Biomedical subjects

Uwe D Neue

Publications and source records attributed to Uwe D Neue.

14 recordsLinked to original sources

PubMed2006

Selectivity in reversed-phase separations Influence of the stationary phase.

The selectivity difference between 15 different stationary phases was measured using a large number of analytes at 2 or 3 different pH values (3, 7 and 10) with acetonitrile and methanol as the mobile phase modifiers. The packings discussed include standard C(8) and C(18) packings, packings with embedded polar groups, a phenyl packing, a pentafluoro-phenyl packing, an adamantylethyl packing and others. The major selectivity differences observed are discussed in detail. Specific effects such as pi-pi interactions on phenyl packings or hydrogen-bond interactions on phases with embedded polar groups are confirmed.

Acetonitriles↗

PubMed2006

Peak compression in reversed-phase gradient elution.

Previous reports suggest that peak widths in linear gradient elution are consistently larger than predicted by theory; however, if gradient compression is ignored, experiment and theory are in reasonable agreement. This suggests that gradient compression might represent an incorrect or poorly understood concept. In the present study, an experimental program was carried out to better understand the role of gradient compression and the reason for past differences between experiment and theory. It is concluded that the concept of gradient compression is correct.

Chromatography, High Pressure Liquid↗

PubMed2006

Changes in the pore character of silicas caused by surface bonding and polymer coating.

The pore character of packings for liquid chromatography, especially reversed-phase (RPLC) packings, has been studied by means of the nitrogen adsorption method (BET method). Micro-spherical silica gels with 9, 12, 30, 40 and 50 nm average pore diameter have been used as carriers. These silica gels have been modified with monochlorodimethyloctadecylsilane and several polymers (polyoctadecylmethacrylate-methylmethacrylate co-polymer, polyacrylamide gel, polyvinyl alcohol, poly-2-hydroxyethyl-methacrylate). A larger decrease in the specific surface area values was observed in the case of the polymer coating with the polyoctadecylmethacrylate-methylmethacrylate co-polymer compared with the derivatization by silanes or the modification with polymers without C18-groups. A new approach has been suggested to explain some questions concerning the interpretation of the data obtained during the measurements of the pore characteristics of the derivatized packings. An attempt has been made to reveal peculiarities connecting values of the measured surface of RP-packings with the specific surface area values of the initial silica, as well as with the chromatographically accessible surface.

Journal Article↗

PubMed2006

A detailed discussion of the influence of the amount of deposited polymer on the retention properties of polymer-coated silicas.

Problems of the modification of the surface of micro-spherical silica gel with layers of polymer and the influence of the quantity of the coated polymer on the chromatographic properties of the resulting sorbents have been considered. The polymer modification of the surface of wide-pore micro-spherical silica gel obtained by means of hydrothermal treatment of the meso-porous silica gel under autoclave conditions is described. The polymer layer itself is formed by an octadecylmethacrylate-methylmethacrylate co-polymer. As a result, packings for RP-HPLC were obtained. An increase of the capacity factor (k) has been observed with an increase of the quantity of the polymer deposited during the polymer modification process. The indicated phenomenon depends on the increase of the number of C18 groups in the packing.

Journal Article↗

PubMed2006

Comparison of mixed-mode anion-exchange performance of N-vinylimidazole-divinylbenzene sorbent.

A newly synthesized copolymer based on N-vinylimidazole-divinylbenzene (NVIm-DVB) was evaluated as a mixed-mode anion-exchange sorbent for SPE, since the NVIm monomer apart from its hydrophilic properties can be protonated at a certain pH, and then performs as an anion-exchanger. To investigate the behavior of the NVIm-based sorbent, the SPE performance was evaluated under reversed-phase (RP), weak anion-exchange, and strong anion-exchange conditions. The results for the NVIm-DVB sorbent were also compared to commercial reference sorbents from each group: Oasis HLB, Oasis WAX, and Oasis MAX, respectively. SPE results from this evaluation showed that NVIm-DVB can be used as an RP material, compared to Oasis HLB. It also has the potential to act as a strong anion-exchange sorbent, compared to Oasis MAX, since under the proper conditions it was able to fraction and quantitatively recover a group of selected solutes.

Journal Article↗

PubMed2005

Theory of peak capacity in gradient elution.

Peak capacity is the best measure of the performance of a gradient separation. In this paper, the theory of peak capacity for the standard operating conditions of reversed-phase and ion-exchange chromatography is outlined. The influence of the operating conditions on the peak capacity of a separation are discussed. Finally, bandspreading phenomena in gradient chromatography are analyzed.

Chemical Phenomena↗

PubMed2005

The combined effect of silanols and the reversed-phase ligand on the retention of positively charged analytes.

