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ISOTACHOPHORESIS

Capillary isotachophoresis is an electrophoretic technique that focuses solute ions between a leading and terminating electrolyte based on their electrophoretic mobilities. The solute ions stack into sharp zones moving at constant velocities. It is commonly used as a sample concentration technique prior to analysis. The instrument consists of a capillary tube placed between compartments containing the leading and terminating electrolytes. Detection methods include thermal and conductivity detectors. Applications include analysis of amino acids, proteins, nucleotides, and inorganic/organic compounds in fields like biomedicine, forensics, and environmental analysis.

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Sesha Madhavi Maruvada
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3K views31 pages

ISOTACHOPHORESIS

Capillary isotachophoresis is an electrophoretic technique that focuses solute ions between a leading and terminating electrolyte based on their electrophoretic mobilities. The solute ions stack into sharp zones moving at constant velocities. It is commonly used as a sample concentration technique prior to analysis. The instrument consists of a capillary tube placed between compartments containing the leading and terminating electrolytes. Detection methods include thermal and conductivity detectors. Applications include analysis of amino acids, proteins, nucleotides, and inorganic/organic compounds in fields like biomedicine, forensics, and environmental analysis.

Uploaded by

Sesha Madhavi Maruvada
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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ISOTACHOPHORESIS

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

Applications Interpretation

Introduction
ITP Isotachophoresis
Iso = equal Tacho = speed Phoresis = migration

Migration of ions under electric field with equal speed. One of the modes of capillary electrophoretic technique suitable for analyzing mixtures of ionogenic substances Comparable to Displacement Chromatography Developed by Martin, Everaerts, Verheggen
Separation Technique, Analyte Concentration Technique

Introduction
Capillary Isotachophoresis (CITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp, focused zones.

Although it is used as a mode of separation, transient ITP has been used primarily as a sample concentration technique.

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

Historical Review
1850s Wideman & Buff
- Charged particles migrate in solution when electric field is applied

Kohlrausch Theory of Ionic Migration


- Regulating function of Kohlrausch

Hardy separated proteins based on pH of electrolyte (IEF) 1942 Martin separated chloride, acetate, aspertate, glutamate by ITP Everarts, Verheggen, Martin designed an instrument for capillary ITP

Principle
Under the influence of an electric field (E), charged particles will move at a velocity acc. to = Mobility is different for different molecules In ITP velocity is kept constant miEi= constant m ITP
Cationic Anionic
1

Carried out in discontinuous electrolyte system

Principle
Analytical ITP Apparatus

Principle

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

Principle
Zone Parameters- Detection of analytes seperated
Effective mobility of sample ion Electric Field Strength Temperature Conductivity
m ( From Electrophoretic Eqn V=mE)
m m
1 (From Joules Heating Effect J=Ei) - Thermodetector 1 ( From Ohms Law E=iR) Conductivity Detector 1

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

Instrumentation- Coupled Column System

Instrumentation- Components
Capillary Tube- Seperation Tube
Material- inert- PTFE is used Length range- 20-100cms Dia of the bore- 0.2- 0.4mm Length and Dia- effects the sensitivity & resolution
1

Length of the Zone

d2

Leading and Terminating Electrolyte System


Anions- Low pH Cations- High pH Conc- 5-10mmol/l Counter ion- regulates pH
shouldnt be detected by UV

Operating Variables
Selection of Leading & Trailing Electrolyte System

Operating Variable- Current & Voltage m I Initial current - 100-150A Just before reaches detector - 40-75A Voltage - 5-20kV

Instrumentation- Detectors
General Detectors
Thermodetectors- Thermistors, Thermocouples Conductivity Detector UV- 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

Instrumentation- Detectors
Thermodetector

Instrumentation- Detectors
UV Detector

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

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

Applications
Amino Acid Analysis Phenylalanine- 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

Applications

Interpretation- Isotachopherogram
Isotachopherogram depicting the seperation of five components

Interpretation
Calibration Curve obtained from a set of standard Isotachopherograms

Qualitative- Step height Quantitative- zone length

Further Developments
On chip ITP

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