Capillary monolithic columns

Ivanov, A.R., Zang, L., Karger, B. L. (2003). Low-attomole electrospray ionization MS and MS/ MS analysis of protein tryptic digests using 20 pm-i.d. polystyrene-divinylbenzene monolithic capillary columns. Anal. Chem. 75, 5306-5316. 

Premstaller, A., Oberacher, H., Walcher, W., Timperio, A.M., Zolla, L., Chervet, J.P., Cavusoglu, N., van Dorsselaer, A., Huber, C.G. (2001). High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies. Anal. Chem. 73, 2390-2396. 

Although Fields already mentioned the possible preparation of monolithic silica-based CEC columns, the lack of experimental data leads to the assumption that this option has not been tested [111]. In fact, it was Tanaka et al. who demonstrated the preparation of monolithic capillary columns using a sol-gel transition within an open capillary tube [99,112]. The trick was in the starting mixture that in addition to tetramethoxysilane and acetic acid also includes poly(ethylene oxide). The gel formed at room temperature was carefully washed with a variety of solvents and heated to 330 °C. The surface was then modified with octadecyl-trichlorosilane or octadecyldimethyl-A N-dimethylaminosilane to attach the hy-... 

Fig. 23. Scanning electron micrograph of monolithic capillary column prepared according to [134]...

Fig. 26. Effect buffer concentration in the mobile phase on EOF velocity (1) and current (2). (Reprinted with permission from [ 110]. Copyright 2000 Elsevier). Conditions monolithic capillary column 75 pm i.d., total length 30 cm, active length 25 cm, containing sol-gel bonded 3 pm ODS/SCX with 80 A pores, mobile phase 70 30 acetonitrile/phosphate buffer pH 3.0, electric field strength 442 V/cm (voltage 15 kV)...

Fig. 28. Effect of pH of the mobile phase on linear flow velocity (1) and electrical current (2) in the monolithic capillary column. (Reprinted with permission from [149]. Copyright 1998 American Chemical Society). Conditions monolithic capillary column 100 pm i. d. 30 cm, mobile phase 80 20 acetonitrile/5 mmol/1 phosphate buffer, pH adjusted by addition of concentrated NaOH, flow marker thiourea 2 mg/ml, UV detection at 215 nm, voltage 25 kV, pressure in vials 0.2 MPa, injection, 5 kV for 3 s...

Since a comprehensive description of all monolithic materials would exceed the scope of this chapter and a number of other monolithic materials are also described elsewhere in this volume, this contribution will be restricted mainly to monoliths for chromatographic purposes and prepared by polymerization of monomer mixtures in non-aqueous solvents. Monolithic capillary columns for CEC are treated in another chapter and will not be presented in detail here. 

Fig. 11. Separation of a mixture of organic solvents using 50 cm long 100 (left) and 320 pm i.d. (right) monolithic capillary columns (Reprinted with permission from [112]. Copyright 2000 Wiley-VCH). Conditions temperature gradient 120 - 300 °C, 20 °C/min, inlet pressure 0.55 MPa, split injection. Peaks methanol (1), ethanol (2), acetonitrile (3), acetone (4), 1-propanol (5), methyl ethyl ketone (6), 1-butanol (7),toluene (8), ethylbenzene (9),propylbenzene (10),butyl-benzene (11)...

Freitag, R. (2004). Comparison of the chromatographic behavior of monolithic capillary columns in capillary electrochromatography and nano-high-performance liquid chromatography. J. Chromatogr. A 1033, 267-273. 

Bedair, M., and El Rassl, Z. (2004). Affinity chromatography with monolithic capillary columns I. Polymethacrylate monoliths with Immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography. /. Chromatogr. A 1044, 177-186. 

Mayr, B., Holzl, G., Eder, K., and Buchmeiser, C. G., Hydrophobic, peUicular, monolithic capillary columns based on cross-linked polynorbomene for biopolymer separations. Analytical Chemistry 74(23), 6080-6087, 2002. 

Lee, D., Svec, F., and Frechet, J. M. J., Photopolymerized monolithic capillary columns for rapid micro high-performance liquid chromatographic separation of proteins, Journal of Chromatography A 1051(1-2), 53-60, 2004. 

