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PubMed2002

Centrosome separation: respective role of microtubules and actin filaments.

In mammalian cells, the separation of centrosomes is a prerequisite for bipolar mitotic spindle assembly. We have investigated the respective contribution of the two cytoskeleton components, microtubules and actin filaments, in this process. Distances between centrosomes have been measured during cell cycle progression in Xenopus laevis XL2 cultured cells in the presence or absence of either network. We considered two stages in centrosome separation: the splitting stage, when centrosomes start to move apart (minimum distance of 1 microm), and the elongation stage (from 1 to 7 microm). In interphase, depolymerisation of microtubules by nocodazole significantly inhibited the splitting stage, while the elongation stage was, on the contrary, facilitated. In mitosis, while nocodazole treatment completely blocked spindle assembly, in prophase, we observed that 55% of the centrosomes separated, versus 94% in the control. Upon actin depolymerisation by latrunculin, splitting of the interphase centrosome was blocked, and cells entered mitosis with unseparated centrosomes. Cells compensated for this separation delay by increasing the length of both prophase and prometaphase stages to allow for centrosome separation until a minimal distance was reached. Then the cells passed through anaphase, performing proper chromosome separation, but cytokinesis did not occur, and binuclear cells were formed. Our results clearly show that the actin microfilaments participate in centrosome separation at the G2/M transition and work in synergy with the microtubules to accelerate centrosome separation during mitosis.

Actin Cytoskeleton↗
PubMed2000

Comparison of different electrokinetic separation modes applicable to a model peptide mixture (collagen type I and III CNBr fragments).

A number of electromigration separation modes were applied to the separation of CNBr-released peptides from rat tail tendon collagen (microemulsion electrokinetic chromatography, methanol- or ethanol-modified background electrolytes and the separation in the presence of molecular sieving effect exerting polymer, both in the presence and absence of SDS). Electrodriven separations with a Hypersil C8 packed capillary were investigated as well. The best separations were obtained with either the molecular sieving effect exerting polymer (polyethylene oxide) in the background electrolyte (whether SDS was present or absent) or with the electrodriven chromatography using the C8 reversed-phase packed capillary. In the latter separation system, it was possible to separate 25-27 peaks of the theoretically expected 24 peptides in the analyzed mixture of which 17 were at least tentatively identified. The additional peaks apparently stem from the incomplete cleavage of the parent collagen alpha chains. Successful separations can be done either with predominating molecular sieving or hydrophobic partitioning mechanism.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed1989

Large DNA separation using field alternation agar gel electrophoresis.

The techniques for large DNA separation have developed from a seminal idea for field alternation which has transformed the field of DNA electrophoresis. This single innovation of pulsed field-gradient electrophoresis (PFGE) and the subsequent modifications have made a significant impact on molecular biology, eukaryote genetics, biopolymer research and diagnostic research. The apparatus types used for large DNA separation are depicted and critically compared with relation to molecular mass separation capabilities, straight-lane migration of samples, band sharpness and ease of operation. With these criteria in mind PFGE and orthogonal field alternation gel electrophoresis systems had a number of drawbacks, the principle one being the inability of these systems to give straight-lane migration. To a large extent this has restricted the widespread use of these systems. Field inversion gel electrophoresis produces straight-lane migration but was subject to an upper molecular mass limitation of 2 megabase pairs and tended to produce broader bands in the higher-molecular-mass areas. Transverse alternating field electrophoresis, rotating gel electrophoresis and contour-clamped homogeneous electric field electrophoresis systems where superior to all the other systems. They gave straight-lane migration, separation of chromosomes up to 10 megabase pairs, good resolution of bands and were all relatively simple to operate. Very little was found to separate these three electrophoresis systems. Field alternation electrophoresis has enabled a 500-fold increase in the size of DNA molecules that can be resolved in agar gels. Consequently, electrophoretic karyotypes of a number of organisms have been produced, while genome maps, gene locations and sequences of large areas of mammalian genomes are now being undertaken. The ability to separate entire chromosomes or large DNA fragments has, in conjunction with novel molecular biology techniques, enabled scientists to work backwards from large purified fragments or entire chromosomes to construct long-range genetic maps. The time saving alone when compared with the old techniques of using very small fragments to construct a picture of the gene or gene complex is commendable. The diagnostic role of large DNA separation and electrophoretic karyotyping is beginning to be explored, while the use of this technique for clinical studies of genetic disorders is well advanced. Very few innovations in nucleic acid separation have had as marked an influence on as many areas as field alternation electrophoresis. These techniques have brought mapping of the mammalian genome into the realms of possibility and is contributing in many sphere

DNA↗
PubMed1998

Application of capillary electrophoresis to the separation of structurally diverse N-(substituted)-glycine-peptoid combinatorial mixtures.

