Markéta Ryvolová

Combined contactless conductometric, photometric, and fluorimetric single point detector for capillary separation methods.

This work for the first time combines three on-capillary detection methods, namely, capacitively coupled contactless conductometric (C(4)D), photometric (PD), and fluorimetric (FD), in a single (identical) point of detection cell, allowing concurrent measurements at a single point of detection for use in capillary electrophoresis, capillary electrochromatography, and capillary/nanoliquid chromatography. The novel design is based on a standard 6.3 mm i.d. fiber-optic SMA adapter with a drilled opening for the separation capillary to go through, to which two concentrically positioned C(4)D detection electrodes with a detection gap of 7 mm were added on each side acting simultaneously as capillary guides. The optical fibers in the SMA adapter were used for the photometric signal (absorbance), and another optical fiber at a 45 degrees angle to the capillary was applied to collect the emitted light for FD. Light emitting diodes (255 and 470 nm) were used as light sources for the PD and FD detection modes. LOD values were determined under flow-injection conditions to exclude any stacking effects: For the 470 nm LED limits of detection (LODs) for FD and PD were for fluorescein (1 x 10(-8) mol/L) and tartrazine (6 x 10(-6) mol/L), respectively, and the LOD for the C(4)D was for magnesium chloride (5 x 10(-7) mol/L). The advantage of the three different detection signals in a single point is demonstrated in capillary electrophoresis using model mixtures and samples including a mixture of fluorescent and nonfluorescent dyes and common ions, underivatized amino acids, and a fluorescently labeled digest of bovine serum albumin.

Chip‐based CE for rapid separation of 8‐aminopyrene‐1,3,6‐trisulfonic acid (APTS) derivatized glycans

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid – lectin affinity enrichment. After fluorescent labeling with 8‐aminopyrene‐1,3,6‐trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470u2009nm) LED. The separation was completed in 40u2009s in a microfluidic channel of 14u2009mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20‐mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip‐based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35u2009cm separation length and microfluidic chips with a separation distance of only 14u2009mm.

Recent significant developments in detection and method development for the determination of inorganic ions by CE

CE has been available as a tool for almost 20 years, but it is only in the past several years that it has been implemented widely. This has been the result of some significant advances in the technique. These fall in the areas of indirect photometric detection (through the use of dyes as probes and LEDs as light sources), the introduction and establishment of capacitively‐coupled contactless conductivity detection (C4D) as a routine, sensitive and commercially available detection method, and in software capable of simulation of separations and the selection of optimal composition of the BGE. These developments are reviewed and their impact illustrated by reference to a case study involving the rapid separation and sensitive detection of 15 anions and 12 cations on a portable CE instrument. It is shown that C4D provides considerably superior detection sensitivity (by a factor of about 8 in comparison with optimised indirect photometry).

Permeability of rapid prototyped artificial bone scaffold structures

In this work, various three-dimensional (3D) scaffolds were produced via micro-stereolithography (µ-SLA) and 3D printing (3DP) techniques. This work demonstrates the advantages and disadvantages of these two different rapid prototyping methods for production of bone scaffolds. Compared to 3DP, SLA provides for smaller feature production with better dimensional resolution and accuracy. The permeability of these structures was evaluated experimentally and via numerical simulation utilizing a newly derived Kozeny-Carman based equation for intrinsic permeability. Both experimental and simulation studies took account of porosity percentage, pore size, and pore geometry. Porosity content was varied from 30% to 70%, pore size from 0.34 mm to 3 mm, and pore geometries of cubic and hexagonal closed packed were examined. Two different fluid viscosity levels of 1 mPa · s and 3.6 mPa · s were used. The experimental and theoretical results indicated that permeability increased when larger pore size, increased fluid viscosity, and higher percentage porosity were utilized, with highest to lowest degree of significance following the same order. Higher viscosity was found to result in permeabilities 2.2 to 3.3 times higher than for water. This latter result was found to be independent of pore morphology type. As well as demonstrating method for determining design parameters most beneficial for scaffold structure design, the results also illustrate how the variations in patients blood viscosity can be extremely important in allowing for permeability through the bone and scaffold structures.

Study of Fluorescently Labelled Peptides and Small Proteins Using Capillary Electrophoresis Combined with Laser Induced Fluorescence and MALDI MS Detection

In this work, we apply CE-LIF, CE-UV and CE - Matrix AssistednLaser Desorption/Ionization Time-of-Flight Mass Spectrometryn(MALDI TOFMS) to investigate derivatization of model peptidesnand small proteins with rhodamine-based fluorescent reagents,nwhich are compatible with the wavelength of frequency doubledndiode pumped Nd:YAG excitation laser (532 nm). While the LIFndetection mode is needed for sensitive detection, the MALDInTOFMS analysis can reveal the mass of analytes. Bothnderivatized and non-derivatized peptides and proteins, thennumber of fluorescent labels per peptide/proteins and othernproduct of derivatization process can be detected directly fromnreaction mixtures or after separation. Thus, MALDI TOFMS servesnfor additional identification of peaks present in CELIF or CEUVnelectropherograms. For off-line coupling of CE with MALDI MS,nsubatmospheric deposition interface was used. An examples ofnfinal sensitive CELIF separation of derivatized peptides andnsmall proteins are given. Main advantages and disadvantages ofnLIF, absorbance and MS detector for capillary electrophoresisnof polypeptides are disscused. Attention is paid to detectionnlimits in practical analysis.