Ultrasonic energy can be provided by different ultrasound devices, such as ultrasonic baths, horns or probes. These devices are equipped with powerful transducers that convert either electrical or mechanical energy into vibrations, which are transferred to the medium in the form of an ultrasonic wave [288]. Three main types of ultrasound transducers are available: (i) gas-driven transducers, which are used in whistles and sirens; (ii) liquid-driven transducers that generate ultrasound by the motion of liquids into confined chambers; and (iii) electromechanical transducers, which are the main type of transducers used in analytical devices, as the ultrasonic bath or the ultrasonic probe [288, 290]. Among electromechanical transducers, the magnetostrictive and piezoelectric transducers are the most
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common to produce ultrasound. In magnetostrictive transducers, short pulses of a magnetic field are applied in a metal to induce modifications in its shape. Nickel, for example, reduces size when a magnetic field is applied, and returns to the original size when the magnetic field is removed. Thus, the metal vibration induced by the magnetic pulse, applied at a given frequency, is transmitted to the media as an ultrasonic wave [288]. On the other hand, in piezoelectric transducers different electric voltages are used to induce structural changes in crystals (e.g. quartz) and ceramics, producing ultrasound [288].
The most common ultrasonic devices used for analytical applications are ultrasonic baths, ultrasonic probes and cup horn reactors [294, 296]. These devices are all based on electromechanical transducers. A brief description of each one is provided in the following sections, and an overview of the main advantages and disadvantages is given in Table I.7.
Table I.7: Overview of the major characteristics of different ultrasonic devices used throughout this
work: ultrasonic bath, ultrasonic probe, and sonoreactor.
Ultrasonic bath Ultrasonic probe Sonoreactor Sample throughput High Low Medium
Sample handling Low High Low
Thermostat Yes No No
Operating frequency 35 kHz – 130 kHz 20 kHz – 30 kHz 20 kHz – 30 kHz
Intensity Low High Medium
Cost € €€€ €€€
Advantages
Available in most laboratories;
No special adaptation is required for the reaction vials.
High ultrasonic power;
Ultrasonic amplitude control.
Higher ultrasonic power than the cleaning bath; Higher throughput than the probe system. No cross-contamination.
Disadvantages
Reduced power; Ultrasonication effects depend on the vial position in the bath.
Sample overheating; Cross-contamination; Tip erosion.
Lower ultrasonic power than the probe system;
Limited sample volume.
I.4.2.1. Ultrasonic bath
Ultrasonic cleaning baths are easily available, relatively inexpensive, and perhaps the most common ultrasonic apparatus present in chemical laboratories. The ultrasonic energy provided is generally of low intensity, 1 to 5 W/cm2, and the operating frequencies are around 40 kHz [293, 294]. A normal ultrasonic bath consists in a stainless steel tank with piezoelectric transducers located at the base. The
51 number and type of transducers are directly related with the intensity of ultrasound provided by the equipment [288]. Operating this ultrasonic device is very simple and straightforward, but a number of variables must be considered: (i) the size of the bath and the position of reaction vessel inside the tank affect the intensity of the ultrasonic energy transmitted to the reaction media, creating reproducibility issues; (ii) the temperature inside the tank increase with the ultrasonication time and is difficult to control, since most ultrasonication baths do not have thermostats; and (iii) the ultrasound frequency varies with the equipment, and must be considered when comparing results obtained from different baths [288].
Many applications of the ultrasonic bath have been reported over the years. Extraction of metal elements from biological samples; extraction of organic compounds, such as pesticides, polymers and pollutants; and the improvement of methodologies for analytical chemistry, are some examples of the wide range of applications of this device [293, 306].
More recently, the ultrasonic cleaning bath was reported as a valuable tool for proteomics applications. The enhancement and improvement of sample processing for protein identification [307], and protein quantitation by 18O-labeling [308] with the ultrasonic bath was performed in our laboratory as part of this dissertation.
The ultrasonic bath used throughout this work has specific characteristics, which make it one of the most advanced equipments of the genera, namely: (i) two different operating frequencies, 35 and 130 kHz; (ii) ultrasound intensity regulation, from 10 to 100 %; (iii) thermostat, for temperature control; (iv) timer; and (v) three different operating modes to regulate how the ultrasound frequency is applied to the bath [294].
I.4.2.2. Ultrasonic probe
The ultrasonic power provided by an ultrasonic probe varies from 50 to 500 W/cm2, with operating frequencies from 20 to 30 kHz. It is at least 100 times higher than the ultrasound intensity of an ultrasonic bath [302, 306]. In general, the design of this kind of equipments consists simply in attaching a probe, also known as sonic horn, to a piezoelectric transducer. The probes, generally made of a titanium alloy, amplify the vibration of the piezoelectric transducer and transfer the ultrasonic energy directly into the liquid medium [288].
Even though the ultrasonic probe is the most reliable source of ultrasound, several factors must be considered when performing ultrasonication with this equipment. First, the ultrasound intensity and performance are largely dependent on the shape, length and diameter of the probe [288]. Nowadays, probes with different specifications are commercially available, and must be chosen according to the
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desired effect and objective of the work. Second, during ultrasonication, the temperature of the liquid media increases and its physical characteristics may change, causing the decoupling of the probe and the decrease of cavitation efficiency, and aerosol formation inside the container. Therefore, the reaction vial must be refrigerated during the ultrasonication procedure, or pulses of ultrasound must be applied to avoid sample overheating [294]. The shape of the reaction vial is another important variable affecting the efficiency of the probe: the vials must have a conical form to ensure a more effective energy transfer [309]. Finally, special care must be taken to avoid sample contamination, because ultrasonication is performed in an open reactor, and the probe, if not efficiently decontaminated between experiments, may introduce contaminants in samples [290].
The ultrasonic probe has been used in many and different works [293, 306], but one of the most recent and promising applications is the use of the ultrasonic probe to accelerate enzymatic digestion [116, 310, 311] and 18O-labeling of proteins [312, 313]. Much of this work was developed in this dissertation, and the results are reported throughout the next chapters.
Recent advances in the ultrasonic probe technology have introduced the silica glass probes, spiral probes and multi-probes [294]. The multi-probe systems provide higher sample throughput, while the spiral probes provide uniform ultrasonic energy across the entire surface, and are especially useful for ultrasound application in lengthy and thin reaction vials. Glass probes are mainly used for metal trace analysis, because they are less prone erosion and, therefore, metal contaminants resulting from the erosion of the probe, as in metal alloy probes, are not introduced in the sample.
I.4.2.3. Cup horn reactors
The sonoreactor technology, available from Hielscher Ultrasonics (www.hielscher.com), is a powerful cup horn reactor. The sonoreactor can be regarded as a small ultrasonic bath, since it provides indirect ultrasonication of samples. Unlike the ultrasonic probe, that delivers ultrasound directly into the liquid media, the ultrasonic waves generated by the sonoreactor have to cross the walls of the reaction vessel, resulting in a lower ultrasonic power, compared to the probe system [294]. However, it is claimed by the manufacturers that the ultrasonic energy provided is 50 times higher than a normal ultrasonic bath. The main advantage of this system is the possibility to perform high-intensity ultrasonication in closed vials, which prevents cross contamination and allows the ultrasonication of hazardous samples. Furthermore, the sample throughput is higher than the ultrasonic probe.
This equipment was used for the first time in proteomics applications during the work developed in this dissertation. Promising results were obtained, namely, the reduction of protein enzymatic digestion time from 24 h to only 1 min [314], and the improvement of protein 18O-labeling [313, 315]. These results are described in detail over the next chapters.
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