PRINCIPLE AND INSTRUMENTATION OF CAPILLARY ELECTROPHORESIS Muhammad Shoaib Khan GM Centre - 1

CONTENTS Introduction Electrophoresis Overview Importance Of separation technique Why capillary Electrophoresis What is CE Types of CE CE The Basics of the instrumentation Theory of Capillary Electrophoresis Electroosmotic Flow Electroosmotic Mobility Flow in CE The Electropherogram Equipment of CE Methods For Improving Efficiency of CE Application Summary and conclusion

Capillary Electrophoresis

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 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=Eq/f v = velocity of molecule E = electric field q = net charge of molecule f = friction coefficient

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 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... Separation Scientists work in many different disciplines, some of these include: Analytical Chemistry Biochemistry Biotechnology Forensic Science Food Science Clinical Science Neuro-Science Medical Research and Production Pharmaceutical and Nutraceutical Science Other Disciplines

Why Capillary Electrophoresis ??????

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).

CEZ Methodology - Multichannel instrument Capillary electrophoresis has been used in research since 1980 for many types of separations (single- capillary). Recent advances in technology have allowed the methodology to be more easily adapted to routine clinical use: - Multichannel instrument - Fast separations - Positive sample ID -Complete automation In 2001, SEBIA launched Capillarys using capillary electrophoresis

Types of Electrophoresis Capillary Native Polyacrylimide Gel Electrophoresis (PAGE) Slab Paper

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. 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

Cont….

Capillary Electrophoresis in Minicap/Capillarys Cathode - Anode + Protein migration EOF Electro migration DETECTION OF PROTEINS INJECTION OF SERUM The Electro-Osmotic Flow (EOF) is a stronger force than the Electrical Field. As a result, all proteins are carried towards the cathodic end of the capillary. Positive charges of the buffer solution Negative charges of capillary wall

Capillary Electrophoresis Apparatus

Capillary Electrophoresis Apparatus

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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 completely analogous. 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) 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.

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 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 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 capillary. 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 %.

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 three component mixture of cationic, neutral and anionic solutes.

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…. Detector UV/Visible absorption Fluorescence Radiometric (for radioactive substances) Mass Spec.

Applications Advantages Analysis of carbohydrates Disadvantages Fast Small Sample Relatively inexpensive Automated Disadvantages Cannot identify neutral species Joule Heating Cannot discern shape Analysis of carbohydrates Analysis of inorganic anions/metal ions DNA profiling Protein identification

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 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.