Adding extra functionality to high-res ultrasonic spectroscopy

High-resolution ultrasonic spectroscopy (HR-US) is a novel technique for non-destructive material analysis based on precision measurements of parameters (velocity and attenuation) of high-frequency sound waves, propagating through analysed samples.

These waves propagate through most materials including opaque samples and allow direct probing of intermolecular forces. HR-US ultrasonic spectrometers provide a broad range of analytical capabilities for research, product development, quality and process control in biotech, pharmaceutical, food, chemical and petrochemical, polymer and other industries.

Applications include analysis of chemical reactions, conformational transitions in polymers and biopolymers, aggregation and gelation phenomena, particle sizing, phase transitions, stability of emulsions and suspensions, formation of micelles and CMC measurements, ligand binding, composition analysis and many others.

Technological advances in the measuring principles of HR-US resulted in a recent launch by Ultrasonic Scientific of new series of HR-US spectrometers and accessories for automatic titrations, which include micro-volume analytical capabilities.

These devices can perform a variety of analytical tasks and require small sample volume, from 30 µL to 1ml. This significantly reduces the cost of analytical work in biotech, pharmaceutical, biomedical and other industries which deal with complex and expensive materials and formulations.

Routine titrations

The HR-US titration system (Fig.1) provides users of the HR-US series of ultrasonic spectrometers with the capability to perform titrations with accuracy and confidence. The system includes the titration module, specially designed stirring systems for fast homogenous mixing, and operating software. It is of particular use for those involved in binding studies, protein studies, critical micelle concentration analysis, molecular association, kinetics of chemical reactions and other measurements.

The titration system delivers the titrant to the ultrasonic cell of the HR-US. The software control package allows users to programme titration sequences.

The software allows easy plotting and analysis of the ultrasonic titration profiles (velocity and attenuation at selected frequencies as function of concentration of the titrant) and kinetic profiles (change of velocity and attenuation at selected frequencies as a function of time after each injection).

Analysis of molecular binding

HR-US spectrometers work as universal detectors of molecular transformations because they probe directly the intermolecular forces. Any change in molecular structure, including molecular binding or formation of molecular aggregates, affects the intermolecular interactions in the sample and therefore can be detected with ultrasonic measurements.

The measured ultrasonic titration profile, dependence of ultrasonic velocity and attenuation on the concentration of titrant, can be recalculated into the binding isotherm (dependence of concentration of bound titrant on the concentration of titrant in solution), which provides binding constants (affinities) and binding stoichometries. Binding constants give the free energy of binding and their temperature dependence allows calculation of entropy and enthalpy of binding.

Because the measurements do not require any optical transparency or other properties of solution and solutes the complex sample preparation procedures, often required by other techniques, become obsolete.

Another advantage of HR-US titration technique is the ability to analyse molecules in their original state without the need for immobilising procedures or transferring them into another environment. In a course of titration the ultrasonic cell is filled with solution of analyte. Then titrant (ligand) is injected stepwise in the solution and then in the reference cell filled with a similar solution, but without analyte.

Chemical kinetics

The titration capabilities allow direct monitoring of chemical reactions by measuring the change in concentration of the substrates and products with HR-US spectrometers. This technology is extremely sensitive, non-destructive, requires no markers and can be used in non-transparent samples, such as emulsions and dispersions. Example applications include determination of enzyme activity and the kinetics of organic reactions. Fig.2 shows ultrasonic monitoring of the enzymatic hydrolysis of cellulose. Cellulose is the most abundant organic source of food, fuel and chemicals. However, its usefulness is dependent upon its hydrolysis to glucose.

Most common technologies of cellulose treatment involve the enzymes (cellulase) from microbial sources. This requires effective direct methods for the measurement of the enzyme activity in various cellulose systems. The standard procedure for monitoring of released glucose in this reaction involves a coupled secondary reaction (hexokinase/glucose-6-phosphate dehydrogenase system), which can be monitored through measurements of optical absorption at 340µm. In contrast to this method, the HR-US technique does not require any secondary reaction and allows direct monitoring of the enzymatic hydrolysis in various cellulose samples including opaque ones.

In this example, the reaction was started by injection (with the titration accessories) of 30mg of cellulase into the 30mL cell containing 5mg/ml solution of cellulose (soluble form) in sodium acetate buffer at 25°C.

Ultrasonic parameters were constantly monitored and recalculated into the concentration of product. The reaction was accompanied by an increase in the ultrasonic velocity, which is attributed to a higher hydration of the glucose in comparison with original cellulose.

The kinetics profile of the reaction (glucose concentration versus time) was obtained directly from the ultrasonic curves, using initial (time zero) and final (at saturation) values of ultrasonic velocity and the initial concentration cellulose. High precision of the ultrasonic measurements allow analysis of the reaction rate profile. u

Evgeny Kudryshov and Breda O'Driscoll are with Ultrasonic Scientific, Dublin, Ireland.

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