Electroosmosis refers to the movement of the liquid adjacent to a charged snrface, in contact with a polar liquid, under the influence of an electric field applied parallel to the solid-liquid interface. The bulk fluid of liquid originated by this electrokinetic process is termed electroosmotic flow. It may be prodnced either in open or in packed or in monolithic capillary columns, as well as in planar electrophoretic systems employing a variety of snpports, such as paper or hydrophilic polymers. The origin of electroosmosis is the electrical donble layer generated at the plane of share between the snrface of either the planar support or the inner wall of the capillary tube and the surronnding solntion, as a consequence of the nneven distribntion of ions within the solid/liquid interface. 

Materials prepared by the ROMP technique find use in monolithic capillary columns (45,46), and monolithic membrane discs (47). A monolithic column is a column in which the stationary phase is... 

Another successful example is the separation of a series of steroids listed in Fig. 6.11 using a monolithic capillary column prepared by redox initiated polymerization of a solution of acrylamide 4, methylene bisacrylamide 5, vinylsulfonic acid 12, and dodecyl acrylate 18 in N-methylformamide/TRIS-boric acid buffer (pH 8.2) to which polyethylene glycol) (MW 10,000) was added (overall composition 5% T, 60% C, 10% vinylsulfonic acid, 15% lauryl acrylate, 3% polyethylene glycol)). The capillary tube was first vinylized and its part beyond the detection window was coated with linear polyacrylamide to avoid band broadening. Since laser induced fluorescence was used to decrease the detection limit of the method to about 100 attomoles for neutral steroids, all of the analytes were first tagged with dansylhydrazine. Fig. 6.12 shows an... 

Although both reproducible preparation and operation of CEC columns are extremely important issues that will further stimulate the development and the acceptance of this technique, only a few groups have reported data on column-to-col-umn, run-to-run, and day-to-day reproducibility of monolithic capillary columns. Palm and Novotny showed reproducibility data for migration times tr, efficiencies, and retention factors for a number of analytes on acrylamide-based monoliths [35], The relative standard deviations (RSD) were smaller for run-to-run compared to day-to-day measurements. For example, the average run-to-run RSD for 6 analytes was... 

Horvath s group has recently reported the preparation of porous rigid monolithic capillary columns for CEC by polymerizing mixtures of chloromethylstyrene 21, divinylbenzene 22 and azobisisobutyronitrile in the presence of various porogenic solvents such as methanol, ethanol, propanol, toluene, and formamide [49]. The capillary wall was silanized using a 50% dimethylformamide solution of 3-(trimethoxysilyl)propyl methacrylate 8 at a temperature of 120°C for 6 hours. In order to avoid the spontaneous polymerization of the functional methacrylate, a stable free radical (DPPH) was added to the solution. The SEM micrographs of Fig. 6.16... 

Fig. 6.20. Schematics for the preparation of monolithic capillary columns. First, the bare capillary is filled with the polymerization mixture (step a) that contains functional monomer, crosslinking monomer, initiator, and porogenic solvent. Polymerization (step b) is then initiated thermally or by UV irradiation to afford a rigid monolithic porous polymer. The resulting monolith within the capillary is washed (step c) with the mobile phase using a pump or electroosmotic flow and used as for the CEC separations.

Fig. 6.21. Electrochromatographic separation of benzene derivatives on monolithic capillary column prepared by UV initiated polymerization. Conditions capillary column, 100 pm i.d. x 25 cm active length stationary phase poly(butyl methacrylate-co-ethylene dimethaciylate) with 0.3 wt. % 2-acrylamido-2-methyl-l-propanesulfonic acid pore size, 296 nm mobile phase, 75 25 vol./vol mixture of acetonitrile and 5 mmol/L phosphate buffer pH 7 UV detection at 215 nm 25 kV pressure in vials, 0.2 MPa injection, 5 kV for 3 s. Peaks thiourea (1), benzyl alcohol (2), benzaldehyde (3), benzene (4), toluene (5), ethylbenzene (6), propylbenzene (7), butylbenzene (8), and amylbenzene (9).

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

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