An electric field Analytical Chemistry Part 16 Summary PDF

Preview

Summary

The samples are spotted in the center of the paper, high voltage is applied, and the spost migrate according to their charges. An electric field is an electrically charged region of space, such as between a pair of electrodes connected to a power supply.  The technique utilizes the varied rates an...

Description





 

An electric field is an electrically charged region of space, such as between a pair of electrodes connected to a power supply. The technique utilizes the varied rates and direction with which different organic ions (or large molecules with charged sites) migrate while under the influence of the electric field. Rates and direction variation Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field of electrical charge.

TYPES OF ELECTROPHORESIS: 1. Paper ( glass plate + cellulose + glass plate) 2. Gel 3. Capillary



 





A power supply is needed for connection to a pair of electrodes to create the electric field. The medium (cellulose or gel) and sample to be separated are positioned between the electrodes



The soaked cellulose sheet is sandwiched between two horizontal glass plates with the ends dipped into vessels containing more electrolyte solution. The electrodes are also dipped into these vessels. The sample is spotted in the center of the sheet, and the oppositely charged ions then have room to migrate in opposite directions on the sheet

Electrophoresis is for separating ions, since only ions will migrate under the influence of an electric field, negative ions to the positive electrode and positive ions to the negative electrode Useful in biochemistry experiments

1. Ions of opposite charge will migrate in different directions and become separated on that basis 2. Ions of like charge, while migrating in the same direction, become separated due to different migration rates  Factors influencing migration rate are charge values (e.g., –1 as opposed to –2) and different mobilities. The mobility of an ion is dependent on the size and shape of the ion as well as the nature of the medium through which it must migrate

The samples are spotted in the center of the paper, high voltage is applied, and the spost migrate according to their charges. 

Qualitative analysis is performed much as with paper chromatography, by comparing the distances the individual components have migrated to those for standards spotted on the same sheet.





The thin gel slab is contained between two glass plates. The slab is held in a vertical position and has notches at the top where the samples to be separated are spotted or streaked. Only downward movement takes place, and thus only one type of ion, cation or anion, can be separated, since there is only one direction to go from the notch.

For this type of electrophoresis – only downward movement takes place meaning only one type of ion is separated either cation or anion A tracking dye can be added to the sample so that the analyst can know when the experiment is completed  The slab can be removed from the glass plates and a staining dye can be applied that binds to the components, rendering them visible  Components with different mobilities through the gel show up as different bands or streaks on the gel.  Qualitative analysis is performed as with paper electrophoresis—standards are applied alongside the samples and the components identified by their positions relative to the standards the leading edge of the sample solvent is visible via the tracking dye). 

  







a useful extension of basic gel electrophoresis in protein analysis In this technique, a series of ampholytes is placed on the slab via electrophoresis An ampholyte is a substance whose molecule contains both acidic and basic functional groups (solution of different ampholytes have different pH values) Different ampholyte molecules differ in size and therefore will have varying mobilities in the electrophoresis experiment Thus, these molecules migrate into the slab, take up different positions along the height of the slab, and create a pH gradient through the height of the slab. Amino acid molecules have different mobilities in different pH environments and also have their charges neutralized at particular pH values,...