Luis A. Colón

Recent progress in capillary electrochromatography.

Capillary electrochromatography (CEC) continues to captivate many separation scientists. A remarkable activity is apparent from the numerous publications in the literature using CEC. A review of the most recent progress in CEC is presented herein, covering an extensive fraction of the literature on CEC published from the year 1997 until the beginning of 2000. Most of the recent developments have concentrated on column technology.

Packing columns for capillary electrochromatography.

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.

Determination of Glucose in Submicroliter Samples by CE-LIF Using Precolumn or On-Column Enzymatic Reactions

Two enzymatic reactions combined with capillary electrophoresis (CE) are used to determine glucose contained in sample volumes of ≤500 nL. In the first enzymatic reaction, glucose is oxidized in the presence of glucose oxidase producing hydrogen peroxide, which reacts quantitatively with the fluorogenic compound homovanillic acid catalyzed by the enzyme peroxidase. The second reaction generates a fluorescent species that is proportional to the glucose concentration. The reaction product is determined by CE using laser-induced fluorescence (LIF) as the detection mode. The overall reaction scheme is faster than commonly used precolumn derivatization procedures and can be performed using very small sample quantities. Alternatively, the enzymatic reactions can be performed on-column, similar to the electrophoretically mediated microanalysis approach, accommodating sample quantities in the nanoliter range. The on-column reaction is a simple and practical approach for the determination of glucose contained in low-volume samples by CE-LIF, where samples are injected directly into the capillary column without any pretreatment. However, sample handling and detectability of the precolumn approach proved to be superior. Determination of glucose using the precolumn and on-column reactions showed detection limits of 50 and 800 nM, respectively. The methods were shown to be linear in the range tested, 1-100 μM and 100 nM-30 μM, for the on-column and precolumn reactions, respectively. The reproducibility for each scheme was <5% RSD. To determine the possibility of using a noninvasive procedure for glucose monitoring, we used the CE-LIF methods to analyze human tear samples for glucose. The tear fluid samples were contained in a volume of ∼200 nL. The concentration of glucose in the human tear samples analyzed using the precolumn and on-column procedures was ∼138 μM.

Fused-core, sub-2 μm packings, and monolithic HPLC columns: a comparative evaluation

Three different HPLC column technologies (i.e., monolith, fused-core particles, and sub-2 microm particles) were evaluated, comparing van Deemter plots, speed of analysis, back pressure, and mobile phase consumption. Very high linear velocities (approximately 12 mm/s) were achieved with the monolithic column using modest pressure (110 bar) at the expense of high mobile phase consumption. The minimum plate height of the monolith was similar to that of a 3 microm-particle packed column (i.e., h = 8 microm), operated at optimal linear velocities; the monolithic column showed substantially lower mass transfer dependence, however. The 2.7 microm fused-core packing material yielded efficiencies closer to the sub-2 microm material than to the 3 microm-particle packed column and could be operated at high flow rates. The fused-core column was able to achieve linear velocities similar to those attained on the sub-2 microm column, staying below 620 bar instead of almost near 1030 bar required by the sub-2 microm material. The lack of pH stability of the monolithic column prevented its use to separate basic compounds (i.e., tricyclic antidepressants) at high pH. Best separation of these components at high pH was achieved using the column packed with 1.7 microm hybrid material.

Rudimentary capillary-electrode alignment for capillary electrophoresis with electrochemical detection.

A capillary-electrode holder was constructed for electrochemical detection in capillary electrophoresis (CE). The device allows for positioning of the working electrode at the end of the capillary column without the aid of micropositioners or microscopes. The design facilitates the exchange of electrodes and capillaries without the need of refabricating the entire capillary-electrode setup. The system can be assembled in a very short period of time. Alignment with the self-guided system proved to be reproducible for the electrodes used (carbon, nickel, copper). The advantages of reduced downtime and low cost, make the device very attractive for the routine analysis of electroactive species by CE with electrochemical detection.

Separation of Bioconjugated Quantum Dots Using Capillary Electrophoresis

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. Under a given set of experimental conditions, the fluorescence intensity of QDSt and QDBi emitting light at 655 nm indicated that about 90% of QDBi complexed with 70% of QDSt. A two-color experiment that made use of two different sizes of QD (i.e., 585 and 655 nm) indicated that 30% of the 655 nm QDBi complexed with 53% of the 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This, in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared.

Allyl-functionalized hybrid silica monoliths

A hybrid organosilica monolith was synthesized containing an allyl functionality. This provided a viable platform for producing silica-based, chromatographic, monolithic columns with the stationary phase bonded through a surface silicon-carbon bond rather than a conventional siloxane bond.

Investigations of a sol-gel derived stationary phase for open tubular capillary electrochromatography

Organic‐inorganic hybrid glass composites were synthesized using sol‐gel processing. These composites were cast onto the inner walls of fused silica capillaries and used as the stationary phase for open tubular capillary electrochromatography (OT-CEC). N-octyl sol‐gel processed bonded phases were prepared using a mixture of n-octyltriethoxysilane and tetraethoxysilane (C8-TEOS/TEOS). The synthesis of the sol‐gel-processed composite incorporates the stationary phase into the glass formation process. This process consists of hydrolysis and condensation reactions of the C 8-TEOS/TEOS mixture. The chemical species present in solution during the sol‐gel process, prior to column casting, dictates the characteristics of the final glass composite. Manipulation of the processing parameters allows control of the retentive properties of the sol‐gel-derived phase. A correlation between the species present in solution at the time of column casting and chromatographic retention has been established. The column preparation procedure was optimized for the C8-TEOS/TEOS hybrid system. A phase containing octadecyl (C18) groups was also fabricated by sol‐gel processing. Using a pressurized coating assembly improved the column-to-column reproducibility. The effect of silanizing the sol‐gel-derived material was also studied. Such a silanization, however, did not cause appreciable changes on the performance of the capillary columns. ©1999 Elsevier Science B.V. All rights reserved.

Hydrolytically stable amino-silica glass coating material for manipulation of the electroosmotic flow in capillary electrophoresis

A hydrolytically stable amino-silica glass coating material was fabricated inside fused-silica capillaries, using sol-gel technology. Aminopropyltriethoxysilane was used as the precursor in the glass formation process. The net charge at the surface of the coating material depends on the degree of protonation of the amino groups and the degree of ionization of the silanol groups, thus enabling manipulation of the magnitude and direction of the electroosmotic flow (EOF). At low pH (< 6.0), the coating bears a net positive charge, which results in an electroosmotic flow from the cathode toward the anode and minimizes the wall-solute interactions of basic species. At high pH (> 6.5), the coating surface bears a net negative charge and the coated capillary behaves like an uncoated one, having an EOF in the cathodic direction. The amino-silica glass coating has also been shown to be extremely stable under both acidic and basic conditions. The reproducibility of the electroosmotic mobility of five different capillaries was found to be 7% R.S.D. The utility of the material is shown with the separation of basic proteins, peptides and basic compounds.

Very high pressure HPLC with 1 mm id columns

Theoretical calculations and experimental data indicate that very high pressure HPLC can be performed using 1 and 1.5 mm id columns, and contrary to previous beliefs, the frictional heating generated does not appear to be detrimental to the separation.