Methacrylates, monoliths

CEC-MS using methacrylate monolith, modified with N-ethylbutylamine... 

CEC-MS using methacrylate monolith, modified with N-ethylbutylamine (investigation of quality of spectra using volatile and nonvolatile buffers)... 

The order of elution of peptides (charged compounds) is governed by a combination of electrophoresis and partitioning, with hydrophobic as well as electrostatic contributions. In this study it was demonstrated that sulfonic acid functionalities in the methacrylate monolith provide high stability and maintain a constant EOF over a wide range of pH (2—12). It was also demonstrated that a better separation of a mixture of therapeutic peptides was obtained at high pH values (Figure 16) due to the suppression of electrostatic attraction. 

Methacrylate monoliths have been fabricated by free radical polymerization of a number of different methacrylate monomers and cross-linkers [107,141-163], whose combination allowed the creation of monolithic columns with different chemical properties (RP [149-154], HIC [158], and HILIC [163]) and functionalities (lEX [141-153,161,162], IMAC [143], and bioreactors [159,160]). Unlike the fabrication of styrene monoliths, the copolymerization of methacrylate building blocks can be accomplished by thermal [141-148], photochemical [149-151,155,156], as well as chemical [154] initiation. In addition to HPLC, monolithic methacrylate supports have been subjected to numerous CEC applications [146-148,151]. Acrylate monoliths have been prepared by free radical polymerization of various acrylate monomers and cross-linkers [164-172]. Comparable to monolithic methacrylate supports, chemical [170], photochemical [164,169], as well as thermal [165-168,171,172] initiation techniques have been employed for fabrication. The application of acrylate polymer columns, however, is more focused on CEC than HPLC. 

Photoinitiated polymerization of the same mixtures at 20°C generally yields monoliths with larger pores compared to those initiated thermally. Thus, reduced contents of dodecanol in the polymerization mixture has to be used for UV initiated polymerizations in order to obtain pore sizes comparable to those of their thermally polymerized analogs. For example, a polymerization mixture containing only 30% dodecanol can be used to produce a 2-hydroxyethyl methacrylate monolith with 1,000 nm pores by UV polymerization at 20°C. These shifts can readily be explained by the effect of the polymerization temperature, since the creation of larger pores is favored at lower temperatures [59],... 

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. 

Ribonuclease A, insulin, a-lactalbumin, myoglobin Methacrylic monolith with tertiary amino functions 30% Acetonitrile in 60 mM aqueous sodium phosphate, pH 2.5 390 mm x 50 pm i.d. 290 mm effective length... 

Krajnc P, Leber N, Stefanec D, Kontrec S, and Podgomik A. Preparation and characterisation of poly(high internal phase emulsion) methacrylate monoliths and their application as separation media. J. Chromatogr. A 2005 1065 69-73. 

Podgornik, A. Bamt, M. Jancar, J. Strancar, A. Isocratic separations on thin glycidyl methacrylate-ethylenedi-methacrylate monoliths. J. Chromatogr., A 1999, 848, 51-60. 

FIGURE 5.17 Effect of ACN concentration in the eluent on the separation of four proteins. Column, 39 cm (effective length 29 cm) x 50 p.m i.d., fused-silica capillary with porous methacrylic monolith having tertiary amino functions mobile phase, ACN (%, v/v) in 60 mM aqueous sodium phosphate, pH 2.5 applied voltage, —25 kV detection, 214 nm sample (1) ribonuclease A, (2) insuhn, (3) a-lactalbumin, and (4) myoglobin. (Reprinted from Zhang, S., et al., J. Chromatogr. A, 887,465-477, 2000. With permission from Elsevier.)... 

This compares well with an RSD of 50% in MALDI signal intensities reported for multiple peptide spots deposited from a single capillary using an optimized matrix seed layer technique. Further improvements in deposition volume uniformity were realized for an eight-channel chip containing a methacrylate monolith developed for reversed-phase chromatography in the second separation dimension, with negligible variations at flow rates down to 200 nL/min per channel. 

FIGURE 47.2 Repeated isocratic HPLC separations of proteins on chip. (Reprinted with permission from Reichmuth, D. S., etal.. Ana/. Chem., 77,2997,2005. Copyright 2005 American Chemical Society.) Conditions lauryl methacrylate monolith mobile phase 24% acetonitrile + 0.16% heptafluorobutyric acid in 5 nunol/L phosphate buffer (pH 2.0) Injections 6,4 nL, 750 ms, pressure 0.2 MPa. Peaks free dye (a), insulin (b), antibiotin (c), a-lactalbumin (d). 

In the following section, details about large volume radial flow methacrylate monolithic columns and the strategy for their preparation are presented. 

DESIGN OF LARGE VOLUME RADIAL METHACRYLATE MONOLITHIC COLUMNS... 

Methacrylate polymerization is a highly exothermic reaction, releasing, in the particular case of methacrylate monoliths, around 190 J/g of heat. Since preparation of the monoliths proceeds through bulk polymerization, the heat generated cannot be dissipated fast enough ... 

Fig. 1 Large volume cylindrical shaped methacrylate monoliths of volume 80, 800, and 8000 ml. The largest, the 8000 ml monolith, has a height of 41.5 cm and an outer diameter of 30 cm.

Fig. 2 Construction of a large volume methacrylate monolithic unit of desired thickness. The monolithic unit (4) consists of three monolithic cylinders (1, 2, and 3). The total thickness of the unit (4) is the sum of the thicknesses of monoliths 1, 2, and 3. Source From Construction of large-volume monolithic columns, in Anal. Chem. ...

CLC) column for the methacrylate monoliths, can be prepared. In this way, extracolumn broadening, which occurs when several columns are connected in series, is significantly reduced. ... 

RADIAL, LARGE VOLUME METHACRYLATE MONOLITHIC COLUMNS AND THEIR PROPERTIES... 

Fig. 5 Separation of lignin peroxidase isoenzymes using the 8 ml radial methacrylate monolithic columns. Stationary phase CIM DEAE 8 ml column. Conditions buffer A 10 mM acetic buffer, pH 6.0 buffer B 1 iVf acetic buffer, pH 6.0 flow rate 8 ml/min gradient 10-70% buffer B in 2.25 min sample supernatant from Phanerochaete chrysosporium cultivation injection volume 500 p.1 detection UV at 409 nm.

Podgomik, A. Jancar, J. Merhar, M. Kozamernik, S. Glover, D. Cucek, K. Barut, M. Strancar, A. Large scale methacrylate monolithic columns design and properties. J. Biochem. Biophys. Meth. 2004,60,179-189. 

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