CZE

Capillary Zone Electrophoresis The simplest form of capillary electrophoresis is capillary zone electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and, after loading the sample, the ends of the capillary tube are placed in reservoirs containing additional buffer solution. Under normal conditions, the end of the capillary containing the sample is the anode, and solutes migrate toward... 

The direction of electroosmotic flow and, therefore, the order of elution in CZE can be reversed. This is accomplished by adding an alkylammonium salt to the buffer solution. As shown in Figure 12.45, the positively charged end of the alkylammonium ion binds to the negatively charged silanate ions on the capillary s walls. The alkylammonium ion s tail is hydrophobic and associates with the tail of another alkylammonium ion. The result is a layer of positive charges to which anions in the buffer solution are attracted. The migration of these solvated anions toward... 

Capillary zone electrophoresis provides effective separations of any charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE has been used to separate a mixture of 36 inorganic and organic ions in less than 3 minutes.Neutral species, of course, cannot be separated. 

Micellar Electrokinetic Capillary Chromatography One limitation to CZE is its inability to separate neutral species. Micellar electrokinetic chromatography... 

The elution order for neutral species in MEKC depends on the extent to which they partition into the micelles. Hydrophilic neutrals are insoluble in the micelle s hydrophobic inner environment and elute as a single band as they would in CZE. Neutral solutes that are extremely hydrophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives. 

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

Procedure. A vitamin B complex tablet Is crushed and placed In a beaker with 20.00 mL of a 50% v/v methanol solution that Is 20 mM In sodium tetraborate and contains 100.0 ppm of o-ethoxybenzamIde. After mixing for 2 min to ensure that the B vitamins are dissolved, a 5.00-mL portion Is passed through a 0.45- xm filter to remove Insoluble binders. An approximately 4-nL sample Is loaded Into a 50- xm Internal diameter capillary column. For CZE the capillary column contains a 20 mM pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer. For MEKC the buffer Is also 150 mM In sodium dodecylsulfate. A 40-kV/m electric field Is used to effect both the CZE and MEKC separations. 

Methanol, which elutes at 4.69 min, is included as a neutral species to indicate the electroosmotic flow. When using standard solutions of each vitamin, CZE peaks are found at 3.41 min, 4.69 min, 6.31 min, and 8.31 min. Examine the structures and p/Ca information in Figure 12.47, and determine the order in which the four B vitamins elute. 

Caffeine in tea and coffee is determined by CZE using nicotine as an internal standard. The buffer solution is 50 mM sodium borate adjusted to pH 8.5 with H3PO4. A UV detector set to 214 nm is used to record the electropherograms. 

Directions are provided for the determination of chloride in samples using CZE. The buffer solution includes pyromellitic acid which allows the indirect determination of chloride by monitoring absorbance at 250 nm. 

Students determine the concentrations of caffeine, acetaminophen, acetylsalicylic acid, and salicylic acid in several analgesic preparations using both CZE (70 mM borate buffer solution, UV detection at 210 nm) and HPLC (C18 column with 3% v/v acetic acid mixed with methanol as a mobile phase, UV detection at 254 nm). 

Janusa and co-workers report the determination of chloride by CZE. Analysis of a series of external standards gives the following calibration curve. 

The analysis of N03 in aquarium water was carried out by CZE using 104 as an internal standard. A standard solution of 15.0-ppm N03 and 10.0-ppm 104 gives peak heights (arbitrary units) of 95.0 and 100.1, respectively. A sample of water from an aquarium is diluted 1 100, and sufficient internal standard added to make its concentration 10.0 ppm. Analysis gives signals of 29.2 and 105.8 for N03 and 104 , respectively. Report the parts per million of N03 in the sample of aquarium water. 

McKillop and associates have examined the electrophoretic separation of alkylpyridines by CZE. Separations were carried out using either 50-pm or 75-pm inner diameter capillaries, with a total length of 57 cm and a length of 50 cm from the point of injection to the detector. The run buffer was a pH 2.5 lithium phosphate buffer. Separations were achieved using an applied voltage of 15 kV. The electroosmotic flow velocity, as measured using a neutral marker, was found to be 6.398 X 10 cm s k The diffusion coefficient,... 

CE. (sometimes CZE), capillary electrophoresis (or capillary zone electrophoresis)... 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

The mode of operation of cze, a technique which is commercially available, is similar to conventional rod or slab electrophoresis, except that the... 