The capillary electrophoresis (CE)-based separation of five N-(substituted)-glycine (NSG)-peptoid mixtures with a wide range of physical and chemical properties was studied. A CE separation, initially developed using a single representative peptoid mixture, with a background electrolyte (BGE) modified by the addition of both methyl-beta-cyclodextrin and heptane sulfonic acid was found to provide good separations of most of the combinatorial mixtures investigated. For those mixtures not separated well by this procedure, the use of SDS micelles in conjunction with methyl-beta-cyclodextrin resulted in dramatic improvements in the separation. While no single set of separation conditions proved sufficient for all of the NSG-peptoid combinatorial mixtures, the two methods were able to provide separation sufficient for characterization of a set of mixtures with a wide range of physical and chemical properties. The efficiency of the CE-based separation of the combinatorial mixtures studied was compared to a reversed-phase liquid chromatographic method using gradient elution.

Buffers↗
PubMedDate not supplied

Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field.

The aim of the present experiment was to study the effects of early postnatal maternal separation on behavioural and adrenocortical responses to novelty in rats tested as adults. Sprague-Dawley rat pups were exposed to daily maternal separation (5 h/day) from postnatal day 2 to 6, during the stress hyporesponsive period. Since this procedure requires physical contact with the animals, a first control group of daily handled pups was introduced. A second control group, consisting of pups never handled or separated from the mother, was also considered. At postnatal day 45, the rats were tested in a two-compartment exploratory apparatus: the maternally separated and the non-handled rats, whose behavioural performance did not differ, showed higher emotional behaviour when compared with the handled rats (P < 0.05), suggesting that the handling procedure but not maternal separation improved the capacity to cope with novelty. Corticosterone plasma levels were found to be higher in the maternally separated rats than in the other two groups (P < 0.05), either at resting conditions or at 30 min after novelty exposure (P < 0.05). Levels of nuclear glucocorticoid receptor immunoreactivity in the CA1 hippocampal field were shown to be regulated by novelty exposure, as expected, in both the handled and the non-handled rats but not in the maternally separated rats. In conclusion, repeated maternal separation periods of 5 h/day during the first week of life produced long-lasting effects on the hippocampal regulation of the hypothalamic-pituitary-adrenocortical axis, which appear to be associated with increased responsiveness to stress stimuli in adulthood.

Adrenal Cortex↗
PubMed2001

Plateletpheresis concentrates produced with the COMTEC cell separator: the French experience.

The latest generation of cell separators such as Trima (Gambro), Amicus (Baxter) and AS-TEC 204 (Fresenius), allow the collection of leucocyte-reduced platelet concentrates without secondary filtration. Fresenius has recently developed the COMTEC cell separator whose performance has been evaluated by several teams in France. This new cell separator is an improved version of the Fresenius AS-TEC 204 cell separator, designed to allow more efficient platelet collections. This study reports on the experience of six French teams (from Bordeaux, Clermont-Ferrand, Creteil, Dijon, Lille and Nancy) who obtained 696 leucocyte-reduced plateletpheresis concentrates in the course of collection using the new Fresenius COMTEC cell separator. All healthy volunteer donors fulfilled French selection criteria for platelet apheresis. Donors were eligible if they had suitable venous accesses, if their bodyweight was *50 kg and if their pre-apheresis platelet count was >150 x 10(9) l(-1). Between 4606 and 5229 ml of blood were processed. The mean volume of the platelet concentrates was between 439 and 493 ml (mean 460 +/- 63 ml). The platelet yield was of the order of 5.18 +/- 1.02 x 10(11) with only one platelet concentrate below the norm of 2 x 10(11) platelets (0.91 x 10(11)). No plausible explanation for this was found. The residual leucocyte levels conform to current norms. The platelet concentrates contained less than 1 x 10(6) leucocytes per concentrate (mean 0.233 +/- 0.150 x 10(6) leucocytes) in more than 97% of the components produced with >95% statistical confidence. The efficacy of the cell separator (52.44 +/- 7.35%) is comparable to that of other separators. The Fresenius COMTEC cell separator makes it possible to obtain leucocyte-reduced platelet concentrates which comply with current standards both in terms of platelet content and residual leucocyte level.

