ISOTACHOPHORESIS

Contents
• • • • Introduction Historical Review Principle Instrumentation
– Single Column System – Coupling Column System

• Applications • Interpretation

Analyte Concentration Technique .Introduction  ITP – Isotachophoresis  Iso = equal  Tacho = speed  Phoresis = migration  Migration of ions under electric field with equal speed. Everaerts. Verheggen  Separation Technique.  One of the modes of capillary electrophoretic technique suitable for analyzing mixtures of ionogenic substances  Comparable to Displacement Chromatography  Developed by Martin.

. focused zones. • Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp.Introduction • Capillary Isotachophoresis (CITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. • Although it is used as a mode of separation. transient ITP has been used primarily as a sample concentration technique.

Preetz  Cons Electrophoresis .Martin  Ionic Migration Technique .Introduction  Other names  Displacement Electrophoreis .Vestermark  Omegaphoresis  Transphoresis  Steady-stack stacking – Ornstein .

acetate.Regulating function of Kohlrausch  Hardy – separated proteins based on pH of electrolyte (IEF)  1942 – Martin – separated chloride. Martin – designed an instrument for capillary ITP .Historical Review  1850s – Wideman & Buff . aspertate. glutamate by ITP  Everarts. Verheggen.Charged particles migrate in solution when electric field is applied  Kohlrausch – Theory of Ionic Migration .

charged particles will move at a velocity acc. to 𝑉 = 𝑚 ∗ 𝐸  Mobility is different for different molecules  In ITP velocity is kept constant  miEi= constant m ∝  ITP  Cationic  Anionic 1 𝐸  Carried out in discontinuous electrolyte system .Principle  Under the influence of an electric field (E).

Principle Analytical ITP Apparatus .

Principle .

Absence of any background electrolyte in separated zone Zone. Regulation of Conc.Principle  STEADY STATE.Sample Analytes.Zones  3 Special Features 1..Seperated Analyte + Counter ion ( LE & TE) 2.By Regulation Function of Kohlrausch.Conc. of LE is a determining factor Ci = 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 mi 𝑖 . Self Sharpening Effect Boundary between the migrating sample zonespermanently sharp 3.

Detection of analytes seperated  Effective mobility of sample ion  Electric Field Strength  Temperature  Conductivity  m ∝ 𝐸 ( From Electrophoretic Eqn V=mE) m∝ m 1 (From Joule’s Heating Effect J=Ei) .Thermodetector 𝑇 1 ∝ ∝ κ ( From Ohm’s Law E=iR) – Conductivity Detector 𝑅 1 .Principle  Zone Parameters.

Principle .

Instrumentation Single Column System  To analyse normal samples Parts  LE Compartment.Earthed Electrode  Separation CompartmentCapillary tube  TE Compartment.HV Electrode  Injection Block  Detectors.Two Detector System .

Coupled Column System .Instrumentation.

effects the sensitivity & resolution 1 – Length of the Zone ∝ d2 • Leading and Terminating Electrolyte System • • • • Anions.Instrumentation.20-100cms Dia of the bore.PTFE is used Length range.0.4mm Length and Dia.5-10mmol/l Counter ion.Low pH Cations.High pH Conc.Seperation Tube – – – – Material.inert.2.regulates pH shouldn’t be detected by UV .0.Components • Capillary Tube.

Operating Variables Selection of Leading & Trailing Electrolyte System Operating Variable.5-20kV .Current & Voltage • m∝ I • Initial current .40-75𝜇A • Voltage .100-150𝜇A • Just before reaches detector .

Thermistors. Thermocouples Conductivity Detector UV.Detectors  General Detectors      Thermodetectors.Instrumentation.Photometric Detectors Fluorescence Detectors Mass Detectors  Specific Detectors  Two detector system is impotant  Sample with identical effective mobility can be seperated  Steady State can be checked by comparing zone lengths in two traces .

Detectors Thermodetector .Instrumentation.

Detectors UV Detector .Instrumentation.

Instrumentation.Detectors Two Detector System .

Applications  Advantages  Fast seperation technique  Minute quantiy of sample.sufficient  Simple to perform  Simultaneous determination of both strong and weak acids  High sensitivity  High seperation efficiency  Sample Pretreatment.not necessary  Seperation of small ions upto 1000Da is possible .

IMP  Aminoacid Analysis . Lipoproteins. AMP. CSF proteins. NADH. cAMP.Applications  Biomedical Field  Protein Analysis Serum proteins. Urinary Proteins  Forensic Investigation Differtiates blood of male from female at crimes of violence Human blood from bovine and ovine  Purine & Pyrimidine Analysis. ADP.Metabolism disorders  Nucleotide Analysis.ATP.

Phenylketonuria • Various Metabolic Disorders  Peptide Analysis  Organic Acids Analysis  Inorganic Compounds Analysis  Pharmaceutical Drug Analysis  Food Analysis.Preservatives  Environmental Analysis • Analysis of air. water. soil .Applications  Amino Acid Analysis • Phenylalanine.

Applications .

Applications .

Isotachopherogram Isotachopherogram depicting the seperation of five components .Interpretation.

zone length .Step height Quantitative.Interpretation Calibration Curve obtained from a set of standard Isotachopherograms Qualitative.

Further Developments On chip ITP .