General Considerations And Method Development In CE,

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capillary electrophoresis

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General Considerations And Method Development In CE, Crown Ether As Buffer Additives In Capillary Electrophoresis,CE-MS Hyphenation Technique:

General Considerations And Method Development In CE, Crown Ether As Buffer Additives In Capillary Electrophoresis,CE-MS Hyphenation Technique By G.Pavani Y17mphpa426 Chalapathi Institute Of Pharmaceutical Sciences Lam,Guntur.

CONTENTS:

CONTENTS DEFINITION PRINCIPLE PROCEDURE INSTRUMENTATION SAMPLE INTRODUCTION TECHNIQUES CE-MS HYPHENATION TECHNIQUE APPLICATIONS.

ELECTROPHORESIS:

ELECTROPHORESIS Electro-Electricity Phoresis-Separation Literally,the term electrophoresis means migration of ions with electricity. “It involves separation of Components of a sample by the differential rate of migration of ions by attractions (or) repulsion in an applied dc electric field”. The technique was first developed by Arne Tiselius in the 1930’s for the study of serum proteins. He was awarded the 1948 Nobel Prize in Chemistry. It is been Principal method for the separation of Proteins (Enzymes Harmones Antibiotics Nucleic Acids)

Capillary electrophoresis:

Capillary electrophoresis “Electrophoresis Performed in a thin tube subjected to Capillary”. Principle:Electrophoretic Mobility Electro Osmotic Flow(EOF) Electrophoretic Mobility The rate of Migration (usually/s)per unit electric field strength (usually V/cm)of a charged particle in Electrophoresis. Symbol-µ It has been found that, the migration Velocity (v)(cm - 1) of a molecule in an electric field is given by, V=µeE Where,E= Strength of the electric field(Vcm-1) µe= Electrophoretic mobility(cm2 V-1 S-1) The value of E depends upon the, Charge of the Analyte ion The frictional retarding forces which includes, - Size and shape of the ion - Viscosity of the medium, in which migration occurs.

Slide 5:

Where in HPLC- No.of Plates – Efficiency of Separation . Plate count= n(16rt)2 W In CE, N= µeV 2D Where,N= No.of Plates µe = Electrophoretic Mobility V = Migration Velocity D = Diffusion co-efficient of the solute(cm-1 s-1) Plate count - resolution High voltage is applied to achieve high resolution separations in CE. In gel slab electrophoresis,joule heating limits the magnitude of applied voltage is about 500 V. Plate heights in CE:

Slide 6:

In Capillary electrophoresis- Due to , small cross sectional area Longer length of the capillary Broad banding is not seen inCE. Electric fields of 100-400V/Cm are typically used. Plate count range of CE- 100,000 to 200,000(1 lakh to 2 lakhs) HPLC-5,000 to 20,000 Plate counts of 3000,000 (3 lakhs ) have been reported for CZE of dansylated amino acids. Plate counts of 10,00000 (1 lakh) have been reported for CGE of Polynuclotides.

Slide 7:

Electro Osmotic flow(EOF): A unique feature in CE is EOF. “ Bulk flow of liquid throuh theCapillary is Called as EOF. i.e, Silica Capillary Walls Contain Surface Silonal (SI-OH)- groups & these are ionized at pH values higher than 3 and the wall becomes (-ve)ly Charged. Wall attracts Cation and double layer forms. Cations inthe diffuse layer are attracted towards Cathod and migrate in that direction as they are in solution, the buffer fluid is also dragged along with them. The direction of flow of EOF may be reversed by the treatement with cetyl trimethyl ammonium bromide.

Slide 10:

CE-STEPS Electrophoresis is done in bufferfilled, narrow bore capillaries. Each Capillary is about 25-100 µm in internal diameter. Small cross sectional area, long length, leading to high resistance, low currents. .Vmax =20-100Kv. N = 100,000-10,000,000 high resolution. When a Voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge .

Slide 11:

Depending on the Charge , the molecules move through at different speeds. Thus Separation is Achieved. A suitable detector is then used to detect the solute as it comes out from the end of the Capillary. The data Obtained are analyzed by a Computer and represented Graphically.

insrumentation:

insrumentation

Slide 14:

The instrumentation of CE is relatively Simple. A buffer filled fused –Silica Capillary,typically 10 to 100 µm in internal diameter& 30 to 100 cm long, extended between two buffer reserviors that also hold platinum electrodes. Like the Capillary tubes used in GC, the outside walls of the fused- silica Capillary are typically coated with Polyimide for durability, flexibility,&Stability. The Sample is Introdused at one end & detection occurs at another end. A Voltage of 5 to 30 Kvdc is applied across the two electrodes.

Slide 15:

The Polarity of this high Voltage canbe as indicated in fig can be reversed to allow rapid separation of anions. High- Voltage Electrophoresis Compartments are usually safety interloked to protect the user. Although the instrumentation is conceptually simple, significant experimental difficulties in sample introduction & detectionarise due to the Very small volumes involved. Because the volume’s must be on the order of a few nl (Or)less.