Adult↗
PubMed2001

Evaluation of opiate separation by high-resolution electrospray ionization-ion mobility spectrometry/mass spectrometry.

The separation of opiates and the primary metabolites was evaluated with ESI-IMS/MS. Seven opiate molecules were analyzed, and spectra were shown for each compound. The IMS separation of two isomers (morphine and norcodeine) was shown with baseline separation. Differences in the mobilities were found for the nonacetylated, monoacetylated, and biacetylated compounds. In this study, two primary findings are reported. First, IMS can easily separate metabolic isomers, and second, the two-dimensional separation capability of IMS/MS can be employed to confidently identify and separate both the opiates and metabolites. Although previous IMS studies have shown the separation of isomers, this is the first example to show the capability of IMS to separate metabolic isomers (within 70 s), a significant advantage in high-throughput screening for pharmacokinetic studies. Second, the monoacetylated and biacetylated compounds were found to form more compact ions for the sodium adducts in comparison to the protonated molecular ions. On the basis of the mobilities, information on structures and conformation can be deduced when sodium and protonated ions are compared.

Indicators and Reagents↗
PubMed2001

Separation of barbiturates and phenylthiohydantoin amino acids using the thermally tuned tandem column concept.

There are many more choices of column type than of eluent type for method development in reversed-phase liquid chromatography. It is common to switch between different column types or between the same type from different suppliers to achieve the desired separations. The key difficulty in modulating band spacing by adjusting the column type is that it is a discontinuous, "hit or miss" proposition. The thermally tuned tandem column (T3C) concept effectively solves this problem by connecting two columns in series and independently controlling the two column temperatures. The columns are chosen to have distinctively different chromatographic selectivities (band spacing), so that the unresolved peaks on one column are separated by the other. The optimized separation in the T3C is achieved by simultaneously tuning the two column temperatures. In this study, we used the T3C combination of a carbon and a conventional bonded phase for the separation of barbiturates and phenylthiohydantoin amino acids (PTH-amino acids). Good peak shapes and comparable retention times were observed on the two phases at room temperature. The selectivities on the two phases are quite different. Baseline separations were easily achieved with the T3C set although neither column could individually resolve all the peaks. We further compared the separation of barbiturates optimized by the T3C approach with that optimized by adjusting the mobile phase. We found that T3C gave a better separation. We believe that the T3C combination of a carbon phase and a bonded conventional reversed-phase material provides a powerful and general method to optimize the separation of various mixtures.

Algorithms↗
PubMed2003

Mechanism for the separation of large molecules based on radial migration in capillary electrophoresis.

We demonstrate a novel separation mechanism for large molecules based on their radial migration in capillary electrophoresis with applied hydrodynamic flow (HDF). The direction of radial migration depends on the direction of the applied HDF relative to the electric field. The radial migration velocities are size-dependent, which could be attributed to the different degree of deformation under shear flow. Analytical separation was demonstrated on a sample plug containing lambda DNA (48 502 bp) and phiX174 RF DNA (5386 bp) with baseline separation. Alternatively, this separation mode can be performed continuously and is thus applicable to preparative separations. Without the need for gel/polymer or complex instrumentation, this separation technique is complementary to capillary gel electrophoresis and field-flow fractionation. Although large DNA molecules were used to demonstrate the separation mechanism here, these protocols could also be applied to the separation of proteins, cells, or particles based on size, shape, or deformability.

Bacteriophage lambda↗
PubMed2006

Rapid separation and quantitative analysis of peptides using a new nanoelectrospray- differential mobility spectrometer-mass spectrometer system.

Differential mobility spectrometry (DMS) (see Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148), also commonly referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS) (see Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszcyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105), is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal-to-noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we investigate the use of our nanoESI-DMS-MS system to demonstrate differential mobility separation of peptides. The formation of higher order peptide aggregate ions (ion complexes) via electrospray ionization and the negative impact this has on DMS peptide separation are examined. The successful use of differential mobility drift gas modifiers (dopants) to reduce aggregate ion size and improve DMS peptide ion separation is presented. Following optimization of DMS peptide separation conditions, we examined next the feasibility of a new analytical platform which uses direct sample infusion with nanoESI-DMS-MS for ultrarapid analyte quantitation. Quantitation of a selected peptide from a semicomplex peptide mixture is presented. Initial feasibility results with this new approach demonstrate good accuracy and reproducibility, as well as an absolute mass sensitivity of 6.8 amol and a minimum dynamic range of 2500 for the peptide of interest. This report offers a first look at utilizing nanoESI-DMS-MS to create an ultrarapid (under 5 s) quantitative analysis platform and its potential in the high-throughput arena. Each ion separation technique, DMS and MS, offers orthogonal ion separation to one another, enhancing the overall specificity for this quantitative approach.