Electrodriven Separation Techniques encompass a wide range of analytical procedures based on several distinct physical and chemical principles, usually acting together to perform the requh ed separation. Example of electrophoretic-based techniques includes capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), and capillary gel electrophoresis (CGE) (45-47). Some other electrodriven separation techniques are based not only on electrophoretic principles but rather on chromatographic principles as well. Examples of the latter are micellar... 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

Figure 9.5 The generic setup for two-dimensional liquid chromatography-capillary zone electrophoresis as used by Jorgenson s group. The LC separation was performed in hours, while the CZE runs were on a time scale of seconds.

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

The HPLC elution time was typieally under 260 min, and the CZE analysis took plaee in 60 s, whieh led to an overall run time of about 4 h. The 1 min CZE sampling interval was problematie, as the LC eolumn was probably slightly undersampled. A shorter CZE analysis time, whieh would provide a more frequent sampling rate, would improve this system a great deal. The seeond-dimension analysis time must be short relative to the first dimension, lest resolution in the first dimension be saeri-fieed. 

In 1993, Jorgenson s group improved upon then earlier reverse phase HPLC-CZE system. Instead of the six-port valve, they used an eight-port electrically actuated valve that utilized two 10-p.L loops. While the effluent from the HPLC column filled one loop, the contents of the other loop were injected onto the CZE capillary. The entii e effluent from the HPLC column was collected and sampled by CZE, making this too a comprehensive technique, this time with enhanced resolving power. Having the two-loop valve made it possible to overlap the CZE runs. The total CZE run time was 15 s, with peaks occurring between 7.5 and 14.8 s. In order to save separation space, an injection was made into the CZE capillary every 7.5s,... 

The only other group to have performed comprehensive multidimensional reverse-phase HPLC-CZE separations is at Hewlett-Packard. In 1996, a two-dimensional LC-CE instrument was described at the Erederick Conference on Capillary Electrophoresis by Vonda K. Smith (21). The possibility for a commercial multidimensional instrument may have been explored at that time. 

A six-port valve was first used to interface the SEC microcolumn to the CZE capillary in a valve-loop design. UV-VIS detection was employed in this experiment. The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse phase HPLC-CZE technique, runs were overlapped in the second dimension to reduce the apparent run time. The main disadvantage of this yu-SEC-CZE method was the valve that was used for interfacing. The six-port valve contributed a substantial extracolumn volume, and required a fixed volume of 900 nL of effluent from the chromatographic column for each CZE run. The large fixed volume imposed restrictions on the operating conditions of both of the separation methods. Specifically, to fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and therefore the SEC efficiency was decreased (22). 

The second interface design that was developed for use with yu-SEC-CZE used the internal rotor of a valve for the collection of effluent from the SEC microcolumn. The volume collected was reduced to 500 nL, which increased the resolution when compared to the valve-loop interface (20). However, a fixed volume again presented the same restrictions on the SEC and CZE operating parameters. An entirely different approach to the interface design was necessary to optimize the conditions in both of the microcolumns. 

The experimental setup for high-speed CZE can be seen in Figure 9.8. Highspeed CZE, or fast CZE (FCZE), yielded 70 000 to 90 000 theoretical plates for the separation of amino acid mixtures. Complete separation was achieved in under 11s, using a capillary length of 4 cm (24). 

In 1995, Moore and Jorgenson used the optically gated CZE system to obtain extremely rapid separations with HPLC coupled to CZE. The rapid CZE analysis made possible more frequent sampling of the HPLC column, thus increasing the comprehensive resolving power. Complete two-dimensional analyses were performed in less than 10 min, with the CZE analyses requiring only 2.5s. A peak... 

Figure 9.8 Schematic illustration of the two-dimensional HPLC/fast-CZE instmmental setup. The argon laser beam was set at 488 nm, which was used to photodegrade and detect the fluorescein isothiocyanate tag.

Moore and Jorgenson eombined the rapid two-dimensional separation aehieved by LC-CZE with SEC to make the first eomprehensive three-dimensional separation involving an eleetrodriven eomponent in 1995. Size exelusion ehromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE eombination separated eomponents in only 7 min. A sehematie diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Eigure 9.9 (18). A dilution tee was plaeed between the SEC eolumn and the reverse phase HPLC injeetion loop in order to dilute the eluent from the SEC eolumn, sinee it eon-tained more methanol than was optimal for the reverse phase HPLC eolumn. 

Figure 9.9 Schematic illustration of the instmmental setup used for three-dimensional SEC-RPLC-CZE.

Figure 9.11 Schematic illustration of the transparent interface used to link the HPLC capillary to the CZE capillary. Reprinted from Analytical Chemistry, 69, T. E. Hooker and J. W. Jorgenson, A transparent flow gating interface for the coupling of microcolumn EC with CZE in a comprehensive two-dimensional system , pp 4134-4142, copyright 1997, with permission from the American Chemical Society.

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