Slide 1: 1 PRINCIPLE AND INSTRUMENTATION OF CAPILLARY ELECTROPHORESIS : 2 PRINCIPLE AND INSTRUMENTATION OF CAPILLARY ELECTROPHORESIS PRESENTED BY
ABIKESH PRASAD KUMAR MAHAPATRA
2 nd YEAR
Regd No.:-0808254026 : 3 Introduction
Importance Of separation technique
Why capillary Electrophoresis
What is CE
Types of CE
CE The Basics of the instrumentation
Theory of Capillary Electrophoresis
Flow in CE
Equipment of CE
Methods For Improving Efficiency of CE
Summary and conclusion CONTENTS Capillary Electrophoresis : 4 Capillary Electrophoresis Introduction : 5 Introduction As in the emerging era separation of different pharmaceutical components are important issue so for that purpose different techniques are used.
Among that one of the most efficiently used separation technique is capillary Electrophoresis.
This describes the basic theoretical concepts and principles of capillary electrophoresis (CE).
The depth of discussion should provide enough background to understand the basic operation of CE instruments and the principles
by which CE separates analytes. Electrophoresis—An Overview : 6 Electrophoresis—An Overview Definition: The differential movement for migration of ions by attraction or repulsion in an electric field.
Separation of components of a mixture using an electric field
v = velocity of molecule
E = electric field
q = net charge of molecule
f = friction coefficient Electrophoresis- overview cont. : 7 Electrophoresis- overview cont. Can determine the size, shape, and charge of a molecule
Different forms of electrophoresis are used for each of these factors independently or in combination. WHY SEPARATION TECHNIQUE : 8 WHY SEPARATION TECHNIQUE Separation Science has grown to become one of the most useful scientific tools.
Used extensively in diagnostic and clinical science.
Separation Science is relied upon by scientists, physicians, law enforcement officials and the general public to provide quantified information about our health, our food, our environment, our products we use and in almost every aspect of our modern society Cont... : 9 Cont... Separation Scientists work in many different disciplines, some of these include:
Medical Research and Production
Pharmaceutical and Nutraceutical Science
Other Disciplines Slide 10: 10 Why Capillary Electrophoresis ?????? WHAT IS CAPILLARY ELECTROPHORESIS : 11 WHAT IS CAPILLARY ELECTROPHORESIS In practical terms, a positive (anode) and negative (cathode) electrode are placed in a solution containing ions.
Then, when a voltage is applied across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive), will move through the solution towards the electrode of opposite charge.
Capillary electrophoresis, then, is the technique of performing electrophoresis in buffer-filled, narrow-bore
capillaries, normally from 25 to 100 pm in internal diameter (ID). Types of Electrophoresis : 12 Types of Electrophoresis Capillary
Native Polyacrylimide Gel Electrophoresis (PAGE)
Paper Capillary Electrophoresis – The Basics Of Instrumentation : 13 Capillary Electrophoresis – The Basics Of Instrumentation Electrophoresis in a buffer filled, narrow-bore capillaries
Each capillary is about 25-100 µm in internal diameter
When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge
Depending on the charge, the molecules move through at different speeds
Separation is achieved Basics cont. : 14 Basics cont. A photocathode is then used to measure the absorbencies of the molecules as they pass through the solution
The absorbencies are analyzed by a computer and they are represented graphically Slide 15: 15 Cont…. : 16 Cont…. Cont…. : 17 Cont…. Capillary Electrophoresis Apparatus : 18 Capillary Electrophoresis Apparatus COMPARISION OF CE WITH HPLC : 19 COMPARISION OF CE WITH HPLC So, if we consider that the power supply is equivalent to an HPLC
pump and the capillary is equivalent to a column, the instrumentation is
This is especially so as the software packages used to control most commercial CE instruments are based heavily on existing HPLC software. Electroosmotic Flow (EOF) : 20 Electroosmotic Flow (EOF) A vitally important feature of CE is the bulk flow of liquid through the capillary.
This is called the electroosmotic flow and is caused as follows. Slide 21: 21 Stern’s model of the double-layer charge distribution at a negatively charged capillary wall leading to the generation of a zeta potential and EOF Electroosmotic Mobility : 22 Electroosmotic Mobility Zeta Potential
The change in potential across a double layer
Proportional to the charge on the capillary walls and to the thickness of the double layer.
Both pH and ion strength affect the mobility Flow Profile in CE : 23 Flow Profile in CE A further key feature of EOF is that it has flat flow profile, which is shown in Figure alongside the parabolic flow profile generated by an external pump, as used for HPLC.
EOF has a flat profile because its driving force (ie., charge on the capillary wall) is uniformly distributed along the capillary, which means that no pressure drops are encountered and the flow velocity is uniform across the
In HPLC, in which frictional forces at the column walls cause a pressure drop across the column, yielding a parabolic or laminar flow profile.
The flat profile of EOF is important because it minimizes zone broadening, leading to high separation efficiencies that allow separations on the basis of mobility differences as small as 0.05 %. Slide 24: 24 The Electropherogram : 25 The Electropherogram The data output from CE is presented in the form of an electropherogram, which is analogous to a chromatogram.
An electropherogram is a plot of migration time vs. detector response.
The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence.
The appearance of a typical electropherogram is shown in Figure for the separation of a threecomponent mixture of cationic, neutral and anionic solutes. Equipment : 26 Equipment Capillary tube
Varied length but normally 25-50 cm
Small bore and thickness of the silica play a role
Using a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage Equipment Cont…. : 27 Equipment Cont…. Detector
Radiometric (for radioactive substances)
Mass Spec. HINTS FOR IMPROVING EFFICIENCY OF CE : 28 HINTS FOR IMPROVING EFFICIENCY OF CE Buffers
Additives for CZE
Additives for HPCE
Ionic Strength in HPCE
PKa Values of Common Buffers
Proteins, Choosing a Proper Buffer
How to Properly Cut A Capillary (Animated)
Migrating Peak Correction Applications : 29 Applications Analysis of carbohydrates
Analysis of inorganic anions/metal ions
Protein identification Advantages
Cannot identify neutral species
Cannot discern shape Summary : 30 Summary CE is based on the principles of electrophoresis.
The speed of movement or migration of solutes in CE is determined by their
size and charge. Small, highly charged solutes will migrate more quickly than large, less charged solutes.
Bulk movement of solutes is caused by EOF.
The speed of EOF can be adjusted by changing the buffer pH used.
The flow profile of EOF is flat, yielding high separation efficiencies.
The data output from CE is called an electropherogram. Conclusion : 31 Conclusion It is the most efficient separation technique available for the analysis of both large and small molecules.
DNA Profiling, protein identification,
inorganic metals and ions can be detected easily by this method.