Electrophoretic mobility sample

FIGURE 20.1 Free-flow electrophoresis separation. Laminar fluid flow carries the organelles perpendicularly to the applied electric held. Organelles migrate in the opposite direction of the electric field according to their electrophoretic mobility. Sample is introduced continually at the inlet and collected in many fractions following... 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

Log k appears to correlate with log P for standards between log P —0.5 to 5.0. One limitation of this method is that solutes must be electrically neutral at the pH of the buffer solution because electrophoretic mobility of the charged solute leads to migration times outside the range of Tm and TEof- Basic samples are therefore run at pH 10, and acidic samples at pH 3, thus ensuring that most weak acids and bases will be in their neutral form. This method has been used in a preclinical discovery environment with a throughput of 100 samples per week [24]. 

The migration or diffusion rate of HpHb complexes or of Hp alone are of importance in several of the methods discussed above. It should be observed that the electrophoretic mobility of the Hp in aged sera may decrease if the sample has been contaminated with any of the common... 

Electrokinetic (also called electromigration) injection is performed by placing the inlet of the capillary and an electrode in the sample vial. Following this a voltage is applied during a defined period of time. The sample constituents are actively carried into the capillary, and when present, the EOF also passively carries them into the capillary. For this reason, neutral compounds are also injected. The active migration is due to the effective electrophoretic mobilities of the constituents. The amount (B), in units of concentration injected into the capillary is expressed by [2,38]... 

Capillary electrophoresis (CE) is a modem analytical technique that allows the rapid and efficient separation of sample components based on differences in their electrophoretic mobilities as they migrate or move through narrow bore capillary tubes (Frazier et al., 2000a). While widely accepted in the pharmaceutical industry, the uptake of CE by food analysts has been slow due to the lack of literature dedicated to its application in food analysis and the absence of well-validated analytical procedures applicable to a broad range of food products. 

Anions and uncharged analytes tend to spend more time in the buffered solution and as a result their movement relates to this. While these are useful generalizations, various factors contribute to the migration order of the analytes. These include the anionic or cationic nature of the surfactant, the influence of electroendosmosis, the properties of the buffer, the contributions of electrostatic versus hydrophobic interactions and the electrophoretic mobility of the native analyte. In addition, organic modifiers, e.g. methanol, acetonitrile and tetrahydrofuran are used to enhance separations and these increase the affinity of the more hydrophobic analytes for the liquid rather than the micellar phase. The effect of chirality of the analyte on its interaction with the micelles is utilized to separate enantiomers that either are already present in a sample or have been chemically produced. Such pre-capillary derivatization has been used to produce chiral amino acids for capillary electrophoresis. An alternative approach to chiral separations is the incorporation of additives such as cyclodextrins in the buffer solution. 

Figure 4.19. Torsion constant a versus buffer concentration for supercoiled M13mp7 DNA in different buffers. All samples except that in 10 mM Tris contain 10 raW NaCl, so all have between 10 and 12 mM univalent positive ions. The sample in the middle contains only 10 mM NaCl. The numbers (1, 2, and 4) in the sample label refer to the gel electrophoretic mobilities, which reflect different tertiary structures, as described in the text. The a samples all contain varying amounts of Tris, while the b samples all contain citrate.

Separations in CE are based on the different velocities of charged species when they encounter an electric field thus a key parameter in CE is electrophoretic mobility. Mobility (pi) is the rate of migration of sample components under a given set of conditions ... 

In CZE, the capillary, inlet reservoir, and outlet reservoir are filled with the same electrolyte solution. This solution is variously termed background electrolyte, analysis buffer, or run buffer. In CZE, the sample is injected at the inlet end of the capillary, and components migrate toward the detection point according to their mass-to-charge ratio by the electrophoretic mobility and separations principles outlined in the preceding text. It is the simplest form of CE and the most widely used, particularly for protein separations. CZE is described in Capillary Zone Electrophoresis. ... 

In electrophoretic injection, the capillary inlet is immersed in the sample solution and a voltage is applied for a determined period of time. The amount of sample introduced into the capillary depends on the voltage and the time it was applied. Sample injection is a compromise between detection and resolution, and its parameters are often best determined experimentally. If detection is not a problem, resolution can be greatly improved by maintaining the sample plug as narrow as possible. If EOF is present, sample ions will be introduced by a combination of electrophoretic mobility and EOF under these conditions, this injection mode is generally termed electrokinetic injection. 

In native gel electrophoresis and CZE, the sample components are resolved by their differences in electrophoretic mobility or mass-to-charge ratios. Electrophoretic analysis under native conditions in gel electrophoresis is not as widely used as SDS-PAGE. In gels, disadvantages of native analysis are the low field... 

Figure 9.8 Ferguson plots for polyethylene oxide used as sieving polymer. The graphs represent plots of the polymer concentration and the log of electrophoretic mobility for six different samples. Graphs A, B, and C represent polymers with MW of 100, 300, and 900 kDa, respectively (From A. Guttman, Electrophoresis, 16 611 (1995). With permission.)...

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