Separation principle, capillary

Fig. 17.14. Separation principle in MECC. A compound (neutral or charged) is partitioned between the micellar and aqueous phase. A fully solubilized neutral compound migrates with the velocity of the micelles. A neutral compound with no affinity for the micelles migrates with the velocity of the EOF. A neutral compound with an affinity for both the micellar and the aqueous phase migrates with an intermediate velocity. (A) Schematic overview of the partitioning of compound (N the EOF moves toward the cathode and the typical SDS micelles toward the anode. (B) Diagram of the zone distribution within the capillary. (C) Reconstructed typical electropherogram.

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. ... 

Electrophoresis has been used for a long time as one of the most important separation principles in analytical biochemistry. In particular, it has been applied to the separation of DNA and DNA components. Electrophoretical techniques have predominated in this field for decades and will continue to do so in the future. The advent of capillary electrophoresis (CE) has boosted the development of electrophoretic techniques, because it opened access to higher sensitivity, better resolution, and greater speed of separation (1-3). 

G Gubitz, MG Schmid. Recent progress in chiral separation principles in capillary electrophoresis. Electrophoresis 21 4112-4135, 2000. 

For packed columns, typical values [701] are h = 3 and v= 3, so that h/ v= 1. For open columns typically h = 1.5 and v= 5, so that /i/ v= 0.3. Consequently, capillary columns will lead to analysis times that are about three times shorter (for dp— dc) for the same separation (N and k constant). Therefore, in principle, capillary columns are superior to packed columns. Unfortunately, capillary columns cannot always be used. This arises from the occurrence of the diffusion coefficient (Dm) in eqn.(7.6). Typically, Dm is 10,000 times larger in gases than it is in liquids. This necessitates the use of very small particles (typically 5-10 pm) in HPLC columns. If we compare packed and capillary columns with dp= which is a reasonable assumption for GC [702], then capillary columns with very small internal diameters need to be considered for LC [703]. Such very narrow columns impose extreme demands on the instrumentation, and at present open tubular columns cannot be used for practical LC separations. 

Capillary electrophoresis combines the separation principles of gel electrophoresis with the throughput and detection methods of HPLC. It overcomes the disadvantages of slab gel electrophoresis, including slow and labor intensive procedures and the difficulty and inaccuracies of quantitation. CE is... 

The separation principle in capillary gel electrophoresis (CGE) is the same as that of slab gel electrophoresis. Most often CGE is used in a denaturing mode with the incorporation of SDS and is referred to as SDS-CGE. As such, separation is based on the protein s molecular mass and, due to the sieving mechanism of the gel, smaller proteins migrate past the detector first. The use of a gel material and SDS decreases the EOF and eliminates protein adsorption to the capillary walls further ensuring that migration is based on molecular mass. This precludes the need for additives and coated capillaries. 

The first applications of CDs as chiral selectors in CE were reported in capillary isotachophoresis (CITP) [2] and capillary gel electrophoresis (CGE) [3]. Soon thereafter, Fanali described the application of CDs as chiral selectors in free-solution CE [4] and Terabe used the charged CD derivative for enantioseparations in the capillary electrokinetic chromatography (CEKC) mode [5]. It seems important to note that although the experiment in the CITP, CGE, CE, and CEKC is different, the enantiomers in all of these techniques are resolved based on the same (chromatographic) principle, which is a stereoselective distribution of enantiomers between two (pseudo) phases with different mobilities. Thus, enantioseparations in CE are commonly based on an electrophoretic migration principle and on a chromatographic separation principle [6]. 

What is the principle of separation by capillary zone electrophoresis ... 

Gtibitz, G. Schmid, M.G. Review chiral separation principles in capillary electrophoresis. J. Chromatogr. A 1997, 792, 179-225. 

A commercial CE system and a micropacked capillary was used to separate N—, O—, and S-containing heterocyclic compounds. Migration time reproducibility, linearity, and detector response was found to be comparable to HPLC. A study of the heterocyclic compound s elution order followed that predicted by the octanol-water partition coefficients (354). While chiral CEC provides improved resolution and higher efficiencies, additional work is needed since chiral CEC capillaries are not available commercially. The separation principles and chiral recognition mechanism for the separation of enantiomers have been reviewed (355). Furthermore, a comprehensive collection of drug applications and other compounds of interest has been reported (356). Direct enantiomeric separations by CEC were studied using a capillary packed with alpha-1-acid glycoprotein chiral stationary phase (357). Chiral resolution was achieved for enantiomers of benzoin, hexobarbital, pentobarbital, fosfamide, disopyramide, methoprolol, oxprenolol, and propanolol. The effects of pH, electrolyte concentration, and con-... 

Another capillary technique, also with significant potential but again just used for analytical enantioseparations, is CEC [173, 174]. This hybrid technique relies on electrophoretic migration and chromatographic separation principles. The enantioseparation of several chiral compounds in non-aqueous CEC using polymethacrylate-type (Chiralpak OP) packing material is shown in Fig. 10 [174]. 

Table 3.2. A number of modes of capillary electrophoresis with their commonly used abbreviations, separation principle and applications.

Chankvetadze, B. Capillary Electrophoresis inChiral Analysis. (John Wiley Sons, New York, 1997). Gubitz G, Schmid M.G, Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography. Electrophoresis, 25,3981-3996 (2004). 

HeUer, C., Principles of DNA separation with capillary electrophoresis. Electrophoresis, 22, 629, 2001. 

The movement of soil colloidal particles was the first description of electrophoresis as early as 1809. However, Arne Tiselius ( 1937) was the first to construct a successful instrument useful for the separation of serum protein by electrophoresis using the boundary separation principle. Because of the clinical significance of this type of separation, many improvements and refinements followed, such as utilizing paper, cellulose acetate, gel, and more recently capillaries in order to speed up and better separate (into distinct zones) the different proteins. The electric current can be utilized in the clinical applications to accomplish not just separation but other tasks ... 

---