Control of EOF

Sometimes more subtle adjustment of the magnitude and direction of the EOF is needed. Yeung and Lucy [3] reported a monotonic alteration of EOF from fully reversed to near zero by coating the capillary with variable nuxtures of cationic and zwitterionic surfactants. Addition of a low concentration of an aliphatic amine salt to the electrolyte is another way to vary the EOF. 

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The control of EOF is critical to the migration time precision of the separation. Among the factors affecting the EOF are buffer pH, buffer concentration, buffer viscosity, temperature, organic modifiers, cationic surfactants or protonated amines, polymer additives, field strength, and the nature of the capillary surface. 

Electroosmotic flow was first identified in the late 1800s when Helmoltz " conducted experiments involving the application of an electrical field to a horizontal glass tube containing an aqueous salt solution. Curious about the ionic character of the inner wall and the movement of ions, he found that the silica imparted a layer of negative charge to the inner surface of the tube, which under an applied electric field, led to the net movement of fluid toward the cathode. More than a century later, this phenomenon still plays the fundamental role in CE analysis. Moreover, the importance of the control of EOF has been realized and has become the focus of several research groups. 

The pH value also affects the ionization of acidic and basic analytes and their electromigration. Since this migration can be opposite to that of the electroos-motic flow, it may both improve and impair the separation. This effect is particularly important in the separation of peptides and proteins that bear a number of ionizable functionalities. Hjerten and Ericson used monolithic columns with two different levels of sulfonic acid functionalities to control the proportion of EOF and electromigration. Under each specific set of conditions, the injection and detection points had to be adjusted to achieve and monitor the separation [117]. Another option consists of total suppression of the ionization. For example, an excellent separation of acidic drugs has been achieved in the ion-suppressed mode at a pH value of 1.5 [150]. 

The zeta potential and the thickness of the double layer (1/k) decrease rapidly with an increase in ionic strength or the valence of the electrolytes in the capillary (Equations (5) and (6)). Therefore, the ionic strength and the nature of the ions in the electrolyte solution are very important parameters determining the strength of the EOF. Careful control of the ionic... 

Impact of control potential, EOF field and frequency for the zeta potential variation... 

Polyelectrolyte multilayers (PEM) have been used to alter surface charges, thus controlling the direction of EOF in chips made of PS [155,216,416], PMMA [216], and PETG [416]. PEM deposition was carried out by exposing the micro-channel alternatively to solutions of positively charged PEM poly(allylamine hydrochloride) PAAH and negatively charged PEM-poly(styrene sulfonate), or... 

The use of CE methods for routine quality control of synthetic or recombinant peptides-proteins necessitates optimization strategies for rapid method development. Ideally, the methods should be simple, fast, and robust. Because capillary electrophoresis in the zone format is the most simplistic, initial efforts should be directed toward the use of a simple buffer system [61]. The high efficiency and reproducibility in protein-pep-tide separations demands that interactions between the analyte and capillary wall be neglible. The use of low-pH buffers generally results in enhanced reproduciblity, and hence ruggedness, as slight variations in the capillary surface will have little impact on the already suppressed EOF. 

Direct control of the EOF in capillary zone electrophoresis can be obtained by using an external electric field. The EOF may be increased, decreased, or even reversed in the fused silica capillaries by the application of a separate potential field across the wall of the capillary. Further, the zeta potential can be changed at any time during the analysis to achieve innovative separation results. 

In the most typical format, cIEF separations are performed in the absence of EOF in three distinct steps the sample is introduced into the capillary, the analytes are focused or separated into distinct zones, and, finally, the zones are mobilized so that they pass by the detector. If the EOF is carefully controlled, it is also possible to perform cIEF separations in the presence of low EOF, in which case the mobilization step becomes unnecessary. To load the analytes, the desalted sample is usually mixed with a 1-2% (w/v) solution of the carrier ampholytes and the entire capillary is filled with the mixture. The procedure, however, requires the use of a relatively large amount of sample. By pretreating the capillary to eliminate the EOF (Section 4.3.3), it is possible to introduce smaller volumes of sample into the capillary.4344... 

Totally packed capillary columns, having one segment packed with the stationary phase and a second segment with bare silica, have been fabricated to control the EOF [56], In this case, the segment that is open in a typical CEC column is packed with bare silica to accelerate and provide a steadier EOF. Such a configuration has allowed an increased EOF that translates into shorter analysis times. Fig. 4.3 and 4 show the EOF mobility as a function of the fractional length of a column packed with bare silica and the effect on analysis time, respectively. As the porosity of the bare silica particles is increased, the EOF is also increased [56], Columns have also been packed... 

Monoliths containing two significantly different percentages of dimethyldiallylam-monium chloride 15 were recently prepared in order to control the EOF component of the overall migration rate of proteins [31]. These charged moieties were incorporated into the monolith during a later stage of the preparation process. This process appeared to be well suited to achieve monolith with properties required for the desired separations (vide supra). 

Capillary zone electrophoresis is another technique which has been used to separate products such as organic acids.26 Separation is based on differences in the mobility of analytes exposed to an electric field. Resolution and separation time in such systems depends on factors including electroosmotic flow (EOF), and a number of approaches for adjusting the EOF have been examined. While some of the approaches (pretreatment of capillaries) are not useful as means of process control, adjusting buffer pH and the electric field27 seem to be possible handles for true feedback control of the separation, although closed-loop operation does not seem to have been attempted. 

The fundamental separation mechanism of capillary zone electrophoresis (CZE) is based on differences in the mobilities of solutes. Mobility is defined as the charge/mass ratio for each solute. Since the charge is often a function of pH, the pH is the most important adjustable parameter for control of resolution. The order of elution on bare silica at high pH is cations, unseparated neutrals, and anions. At low pH, where the EOF is very low, the anions may migrate toward the positive electrode and may not be seen using normal polarity. 

Another way to control the EOF is to vary the composition of the surface-bonded sol-gel stationary phase [60]. A sol-gel ODS-coated column was prepared with significantly stronger EOF mobility than the uncoated column. It was found that the magnitude of EOF increases (shifts toward more negative values) when the percentage of organic modifier in the mobile phase was increased. 

As is commonly known, the EOF velocity depends on the value of the gradient electric voltage and thus, EOF can be controlled by on/off control of applied... 

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