Pavel A. Levkin

Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200micromx200microm and a length of 6.8cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.

Monolithic Superhydrophobic Polymer Layer with Photopatterned Virtual Channel for the Separation of Peptides Using Two-Dimensional Thin Layer Chromatography-Desorption Electrospray Ionization Mass Spectrometry

Superhydrophobic monolithic porous polymer layers with a photopatterned hydrophilic channel have been prepared. These layers were used for two-dimensional thin layer chromatography of peptides. The 50 microm thin poly(butyl methacrylate-co-ethylene dimethacrylate) layers supported onto 4.0 x 3.3 cm glass plates were prepared using UV-initiated polymerization in a simple glass mold. Photografting of a mixture of 2-acrylamido-2-methyl-1-propanesulfonic acid and 2-hydroxyethyl methacrylate carried out through a mask afforded a 600 microm wide virtual channel along one side of the layer. This channel, which contains ionizable functionalities, enabled the first dimension separation in ion exchange mode. The aqueous mobile phase migrates only through the channel due to the large difference in surface tension at the interface of the hydrophilic channel and the superhydrophobic monolith. The unmodified part of the layer featuring hydrophobic chemistry was then used for the reversed phase separation in the orthogonal second dimension. Practical application of our technique was demonstrated with a rapid 2D separation of a mixture of model peptides differing in hydrophobicity and isoelectric point using a combination of ion-exchange and reversed phase modes. In the former mode, the peptides migrated 11 mm in less than 1 min. Detection of fluorescently labeled peptides was achieved through UV light visualization. Separation of the native peptides was monitored directly using a desorption electrospray ionization (DESI) source coupled to a mass spectrometer. Unidirectional surface scanning with the DESI source was found suitable to determine both the location of each separated peptide and its molecular mass.

Visible light initiated polymerization of styrenic monolithic stationary phases using 470 nm light emitting diode arrays

Poly(styrene-co-divinylbenzene) monolithic stationary phases have been synthesized for the first time by photoinitiated polymerization. An initiator composed of (+)-(S)-camphorquinone/ethyl-4-dimethylaminobenzoate/N-methoxy-4-phenylpyridinium tetrafluoroborate was activated using a 470 nm light emitting diode array as the light source. Spatially controlled polymerization of styrenic monoliths has been achieved within specific sections of a 100 microm id polytetrafluoroethylene-coated fused-silica capillary using simple photo masking. The sharpness of the edges was confirmed by optical microscopy, while SEM was used to verify a typical porous, globular morphology. Flow resistance data were used to assess the permeability of the monoliths and they were found to have good flow through properties with a flow resistance of 0.725 MPa/cm at 1 microL/min (water, 20 degrees C). Conductivity profiling along the length of the capillary was used to assess their lateral homogeneity. Monoliths which were axially rotated during polymerization were found to be homogeneous along the whole length of the capillary. The monolithic stationary phases were applied to the RP gradient separation of a mixture of proteins. Column fabrication showed excellent reproducibility with the retention factor (k) having a RSD value of 2.6% for the batch and less than 1.73% on individual columns.

Apparent and true enantioselectivity of single- and binary-selector chiral stationary phases in gas chromatography.

Almost all gas-chromatographic chiral stationary phases (CSPs) are complex systems containing one or more chiral selector(s) dissolved in, or bonded to, an achiral solvent such as squalane or poly(dimethylsiloxane). The presence of different components in the total CSP, interacting independently with the analyte enantiomers, impairs the elucidation of enantiorecognition mechanisms and complicates the optimization of enantioseparations. In the present work a quantitative analysis of the influence of different factors on the observed enantioselectivity is performed. The parameters varied in this study were the composition of the CSP, the concentration and the enantiomeric excess of the chiral selector(s) and the presence of achiral selectors (including racemic compositions). Special attention is given to the determination of distribution and association constants, as well as apparent and true enantioseparation factors.

