Ultrasonic spectroscopy moves into laboratory for dynamic monitoring

1st April 2013

Ultrasonic spectroscopy is all set to become a routine analytical technique in the laboratory. It provides a non-destructive way to measure the properties of materials, using sound waves rather than the more usual electromagnetic waves. As Vitaly Buckin and Breda O'Driscoll show, it can also be used for monitoring the course of dynamic processes such as biochemical reactions.

Until recently, although the ways in which the properties of ultrasonic waves are changed as they passed through materials were well known, they could not easily be applied to material analysis. The sample sizes required were impractically large, the measuring techniques were overly complicated, and it was impossible to make measurements that were sufficiently accurate to give meaningful data.

These difficulties have now been overcome with the launch of what is believed to be the world's first high-resolution ultrasonic spectrometer. Developed by Dublin-based Ultrasonic Scientific, the HR-US101 won the silver award at Pittcon earlier in the year as the best new product at the show.

Ultrasound waves are already used routinely in a number of applications. Perhaps the most familiar is medicine, where their ability to penetrate the human body without causing damage is exploited in the scanning of unborn babies and the diagnosis of a range of medical conditions. Existing laboratory applications include particle sizing, and ultrasonic cleaning baths. And the sonar systems submarines use to establish their location rely on sound waves.

Electromagnetic waves in various forms are commonly used in analytical techniques, from IR and UV spectroscopy through to NMR. Sound waves are very different as they consist of a series of compressions and decompressions. The ultrasound waves used in ultrasonic spectrometry have a frequency above 100KHz, a higher frequency than the sound waves we can hear. The big advantage of ultrasound waves is that they can penetrate most materials, including those that are opaque to electromagnetic waves.

Measuring two parameters

The new spectrometer measures two separate parameters: the velocity and the attenuation of the waves as they pass through a sample. The waves travel most rapidly through solids, more slowly through liquids, and slowest of all through gases. The density of the material has some bearing on the velocity, but more important is the elastic response of the sample, which is extremely sensitive to molecular organization and intermolecular interactions.

The velocity can thus be expressed in terms of compressibility or storage modulus. Velocity measurements must be carried out at extremely high resolution if useful information is to be gleaned from them, and the minimum practical resolution is around 10­4 per cent.

The second parameter that is measured, attenuation, is a measure of the loss of energy in the compressions and decompressions as the ultrasound wave passes through the sample, and can be measured in terms of viscosity of the medium, or its longitudinal loss modulus.

Attenuation is caused largely by two factors: ultrasonic scattering in non-homogeneous samples, and fast chemical relaxation. The ultrasonic wave's periodical changes of temperature and pressure lead to a periodical shift in the equilibrium position of a chemical reaction, and relaxation back to the equilibrium position leads to further energy losses.

This means that fast chemical kinetics can be analysed using ultrasound. Attenuation measurements do not require as high a resolution as velocity measurements, and hence most earlier applications of ultrasound in material analysis, such as particle sizing in suspensions and emulsions, relied on attenuation.

The high resolution required for spectrometry has been achieved by Ultrasonic Scientific with the introduction of a novel method of generating and measurements of the ultrasonic waves.

In the past, ultrasonic characteristics were generally measured using the pulse technique, where an ultrasonic pulse, generated at a specific frequency, was passed through a sample, with the wave's amplitude allowing the attenuation to be determined, and the propagation time characterising the velocity. However, the resolution that could be achieved was limited by the path length of the pulse ­ in other words, the size of the sample. But, in a large sample, it is very difficult to control the temperature well enough to allow accurate velocity measurements to be made.

This problem has been solved by applying a different principle to the measurement of the ultrasonic parameters. The path length of the wave through the sample is larger than the size of the sample, which is achieved by applying recent advances in ultrasonic design, electronics and digital processing. This means much smaller samples can be analyzed, and a high resolution can be achieved.