Mass Spectrometry↗
PubMed2006

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode.

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.

Journal Article↗
PubMed2000

Application of the thermally tuned tandem column concept to the separation of several families of environmental toxicants.

Separations of several families of environmental toxicants were optimized by means of the thermally tuned tandem column (T3C) concept. We use a tandem combination of an octadecylsilane (ODS) and a carbon-coated zirconia (C-ZrO2) column; and tune the selectivity by independently adjusting the isothermal temperatures of the two columns. This results in the change in the contribution that each column makes to the overall retention and selectivity. The separation was optimized by locating the optimum pair of column temperatures which give the best separation of the critical solute pair. For both triazine herbicides and carbamate pesticides samples, dramatically different selectivities and different critical pairs were observed for the two types of phases. Although neither individual phase gave adequate separation, the T3C approach provided baseline separations using only four preliminary trial separations. We also showed that, for the triazine samples, the T3C approach gave a better separation than did conventional mobile phase optimization with an ODS column. The combination of superior selectivity of T3C and high flow rate allows the baseline separation of complex mixtures in just a few minutes.

Carbamates↗
PubMed1997

Changes in vesicle morphology induced by lateral phase separation modulate phospholipase A2 activity.

The action of phospholipase A2 (PLA2) toward zwitterionic bilayers is modulated by lateral phase separation of reaction products and substrate. The experiments here address the mechanism of this modulation. PLA2 is particularly active toward lipid dispersions containing reaction products and substrates in which lateral phase separation has occurred. Here, we study PLA2 activity in two related model systems: first in a system in which lateral phase separation can be produced a priori, and second in a system in which the action of PLA2 produces sufficient reaction product in situ such that lateral phase separation occurs. The dispersions in which lateral phase separation occurs a priori form either disk micelles or disk vesicles, not canonical vesicles. When lateral phase separation occurs due to in situ PLA2 activity, there is an abrupt change in vesicle structure and a simultaneous profound increase in catalytic rate. This observation is surprising in light of several reports that vesicles remain intact even when the entire outer monolayer has been hydrolyzed. Membrane curvature and the associated structural defects and dynamic fluctuations in membrane structure have been proposed to modulate PLA2 activity. The mechanism by which lateral phase separation modulates PLA2 activity has been unclear. The data presented here indicate that lateral phase separation affects PLA2 activity by altering membrane curvature and/or inducing defects in the membrane structure.

Kinetics↗
PubMed2005

Hidden minima of the gibbs free energy revealed in a phase separation in polymer/surfactant/water mixture.

We observed a very unusual kinetic pathway in a separating C(12)E(6)/PEG/H(2)O ternary mixture. We let the mixture separate above the spinodal temperature (cloud point temperature) for some time and next cool it into a metastable region of a phase diagram, characterized by two minima of the Gibbs potential, one corresponding to the homogeneous mixture and one to the fully separated PEG-rich and C(12)E(6)-rich phases. Despite the fact that in the metastable region the thermodynamic equilibrium corresponds to the separated phases (global minimum of the Gibbs free energy), we observe perfect mixing of the initially separated phase. The homogeneous state, obtained in this way, does not separate, if left undisturbed. However, many cooling-heating cycles or full separation with visible meniscus above the cloud point temperature induce the phase separation in the metastable region. The metastable region can exist tens of degrees below the cloud point temperature. This effect is not observed in the binary mixture of C(12)E(6)/H(2)O.

Journal Article↗
PubMed2005

Relationship between scattered intensity and separation for particles in an evanescent field.