Strong detrimental effect of a minute enantiomeric impurity of a chiral selector on the enantioselectivity factor.

Separation of racemates into pure enantiomers by chiral chromatography is an essential process both in university laboratories and in the pharmaceutical industry. Up to 75% of small-molecule drugs approved by the US Food and Drug Administration (FDA) are single enantiomers, some of which undergo separation by chiral chromatography at the multi-ton scale during the manufacturing process. The high enantioselectivity of chiral chromatography is important for producing pure enantiomers. The enantioselectivity of a chiral stationary phase (CSP), which is composed of a chiral selector covalently or adsorptively immobilized on the surface of a porous support material, is commonly represented by the enantioseparation factor a. It is anticipated intuitively that a depends on the enantiomeric excess (ee) of the chiral selector and is reduced upon decrease of the ee value. It was theoretically predicted, however, that this reduction significantly depends on the enantioselectivity of the selector and becomes very substantial for chiral selectors with higher enantioselectivity. Herein, we report the first experimental confirmation that for highly enantioselective chiral selectors even trace amounts of the opposite enantiomer of the selector present in the stationary phase can lead to a drastic decrease of the observed enantioseparation factor. Our results provide an important additional criterion for the design of artificial, highly enantioselective, receptor-like chiral selectors and chromatographic systems. Chiral anion-exchange-type stationary phases based on modified cinchona alkaloids developed by Lindner et al. show extremely high enantioselectivity towards different classes of chiral acids and are suitable for our study. For the present work we chose the adamantyl, neopentyl derivative of quinine (AN-QN) and quinidine (AN-QD; Figure 1), which showed exceptionally high enantioselectivity for the enantioseparation ofN-3,5-dinitrobenzoyl (DNB) a-amino acids (Figure 2). It should be noted that natural quinine and quinidine are diastereomers having two (C8 and C9) out of five stereogenic centers of opposite configuration. Nevertheless, they represent a good model for our study because they show almost completely opposite enantioselectivity and thus behave quasi pseudo-enantiomerically. Moreover, to our knowledge, another chiral selector possessing very high enantioselectivity and being easily accessible in both enantiomeric forms is unavailable. The chiral selectors AN-QN and AN-QD (Figure 1) were synthesized from natural quinine and quinidine, respectively (for experimental details see the Supporting Information). The enantiomeric excess of each selector was at least 99.7% according to HPLC analysis. The diastereomerically pure selectors were immobilized onto g-mercaptopropyl-modified silica (5 mm particle size) using 2,2’-azobisisobutyronitrile as an initiator, thus generating the AN-QN100andAN-QD100type CSPs. Selector loadings measured by elemental analysis were found to be (145 0.5) and (158 0.5) mmolg 1 for silica modified with AN-QN and AN-QD, respectively. Portions of Figure 1. Structure of quinineand quinidine-based chiral selectors (AN-QN and AN-QD, respectively) possessing exceptionally high and opposite enantioselectivity.

Temperature-dependent racemic compound-conglomerate crystallization of 2,3 :6,7-dibenzobicyclo[3.3.1]nona-2,6-diene-4,8-dione

Abstract 2,3:6,7-dibenzobicyclo[3.3.1]nona-2,6-diene-4,8-dione 1 is a new example of a compound capable of temperature-dependent racemate-conglomerate crystallization: at temperatures below 90°C crystals of the racemic compound (space group P 1, Z =4) can be obtained, whereas above 100°C a conglomerate of (+)- and (−)-homochiral crystals (space group P 2 1 2 1 2 1 , Z =4) forms and therefore it undergoes spontaneous resolution upon crystallization. Enantioselective analytical gas chromatography on a single crystal has been proposed as a simple method for detection of conglomerate formation. The 1 H and 13 C NMR spectra of 1 are analyzed in detail and the crystal structures of both species (racemic compound and single enantiomer) have been solved by X-ray structural analysis.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.