A new laboratory tool

High-resolution ultrasonic spectroscopy has many potential applications. These include particle sizing, structural analysis, thermal analysis and phase transitions, analysis of the quality of liquids, and the detection and analysis of chemical reactions. Conformational changes in both polymers and biopolymers can be studied, as can aggregation, gelation, ligand­polymer binding and antigen­antibody binding, micellisation, hydration and composition analysis.

The standard cells for the HR-US 101 have a capacity of 1ml, and their construction allows the sample inside to be stirred. They have an optimal geometry, with no sharp corners or crevices, making them easy to fill, refill, clean and sterilise. Their screw caps prevent samples evaporating, and they are safe to use with aggressive liquids including strong acids or organic solvents.

As well as the standard cells, several special cells are available, including a small volume version with a capacity of 0.03ml, cells that accommodate non-liquid samples, flow through cells, and cells that allow an extended frequency of 0.1­20MHz to be used.

The sensitivity of the instrument means low concentration samples, typically 1mg/ml can be analysed, and in some cases samples as dilute as the mg/ml scale can be studied. Concentrated mixtures also pose no problems.

All measurements are computer-controlled, and output options include both graphical and digital formats, the latter being compatible with Excel and most forms of data analysis software. Ultrasonic velocity can be measured at a resolution down to 10­5 per cent, and the ultrasonic attenuation to 0.2 per cent, all across a temperature range of ­20°C to 120°C.

Several different measurement regimes can be used, allowing a wide variety of different samples and processes to be analysed. The kinetic regime measures the velocity and attenuation as a function of time so the kinetics of a chemical reaction can be followed. The titration regime measures velocity and attenuation during a titration, allowing processes such as molecular adsorption, ligand binding and complexation to be studied.

And in the temperature ramp regime, velocity and attenuation are measured as a function of temperature so processes like phase transitions and polymer conformational transitions can be monitored. All three of these regimes can be carried out at a range of frequencies.

Application examples

An example of the application of the temperature ramp regime is in the analysis of heat transition in an aqueous solution of poly(N-isopropylacrylamide), a polymer whose applications include thermoresponsive gels.

As temperature rises, the polymer coil collapses into a compact globule, and aggregates are formed. Ultrasonic velocity decreases during the process, reflecting the dehydration of the polymer, and the intrinsic elasticity of the globules and aggregates.

Attenuation increases as the aggregates cause increased scattering of the ultrasonic waves. Ultrasonic spectrometry can be used to pinpoint the temperature and width of the phase transition and analyse the transformations in the polymer structure, both of which are illustrated by the changes in velocity. The differences in attenuation mean the structure of the aggregates can be characterised.

Another example is the monitoring of enzyme activity. A 5µg sample of beta-amylase was added to a 3.5µM solution of maltoheptose at 25°C. The resulting hydrolysis reaction could be followed by monitoring the changes in ultrasonic velocity, which increases as the reaction proceeds. The resulting curve can be recalculated into the time dependence of the amount of substrate hydrolysed, giving the kinetic profile of the reaction, and the activity of the enzyme can be calculated.

A further example is the monitoring of the calcium fortification of low-fat milk. Adding calcium ions to low fat milk reduces the stability of the milk's casein micelles, causing it to coagulate on heating or when it is added to hot beverages. Additives can be used to stabilise the system, and ultrasonic spectroscopy has been used to establish the best stabiliser to use.

When the milk coagulates, ultrasonic attenuation increases as aggregation increases absorption and scattering, and ultrasonic velocity decreases as the aggregates have a compressible core which slows the waves down. Potassium citrate was shown to be the most effective, followed by sodium carbonate, and sodium acetate was the least effective.

The technical advances that have been made in the measurement of ultrasonic velocity and attenuation have transformed ultrasonic spectrometry from a mere idea into a practical laboratory technique. Its wide range of applications and ability to analyse samples that were previously very difficult to investigate mean it has the potential to become an important routine analytical tool in the laboratory. u

Breda O'Driscoll is Managing Director with Ultrasonic Scientific Ltd. Dr Vitaly Buckin is Head of Laboratory of Physical Chemistry of Biocolloids at University College Dublin. For more information call +353 1 4100847, or visit





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