The nature of the interaction of positively charged analytes with the surface of reversed-phase bonded phases has been investigated as a function of both pH and volume fraction of organic modifier. Studies of the combined effect of both the parameters have been previously reported by us, and the data presented here further demonstrate a multiplicative interaction between pH and the concentration of organic modifier in the mobile phase. Fitting of the data as functions of pH and eluent composition clearly shows that the hydrophobically assisted ion-exchange process dominates over a purely reversed-phase or a pure ion-exchange retention mechanism. The underlying theory is developed in detail, and the mechanism is elucidated using several reversed-phase packings of substantially different character.

Ligands↗

PubMed2004

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography.

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.

Chromatography, High Pressure Liquid↗

PubMed2004

Development of an accelerated low-pH reversed-phase liquid chromatography column stability test.

The important experimental design criteria for an accelerated low-pH RPLC column stability test are discussed. The influence of method variables on the amount and rate of retention-loss and the final optimized parameters for the accelerated low-pH RPLC stability test are presented. The retention-loss curves for selected C8 and C18 stationary phases are compared. These studies indicate that ligand chain length, functionality and bonding density play an important role in determining the low-pH stability of a stationary phase. Additionally, elemental analysis data are used to infer the mechanism responsible for the observed retention-loss under low-pH conditions.

Chromatography, High Pressure Liquid↗

PubMed2003

Fast LC/MS in the analysis of small molecules.

Liquid chromatography-mass spectrometry (LC/MS) has become one of the most widely used analytical techniques in both qualitative and quantitative analysis of small molecules. Recently, with the increasing demand for ever-higher sample throughput, the use of faster chromatographic separations has become popular, along with other LC/MS methods that decrease analytical cycle-time. The burgeoning use of LC/MS has meant that the primary expertise of many practitioners today is not in the field of LC/MS, which has been facilitated by the ease-of-use of modern LC/MS systems. An examination of the current state of the literature, relating to "fast LC/MS", should serve well to those new to LC/MS, and should help them in the development of fast LC/MS methods that are effective in terms of both the chromatography and the utilization of the mass spectrometer. This review paper focuses on fast LC/MS analyses of small molecules that have been reported in peer-reviewed publications.

Animals↗

PubMed2003

Role of mass spectrometry in the purification of peptides and proteins.

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.

Mass Spectrometry↗

PubMed2003

Comparison of the acidity of residual silanol groups in several liquid chromatography columns.

The silanol acidity of Waters Resolve C18, Waters Resolve silica, Waters Symmetry C18, Waters Symmetry silica, Waters XTerra MS C18 and underivatized XTerra columns has been measured from the retention of LiNO3 with a methanol/water (60:40) mobile phase buffered to different pH values. The Li+ cation is retained by cationic exchange with the background cation of the mobile phase (Na+) through the ionized silanols. The number of active silanols increases in the order: XTerra MS C18 << Symmetry C18 < underivatized XTerra << Resolve C18 < Resolve silica approximately equal to Symmetry silica. XTerra MS C18 does not present any residual silanol acidity up to s(s)pH 10.0 (pH in 60% methanol) as measured by LiNO3. The underivatized XTerra packing and Symmetry C18 present active silanols only at s(s)pH values higher than 7.0. For the other three columns, two different types of silanols with different acidity (s(s)pKa values about 3.5-4.6 and 6.2-6.8, respectively) have been observed. Symmetry C18 shows evidence of the presence of active basic sites that retain NO3- by anionic exchange.

Acids↗

PubMed2003

Performance of an ultra-low elution-volume 96-well plate: drug discovery and development applications.

Recently, sample preparation has been considered to be the major cause of bottlenecks during high-throughput analysis. With the assistance of robotic liquid handlers and the 96-well plate format, more samples can be prepared for subsequent liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Protein precipitation is still widely used despite potential loss of sensitivity or variable results due to ion suppression. The use of solid-phase extraction (SPE) clearly gives superior results but may not be as cost effective as protein precipitation due to the labor and material costs associated with the process. Here, a novel 96-well SPE plate is described that was designed to minimize the elution volume required for quantitative elution of analytes. The plate is packed with 2 mg of a high-capacity SPE sorbent that allows loading of up to 750 microL of plasma, while the novel design permits elution with as little as 25 microL. Therefore, the plate offers up to a 15-fold increase in sample concentration. The evaporation and reconstitution step that is typically required in SPE is avoided due to the concentrating ability of the plate. Examples of applications in drug discovery/development are shown and results are compared to protein precipitation. Excellent sensitivity and linearity are demonstrated.

Amitriptyline↗

PubMed2002

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Base Sequence↗