unfocused bath sonication requires still water during the sonication process to maintain the standing wave patterns so that samples receive uniform acoustic energy. One of a simple method is to add crushed ice to the water bath. This method is marginally effective, because the continuously changing water level and water/ice ratio caused by the melting ice interferes with the wave pattern and introduces uncertainty to the sample processing. Another commonly used method is to circulate cooling water with limited flow rate during the process. This method enables a more consistent pressure field during the process, but does not allow for tight temperature control of the sample during processing. During the sonication process, large amounts of acoustic energy are absorbed by the water bath, samples, and vessels. With limited circulation of cooling water, heat is trapped in the system (bath, sample, and vessel) and the sample temperature will increase.
The figure above shows that the temperature of the sample in a bath sonicator increases after thirty seconds and the heat generated from the transducer is transmitted to the water bath. The high power output required by Bath sonicators heats the water bath and reduces the temperature gradient between the sample and the cooled bath further reducing the heat flux from the over-heated sample to the surrounding water bath. The temperature in vessels may even reach 40 degree Celsius in prolonged processing, causing thermal molecular damage to the sample. Furthermore the plastic sample tubes typically used in the laboratory are poor thermal conductors and exacerbate heat build-up in the sample.
Covaris AFA technology processes samples isothermally (for both individual and batched samples) as shown in the following figure. There are three principal features that enable this. First, the total power required for sample processing is much less with Covaris AFA than with low frequency unfocused bath and probe sonicators. Second, the convergent acoustic field enables the water cooling system to be integrated directly within the water bath without adversely affecting the Covaris AFA energy while simultaneously controlling and monitoring the temperature in real time. Covaris AFA circulates cooling water continuously and because of the intrinsic difference of energy delivery mechanisms, the circulation of cooling water does not affect the delivery of the acoustic energy into the sample. Third, the Covaris sample vials are engineered for optimal sample processing with Covaris AFA. For example, with efficient acoustic transmission (and very low acoustic absorption), the AFA sample glass vials do not heat up even during extremely intense AFA sample processing. In addition, the glass vials, with much higher thermal conductivity (approximately 10 folds of the plastic vessels), are superior thermal conductors compared to plastic vessels.
Focused-ultrasonication versus sonication
Power input to generate 1MPa pressure in the sample
Thermal control during process
Covaris AFA and unfocused sonication are considerably different from each other because of the intrinsic difference in wavelengths, source power, and patterns of wave propagation.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.