The Ultrasonic All Chemical Reaction Monitor
New developments in ultrasonic detection and data processing have enabled Ultrasonic Scientific to develop a novel analytical instrument - Titration Kinetic Analyser (TKA)
This device allows analysis of most chemical reactions and molecular bindings without or with minimum sample preparation. It does not require optical markers and optical transparency and uses only 0.04 ml of sample. The device works in two modes, titration and kinetics.
Titration comes from the French word “titer” and means the concentration of a substance in solution or the strength of such a substance determined by titration (the word “titer” takes its origins from Latin titulus “Something that provides a basis for or justifies a claim”). Today titration is well known as an analytical method, which allows quantitative determination of a specific substance (analyte) in a sample by adding to it a second reactant solution of known concentration in carefully measured amounts until a reaction of definite and known proportion is completed (as shown by a colour change, precipitation, conductivity change, pH, etc.). Another important application of titration is analysis of molecular binding and determination of binding stoicheometries and affinities. In this case an appropriate detection system shall provide quantitative information on the amount of titrant bound to its target (analyte), thus allowing measurement of the binding isotherm, which represents the dependence of concentration of bound titrant on the concentration of tritrant in solution. The binding isotherm is then used to calculate binding constants (affinities) and stoicheometries. Binding constants allow calculation of free energy of binding and their temperature dependence is used to calculate the entropy and enthalpy of binding.
Titration Analysis Detectors
The key element of titration analysis is selection of an appropriate detector, which can provide quantitative information on the amount of titrant bound or reacted with the analyte. There is a range of techniques, which can be employed in titration analysis. However none of these can serve as a universal detector of the binding of titrant to analyte. For example, fluorescence and UV/VIS absorbance require a change in optical activity titrant or analyte in the binding as well as optical transparency of the solution. Therefore titrations are often accompanied with a complex sample preparation procedure such as extraction of analyte to a solution with requires optical transparency or other properties, attaching optical markers to titrant molecules etc.
Because High-Resolution Ultrasonic Spectroscopy allows direct probing of intermolecular forces it has the potential of a universal detector for chemical reactions. Any change in molecular structure, including molecular binding, affects intermolecular interactions in the sample and therefore can be detected with ultrasonic measurements. Because the measurements do not require any optical transparency or other properties of solution and solutes the complex sample preparation procedures in many cases become obsolete.
Figure 1 shows the principles of ultrasonic titration. At time zero the ultrasonic cell is filled with solution of analyte (polymer in the figure). Then titrant (ligand) is injected stepwise in the solution and the reference cell filled with a similar solution, but without analyte. Ultrasonic parameters of the solution are constantly monitored and the contribution of the titrant is subtracted using the reference cell data. The curve on the screen of the monitor represents a change in ultrasonic parameters of the solution, where each step corresponds to the addition of titrant to the solution. This curve than is recalculated into the binding isotherm, and binding constants and stoicheometries or the equivalent point is obtained.
Figure 1: The principles of ultrasonic titration
Analysis of speed of chemical reactions is an important factor for optimisation of chemical processes in various industries. This analysis produces a change in concentration of the products of the chemical reaction against time and allows calculations of kinetic constants, activities of enzymes etc. The key element of analysis of kinetics of chemical reactions is an appropriate detector, which can measure the concentration of product.
The most common approach for monitoring of chemical reactions is based on standard spectroscopic UV/visible and fluorescence assays. However, this requires optical activity of the reactants or products and in their absence the analysis is more complicated as optical markers are needed. In addition, these techniques require an optical transparency of the sample. An alternative approach is to take samples from the reaction chamber at different time intervals, stop the reaction in the samples and analyze them using HPLC or other techniques. This analysis is more complicated and costly and does not provide data in real time.
The TKA allows direct detection of chemical reactions by monitoring the change in concentration of the substrates and products. This technology is extremely sensitive, non-destructive, requires no markers and can be used in non-transparent samples, such as emulsions, dispersions etc (e.g. blood, milk).
Figure 2. shows the principles of ultrasonic analysis of kinetics of depolymerisation of polysaccharide. The sampling module provides filling the cell with solution of polymer and beginning of the reaction by injection of enzyme. Ultrasonic parameters (velocity and attenuation) are constantly monitored and recalculated into the concentration of product and the effective molecular weight of the polymer.
Figure 2: The ultrasonic analysis of kinetics of depolymerisation of polysaccharide
What is novel about the Titration Kinetic Analyser (TKA)?
The Ultrasonic TKA can ‘see’ nearly all reactions in real time, it measures all solutions, requires no optical markers or optical transparency, requires very small amounts of sample (one droplet) and has flexible automated sample preparation procedures. Previously, analysts had a choice of wet chemistry or spectroscopy but now with the advances made in Ultrasound, the TKA combines the benefits of both in one device.
In effect, the TKA is an‘All Chemical Reactions Monitor’. This is because ultrasonic waves propagate through most materials and as ultrasonic waves measure directly the intermolecular forces, any change in molecular structure and molecular arrangement can potentially be detected.
The development of the TKA was made possible by accumulated experience in the application of HR-US range of ultrasonic spectrometers in various industries, a breakthrough in ultrasonic detection that allows the fast simultaneous measurement of parameters of ultrasonic waves propagating in small sample volumes in “real time”, and advanced signal processing algorithms coupled with new software and electronics development leading to fast data collection and processing.
The TKA can be applied to the following application areas; molecular binding (proteins, nucleic acids, chelators, etc.), enzymes (activity, kinetics), drug screening, medical diagnostics (blood analysis), and polymer synthesis. The TKA is designed to be applied in the following market segments; pharmaceutical, biotech, biomedical, environmental, polymers, personal care, and food and beverage.
How Does the TKA Save Time in the Laboratory and Benefit Users?
The TKA overcomes the limitation of wet chemistry, which included multistage sample preparation routine and finding an appropriate detector, and spectroscopy, which required facilitation of the ‘visibility’ of the analysed process - optical markers or coupled reactions - or optical transparency (blood, etc.). The TKA combines the benefits of both techniques in one device.
The result is minimum sample preparation, the use of one detector for broad range of reactions. By using only one droplet of sample TKA enables cost-effective analysis of expensive samples such as proteins (antibodies), nucleic acids, drugs, etc. In addition, it can analyse reactions, which are ‘invisible’ to other techniques, no optical markers are required anymore and opacity is no longer an issue, thereby saving time on sample preparation. As the TKA has an integrated approach (synchronised sampling and measuring), this allows fast automatic/robotic analysis of a large number of samples. One device for analysis of a broad range of reactions and samples also means savings on the number of instruments, operator training and space.