SAMPLE INTRODUCTION:

SAMPLE INTRODUCTION By 2 methods,-Electro kinetic injection Pressure injection Electro kinetic injection:- One end of the Capillary & its Electrode are removed from their buffer Compartment &Placed in a small cup containing the Sample. A Voltage is then applied for a measured time,Causing the Sample to enter the Capillary, by a combination of ionic migration &EOF.

Slide 17:

The Capillary end & electrode are then returned to the regular buffer solution for duration of the separation. This injection technique discriminates by injecting larger amounts of the more mobile ions relative to the slower moving ions.

ii.Pressure injection:

ii.Pressure injection The sample introduction end of the capillary is also placedin small cup containing the sample,but here a pressure difference drives the sample solution into the capillary. The pressure difference can be produced by applying a vaccume at the detector end, by pressurizing the sample, (or) by elevating the sample end (hydrodynamic injection) Pressure injection does not discriminates because of ion mobility, but it can’t be used in gel-filleed capillaries.

Slide 19:

For both Electrokinetic injection & Pressure injection the Volume injected is Controlled by the duration of the injection. Injections of 5 to 50nl are common and volumes below 100pl have been reported. For a buffer with density & Viscosity near the values for water, a height differential of 5 cm for injects about 6nl with a 75 µm inside diameter capillary.

Slide 20:

Micro injection tips constructed from capillaries drawn to very small diameters allow sampling from picoliter environments such as single cells (or) substructures with in single cells. This technique have been to study aminoacids & neurotransmitters from single cells. Other novel injection techniques have been described in the literature, commercial CE systems are available with thermostatted multiplication for automated sampling.

DETECTION FOR CE:

DETECTION FOR CE TYPE OF DETECTOR REPRESENTATIVE DETECTION LIMIT (attamoles detected) Spectrometry Absorption 1-1000 Flourescence 1-0.01 Thermal lens 10 Raman 1000 Chemoluminiscence 1-0.0001 Mass spectrometry 1-0.01 Electrochemical Conductivity 100 Potentiometry 1 Amperometry 0.1

Slide 22:

CE-MS HYPHENATED TECHNIQUE

Slide 23:

The very small volumetric flow rates of less than 1 µl/min from electrophoresis capillaries make it feasible to couple the effluent directly to the ionization sources of a mass spectrometer . The most common sample introduction and ionization interface for this purpouse is currently, electronspray fast atom bombardment, matrix-assisted laser desorption ionization (MALDI) spectrometry, Inductively coupled plasma mass spectrometry (ICPMS) have also been used. Because the liquid sample must be vapourised before entering the mass spectrometry(MS) system, It is imp to that volatile buffers be used. CE-MS systems have become quite imp in the life sciences for determining large biomolecules

Slide 24:

Proteins DNA fragments Peptides FIG-a typically electron spray interfacecoupled to a quadrapole mass spectrometer, Note that the capillary is positioned b/w the isolated high-voltage region and the electronspray source. The high-voltage end of the capillary was at 30 to 50 kV with respect to common. The low-voltage end was maintained at 3-5kv and charged the droplets. Similar electronspray instruments are available commercially coupled with either quadrapole or ion-trap mass spectrometers. Ion trap mass spectrometers can allow CE-MS/MS(Or) MSn operation.

Slide 26:

Spectrum-For vasotocin, A polypeptide having mass of 1050. Note the presence of doubly and triply charged species. Wiith higher molecular mass species, ions are often observed with charges of +12 or more. Ions with such charge make it possible to detect high molecular mass analytes with a quadrapole instrument with a relatively modest mass range. Typical detection limits for CE-MS are of a few tens of molecules with molecular massess of 1000,000 (1 lakh) or more.

Applications:

Applications The vast applications of electrophoresis include, Vaccine analysis Protein and DNA analysis Used in forensic investigations Determination of impurities Chiral analysis Analysis of Carbohydrates and other macromolecules Analysis of inorganic anions /metal ions.

Vaccine Analysis:

Vaccine Analysis Vaccine Analysis is one of the many important applications of electrophoresis. There are Several Vaccines that have been Purified, Processed and analyzed through Electrophoresis ,such asthe, Influenza vaccine,Hepatitis vaccine,Poli vaccine.

PROTEIN ANALYSIS:

PROTEIN ANALYSIS Electrophoresis has advanced our understanding on the structure and function of proteins. These molecules are needed by our body cells and may be analyzed, for instance, by getting blood and urine samples. Then through electrophoresis, the amount of Proteins in your blood or in your urine is measured and compared to establish normal values ---lower or higher than the normal levels usually indicates a disease.

Assay of drugs:

Assay of drugs Atropine Sulphate i.v. Solution. Codeine Phosphate Syrup. Ketamine HCLi.v. Solution. Have Successfully been assayed by this Process.

THE END PREPARED BY PAVANI.G:

THE END PREPARED BY PAVANI.G

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