capillary electrophoresis and application

ELECTROPHORESIS

Presenter Dr Anurag YadavPost-graduate, biochemistryFather Muller Medical collegeDr Anurag yadav,Bio-FMMC1ELECTROPHORESIS

Dr Anurag yadav,Bio-FMMC2Technique first described by- Jorgensen and Lukacs (1980s) Also referred as High performance capillary electrophoresis(HPCE)Capillary zone electrophoresis (CZE)Free solution capillary electrophoresis (FSCE)Capillary electrophoresis (CE)

Upadhyay book2

Dr Anurag yadav,Bio-FMMC3The sensitivity has made it as one of the choice for many biomedical and clinical analyses.

Clinical application: estimation of 1. urine protein. 2. serum protein. 3. HbA1c estimation3

Dr Anurag yadav,Bio-FMMC4

Dr Anurag yadav,Bio-FMMC5Reservoirs.Electrical buffer.Power source.Capillary tube.Detector and computer.Sample applicator.


High voltage injectionPressure injectionDr Anurag yadav,Bio-FMMC7The buffer reservoir is replaced by the sample reservoirthe high voltage is applied (+ electrode)buffer reservoir is placed again and voltage applied for the separation.Anodic end of capillary is removed from buffer and placed in air tight sample solwith pressure sample is pushed into capillarykept back in the buffer sample and voltage is applied.

Dr Anurag yadav,Bio-FMMC8As the name suggest, the separation is carried in a narrow bore Capillary

Dr Anurag yadav,Bio-FMMC9High voltage is applied (up to 50 kV)The components migrate at different rate along the length.Although separated by the electrophoretic migration, all the sample is drawn towards cathode by electroendosmosis.Since this flow is strong, the rate of electroendosmotic flow is greater than the electrophoretic velocity of the analyte ion, regardless of the charge.


Dr Anurag yadav,Bio-FMMC11DETECTION:near to cathode end, viewing window- Detected by the ultraviolet monitor, transmit signal and integrated by computer.Refractive indexFluorescenceCE-MS

Dr Anurag yadav,Bio-FMMC12Troubleshooting :Adsorption of protein to the wall of capillary leading to smearing of protein viewed as peak broadening or complete loss of protein.- Use of neutral coating group to the inner surface of the capillary.

Dr Anurag yadav,Bio-FMMC13Advantage over slab type:Reduce the problem of heating effect.Large surface to volume ratio.Less diffusion of the separated bands.

Dr Anurag yadav,Bio-FMMC14Variations in technique:Add of surfactant to buffer i.e., SDS (for Neutral molecules).Micellar formation In MECC- electrophoresis + chromatography.

Dr Anurag yadav,Bio-FMMC15Capillary zone electrophoresis :Separation principle based on charge to mass ratio of molecule.Separation is faster.Due to High EOF, the molecules regardless of the charge, they are moved to cathode.

Dr Anurag yadav,Bio-FMMC16Micellar electrokinetic chromatography:It is an hybrid.Used for separation of the neutral and charged solutes.The separation is accomplished by micelles formation. (8-9mmol/L for SDS)During migration, micelle interact with analyte as chromatographic manner and the separation is brought about.


Dr Anurag yadav,Bio-FMMC18Capillary gel electrophoresis: Identical to the slab.Separation based on the sieving.The capillary is filled with sieving matrix or soluble polymer network.Low viscosity, self entangling for formation of pore size.Variety of polymeric matrices are available for DNA and Protein.Cross linked polyacrylamide- choice of polymer.


Dr Anurag yadav,Bio-FMMC20Capillary isoelectric Focussing Electrophoresis:Is comparable to tube IEF.Carried out in the capillary.The focused zone migrate to the detector with the separated sample.cIEF is completed in ~15 min.

Dr Anurag yadav,Bio-FMMC21Capillary Isotachophoresis:Same feature as ITP.Except condition of pure ITP not achieved.Typically used for online sample preconcentration.CZE, MEKC, CGE.





Dr Anurag yadav,Bio-FMMC26Current advanced method.Development in technique includeIntegrated microchip designAdvanced detection systemNew applicationProtein and DNA separation can be done

Dr Anurag yadav,Bio-FMMC27Similar to the capillary electrophoresis.Separation channelSample injection (50-100pL)ReservoirsVoltage (1-4kV)sample preparationPrecolumn or postcolumn reactors.Classical Cross-T design.Time period of 50-200sec.


Dr Anurag yadav,Bio-FMMC29An alternative for the DNA analysis.Herpes simplex virus DNA in CSF for diagnosing encephalitis.Gene rearrangement correlative with lymphoproliferative disorders.Polymorphisms in gene.Tetranucleotide associated with hypercholesterolemia.Diagnosing fragile X syndrome.Muscular dystrophy.Anthracis specific PCR product.

Dr Anurag yadav,Bio-FMMC30Thank you

Dr Anurag yadav,Bio-FMMC31Keith Wilson- Principles and techniques of biochemistry and molecular biology.Upadhyay- biophysical chemistry.Tietz- Text book of clinical chemistry.Kaplan- clinical chemistry.YouTube and Google images.

capillary electrophoresis and application

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