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

𝑉 = 𝑚 ∗ 𝐸

Mobility is different for different molecules

In ITP velocity is kept constant

miEi= constant m ∝1

𝐸

ITP

Cationic

Anionic

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 zones-

permanently sharp

3. Regulation of Conc.- By Regulation Function of

Kohlrausch- Conc. of LE is a determining factor

Cimi= 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡

𝑖

Principle

Zone Parameters- Detection of analytes seperated

Effective mobility of sample ion

Electric Field Strength

Temperature

Conductivity

m ∝1

𝐸 ( From Electrophoretic Eqn V=mE)

m ∝1

𝑇 (From Joule’s Heating Effect J=Ei) - Thermodetector

m ∝1

𝑅 ∝ κ ( From Ohm’s Law E=iR) – Conductivity Detector

Principle

Instrumentation

To analyse normal samples

Parts LE Compartment- Earthed

Electrode

Separation Compartment-

Capillary tube

TE Compartment- HV Electrode

Injection Block

Detectors- Two Detector System

Single Column 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

– Length of the Zone ∝ 1

d2

• Leading and Terminating Electrolyte System

• Anions- Low pH

• Cations- High pH

• Conc- 5-10mmol/l

• Counter ion- regulates pH

shouldn’t be detected by UV

Operating Variables

Selection of Leading & Trailing Electrolyte System

Operating Variable- Current & Voltage

• m∝ I • Initial current - 100-150𝜇A

• Just before reaches detector - 40-75𝜇A

• Voltage - 5-20kV

Instrumentation- Detectors

General Detectors

Thermodetectors- Thermistors, Thermocouples

Conductivity Detector

Specific Detectors

UV- Photometric Detectors

Fluorescence Detectors

Mass 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