We describe measurements of the scattering of visible light from an evanescent field by both spherical particles (R = 1-10 mum) that are glued to atomic force microscopy (AFM) cantilevers, and by sharp tips (R < 60 nm) that were incorporated onto the cantilevers during manufacture. The evanescent wave was generated at the interface between a flat plate and an aqueous solution, and an atomic force microscope was used to accurately control the separation, h, between the particle and the flat plate. We find that, for sharp tips, the intensity of scattered light decays exponentially with separation between the tip and the plate all the way down to h approximately 0. The measured decay length of scattered intensity, delta, is the same as the theoretical decay length of the evanescent intensity in the absence of the sharp tip. For borosilicate particles, (R = 1-10 mum), the scattering also decays exponentially with separation at large separations. However, when the separation is less than roughly 3delta, the measured scattering intensity is smaller in magnitude than that which would be predicted by extrapolating the exponential decay observed at large separations. For these particles, the scattering approximately fits the sum of two exponentials. The magnitude of the deviation from exponential at contact was roughly 10-15% for R = 1 mum particles and about 30% for larger particles and is larger for s-polarized light. Preliminary experiments on polystyrene particles shows that the scattering is also smaller than exponential at small separations but that the deviation from exponential is larger for p-polarized light. In evanescent wave AFM (EW-AFM) the scattering-separation can be calibrated for situations where the scattering is not exponential. We discuss possible errors that could be introduced by assuming that exponential decay of scattering continues down to h = 0.

Journal Article↗
PubMed2007

Phase separation in liquid crystalline mesophases of [Co(H2O)6]X2:P65 Systems (X = NO3-, Cl-, or ClO4-).

Transition-metal aqua complex salts [M(H2O)6]X2 (where M is Mn(II), Co(II), Ni(II), Zn(II), or Cd(II) and X is NO3-, Cl-, or ClO4-) can be dissolved in triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers (Pluronics, such as P65) to form homogeneous liquid crystalline (LC) mesophases. However, the [Co(H2O)6]X2:P65 LC mesophases slowly undergo phase separation into a disordered ion-free phase and an ordered ion-rich LC mesophase. The phase separation also takes place in the two-salt systems [Co(H2O)6](NO3):[Co(H2O)6](ClO4)2:P65 in which the ion-free disordered domains separate out from the initially ordered homogeneous mesophase. The phase separation results in a physical mixture of a hexagonal nitrate-rich and cubic perchlorate-rich LC and disordered ion-free domains in the mixed salt systems. The driving force in the phase separation in the [Co(H2O)6]X2:P65 system is Co(II)-catalyzed aerobic oxidation of P65 into ester and/or other oxidation products. The separation of ions in the [Co(H2O)6](NO3)2:[Co(H2O)6](ClO4)2:P65 system is related to the mesostructures of the two-salt systems that are different, hexagonal in the [Co(H2O)6](NO3)2:P65 system and cubic in the [Co(H2O)6](ClO4)2:P65 system. There is no visible phase separation in the other transition-metal salt:P65 systems. The phase separation in the [Co(H2O)6]X2:P65 systems can also be eliminated by keeping the mesophase under a N2 atmosphere.

Journal Article↗
PubMed2006

Two-dimensional microcolumn separation platform for proteomics consisting of on-line coupled capillary isoelectric focusing and capillary electrochromatography. 1. Evaluation of the capillary-based two-dimensional platform with proteins, peptides, and human serum.

In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.

Amino Acid Sequence↗
PubMed2006

The effect of photomask resolution on separation efficiency on microfabricated devices.

Separation quality on glass microfluidic devices fabricated from photomasks of different optical resolutions was compared by measuring the dispersion (apparent diffusion) coefficients of a set of standard compounds separated on these devices. Currently, the channel manifolds of most microfluidic devices are patterned using chrome photomasks. A much cheaper, more robust alternative to chrome photomasks are laser photoplotted masks. The primary disadvantage to using laser photoplots is that the optical resolution of these masks is not as high as that of chrome masks, and this feature increases the side-wall roughness of etched channel manifolds patterned using such masks. The increased wall roughness may affect the fluid flow within the channels and, therefore, the separation quality. To determine the effect of increased sidewall channel roughness, microchip channel manifolds were patterned in soda lime glass using a chrome photomask and laser photoplots printed at resolutions of 620, 1240, 3100 and 6200 dots per centimetre (dpc). Separations were performed on these devices using dilute solutions of fluorescently labeled amino acids. The peak variances of the amino acids were calculated at increasing distances down the separation channel and plotted as a function of migration time. From this plot, dispersion coefficients of the analytes were measured. This allowed for a reliable, relatively easy, direct separation analysis among microchips fabricated from the various photomasks. After multiple separations using microchips fabricated from each resolution mask, we found that the change in sidewall surface roughness did not significantly affect the dispersion coefficients measured, and thus the separation quality. The lower mask resolution limit, rather, was governed by the fidelity to which the mask could capture the original CAD design.

Journal Article↗