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AnalysisofMolecularBindingwithoutmarkers
WetChemistryorUltrasonicSpectroscopy?
FastMixingandReactionKinetics
Homogenousstirringinsedimentingdispersions
HRUSTITRATIONSYSTEM
ForHRUSSeriesofHighResolutionUltrasonicSpectrometers
PROVIDESADDEDFUNCTIONALITYFORHIGHRESOLUTIONULTRASONICSPECTROSCOPY
HighResolutionUltrasonicSpectroscopyisanoveltechniquefornondestructivematerialanalysisbasedon
precision measurements of parameters (velocity and attenuation) of highfrequency sound waves,
propagating through analysed samples. These waves propagate through most materials including opaque
samples and allow direct probing of intermolecular forces. The award winning range of HRUS ultrasonic
spectrometers from Ultrasonic Scientific provide an unprecedented set of new 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,ligandbinding,compositionanalysisandmanyothers.

Titration is a routine analytical method in the modern laboratory, which allows analysis of molecular
binding as well as a quantitative determination of a specific substance (analyte) in a sample by stepwise
addingasecondcomponent(titrant)whichreactswiththesubstanceofinterest.

The HRUS Titration System (left on Figure 1) provides users of the HRUS Series Ultrasonic
Spectrometerwiththecapabilitytoperformtitrationswithaccuracyandconfidence.Thesystemincludesthe
titrationmodule,speciallydesignedstirringsystemsforfasthomogenousmixingandoperatingsoftware.It
is of particular use for those involved in binding studies, protein studies, critical micelle concentration
analysis,molecularassociation,kineticsofchemicalreactionsandothermeasurements.
The Titration System delivers the titrant to the ultrasonic
cell of the HRUS 102 High Resolution Ultrasonic
Spectrometer. The easy to use, intuitive software control
package allows users to program titration sequences in
simple step by step instructions. The user can specify the
volumeoftitrantdispensed,dispensingandmeasuringtime
intervals, pauses and the stirring profiles. Titration
sequences for different titrations can be stored in method
files and recalled as required. The software allows an easy
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plotting and analysis of the ultrasonic titration profiles


(velocityandattenuationatselectedfrequenciesasfunction
of concentration of the titrant) and kinetic profiles (change
of velocity and attenuation at selected frequencies as a
functionoftimeaftereachinjection.

Figure1:TheHRUSTitrationSystem

Analysisofmolecularbindingwithoutmarkers
HRUSspectrometersworkasuniversaldetectorsofmoleculartransformationsbecausetheyprobedirectly
the intermolecular forces. Any change in molecular structure, including molecular binding, formation of
molecular aggregates, etc 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 HRUS titration technique is the ability to analyse molecules in their original state without
immobilisingprocedures,transferringintoanotherenvironmentetc.

Figure 2 shows the principles of ultrasonic titration. The ultrasonic cell is filled with solution of analyte
(polymer in the figure). Then titrant (ligand) is injected stepwise in the solution and then in 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 velocity caused by the binding of ligand with
polymer.Thiscurvethanisrecalculatedintothebindingisotherm,andbindingconstantsandstoichometries
ortheequivalentpointisobtained.
Example applications: binding of ions with chelators (EDTA, etc), polymerligand binding, antigenantibody
binding,equivalencepointdetermination.

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WetChemistryandUltrasonicSpectroscopy
Wet chemistry is a classical technique for measurements of concentrations of various components in
analysedsamples.Thistechniqueincludesaseriesofchemicaltreatmentsofthesample(extractions,etc),
which allow transferring the component of interest in the environment where its concentration can be
measured. The concentration is often measured through a titration by another component (titrant), which
reactwiththecomponentofinterest(analyte)andproducesavisiblechangesincolour(opticalspectrum),
pH or other parameters of the solution, which can be analysed quantitatively. The equivalence point on the
titration curve gives the concentration of the component of interest, which than is recalculated into the
concentrationintheoriginalsample.Thekeyelementofthisanalysisisanappropriatedetector,whichcan
provide quantitative information on the amount of titrant bound or reacted with the analyte and which
determines the complexity of the wet chemical routine. Because HRUS titrations allow analysis of any
moleculartransformationsandbecausethemeasurementsdonotrequireanyopticaltransparencyorother
properties of solution and solutes many steps in we chemical routines can be excluded. In addition one
device,HRUSspectrometer,canbeusedforvariousanalytesandtitrantsthose,whichcannotbeusedwith
anotherdetectors.
Applicationlistincludesdeterminationofconcentrationofabroadrangeofcomponents(metalions,sugars,
proteins,water,etc).

KineticsofChemicalReactions
The Titration Accessories allow direct monitoring of chemical reactions by measuring the change in
concentration of the substrates and products with HRUS spectrometers. This technology is extremely
sensitive, nondestructive, requires no markers and can be used in nontransparent samples, such as
emulsions,dispersionsetc(e.g.blood,milk).Figure3showstheprinciplesofultrasonicanalysisofkinetics
of depolymerisation of polysaccharide. The reaction is started by injection of enzyme with the Titration
Accessories. Specially designed stirring system (part of the Accessories), provides fast and uniform mixing
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of the injected enzyme. Ultrasonic parameters (velocity and attenuation) are constantly monitored and
recalculatedintotheconcentrationofproductandtheeffectivemolecularweightofthepolymer.
Exampleapplications:determinationofactivityofenzymes,kineticsoforganicreactions,etc.

Doublestirringsystem,sedimentation,creaming,etc.
Titration accessories include specially designed double stirring system, which allows fast and homogenous
mixing of the sample. It consist of the top and the bottom stirrers with adjustable speed. The software
allows to program the ON and OFF state if the stirrers as well as perform the measurement under
continuous stirring. The last option allows analysis of sedimenting or creaming samples (emulsions,
suspensions, gas bubbled samples, etc) by providing their homogenous consistency through an effective
stirring. Effective stirring allows performance of temperature ramps, titrations and other measurements in
sedimenting samples. Kinetics of sedimentation and creaming processes can simply be analysed by
monitoringofthechangeofultrasonicparametersaftertheprogrammedstopofstirring.
Example applications: gelation in sedimenting samples (starches, etc), stability of emulsions and
suspensions,kineticsofcreaming/sedimentation,etc.
Specifications
Titrationsyringesizes

5Lto500L

SyringeSpeed

From0.5secondsto60minutesperstroke

Resolution

2000stepsstandard/fullresolution

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Accuracy

+/1%(reproducibilitywithin0.2%)

Stirringspeeds

100to1400RPM

OperatingVoltage

110to240VAC,50/60Hz

Size

W26cmsXH14cmsXD30cms

OrderingInformation
TOption
HREXTS

forHRUS102SeriesSpectrometers
ExternalTitrationAccessory,includingsoftware,25Lsyringe(orotheron
request)andaccessories.

Thistechnologyisprotectedbygrantedpatentsandpendingpatentapplications

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