Tutorial 11 Modes and Methods in Capillary Electrophoresis

By the end of the tutorial , students should be able to Define principles of 3 types of capillary electrophoresis. Discuss principle of CIEF Electrophoresis. Compare different injection mode.

Modes of capillary electrophoresis CZE Capillary Zone Electrophoresis 2. CGE Capillary Gel Electrophoresis 3. CIEF Capillary IsoElectric Focusing

I. CAPILLARY ZONE ELECTROPHORESIS Briefly describe the mechanism of separation in capillary zone electrophoresis. What parameters can be changed to optimize separation conditions in CZE? Separation in capillary zone electrophoresis results from two phenomena, electrophoretic mobility and electroosmotic mobility. Electrophoretic flow occurs as a result of movement of charges species in the electric field. cations are attracted to the cathode at the negative end of the capillary, and anions are attracted to the anode at the positive end. It depends on their charge and their mass , Neutrals are unaffected by the electric field and do not move as a result of electrophoretic flow. Electroosmotic flow results from the general flow of solvent (water) toward the cathode end of the capillary. Cations tend to "drag" solvent along as they move toward the cathode , bulk solvent flow results, carrying all species (anions, cations, and neutrals) toward the cathode. Elution order is: cations, neutrals, anions.

Reversed Mobility 2- TOWARD ANODE via reversal of polarity of the wall by addition of alkyl ammonium salt: Normal Reversed

II. Capillary Gel Electrophoresis CGE

III. CIEF separation principles A technique where proteins are separated according to their pI Proteins carry different charges based on pH (+ve, -ve or neutral) Proteins are +ve charged at pH < pI Proteins are -ve charged at pH > pI Proteins at pH = pI Protein in a medium with linear pH gradient / applied field Travel to cathode Travel to anode No movement under electric field Move toward electrode of opposite charges During movement , it will pick up or loose protons , slowing down its mobility At pH = pI , it will be uncharged and stop migrating Proteins focus into sharp bands in the pH gradient zone equal to their pI pI ranges 3-12.

Hydrodynamic injection in CE can be performed in two ways: Describe each of them

Choose the correct answer: In Isoelectric focusing, proteins: A. Precipitate out of solution when pH of gradient is above the protein pI B. Precipitate out of solution when pH of gradient is below the protein pI C. Precipitate out of solution when pH of gradient is the same as the protein pI Isoelectric focusing is a technique in which proteins are separated: A. By charge alone B. By size alone C. By pH and charge D. By pH and size

The isoelectric point (pI) of a protein is the pH at which: A. The protein has no net charge B. The protein has a negative charge C. The protein has a positive charge D. A protein migrates best in electrophoresis

In capillary electrophoresis, hydrodynamic (pressure) injection means: using an injection valve before the capillary using pressure to force the sample into the capillary using a voltage to migrate sample into the capillary All is true regarding number of theoretical plates (N) achieved in CZE Except: N Values reach 100,000 Larger N means better resolution N value depends on the capillary length Voltage can be increased unlimitedly to improve resolution.

2- 1- 3- The above chart represents an electropherogram (normal CZE), where X axis is 1- ---------------, whereas Y axis is 2- --------------------------------------; where ions appearing at part 1 of the electropherogram represents 3- ------------------- , ions appearing at part 2 of the electropherogram represents 4- ------------------, ions appearing at part 3 of the electropherogram represents 5- --------------------. 1- MIGRATION TIME 2- DETECTOR SIGNAL 3- CATIONS 4- NEUTRALS 5-ANIONS

Quiz 3 Hall Date / Time Groups H3 Wednesday 27-04-2016, 12:00-12:30 Quiz 3 will take place on Wednesday, April 27, 2016 from 12:00-12:30. It will involve lectures 9 and 10 + tutorials 8 and 9 No make up quiz will be offered. Kindly attend in your allocated locations as per the next slide. Hall Date / Time Groups H3 Wednesday 27-04-2016, 12:00-12:30  1-4 H5  5-8 H15  9-12 C7.01  13-16 C7.02  17-20 Lecture 11, PHCM662-SS2016