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