Gradient Pump Research Articles

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Development of miniaturized multi-channel high-performance liquid chromatography for high-throughput analysis

We have developed miniaturized multi-channel high-performance liquid chromatography (HPLC) system. With this system, we can simultaneously separate multiple samples, using a single high-pressure gradient pump, a chip-based sample injection unit, a monolithic silica capillary column array, and a multi-channel UV detection unit based on fiber optics. The injection unit has a simplified structure composed of brass housing and a quartz microchip having microchannels and access ports, which enable a direct injection of sample to multi-channel by commercial multichannel micropipette. Moreover, that possesses a function of microvalve, and on-chip definition of sample injection plugs achieved with a cross channel injection method, providing each column of monolithic silica capillary array. The substances in channels were simultaneously detected with UV having multiple cells. Standard samples were analyzed for characterizing newly developed system, and sharp peaks were obtained with reproducibility data of < 0.9% (R.S.D.). Analysis of tryptic digestion of casein was also employed. These results show that the novel multi-channel HPLC system has the benefits for the high-throughput analysis in the post-genomic analysis/combinatorial chemistry.

Investigation of new chelation ion chromatography procedure to determine the surface composition of powdered metal oxide samples in the solid state

Numerous tests have been conducted on the feasibility of characterizing the surfaces of metal oxide powders using HPLC. An in-line filter housing was modified to serve as a sample chamber to replace the sample loop. A gradient pump was used to gradually increase eluent acidity to find the conditions at which the surface of a metal oxide powder began to dissolve. The theoretical masses of surface monolayers of metal oxide powders were compared with the experimentally determined masses of dissolved material thought to be from the surface to test whether surface and bulk dissolution phenomena in acidic conditions are separable and quantifiable. A set of methods was tested that could first dissolve a metal oxide sample’s surface, then separate and detect analyte species by chelation ion chromatography. Surface characterization by ion chromatography could be more cost-effective than existing methods, and reveal chemical properties of the sample where existing methods only give physical composition and properties.

Investigation of new chelation ion chromatography procedure to determine the surface composition of powdered metal oxide samples in the solid state

Numerous tests have been conducted on the feasibility of characterizing the surfaces of metal oxide powders using HPLC. An in-line filter housing was modified to serve as a sample chamber to replace the sample loop. A gradient pump was used to gradually increase eluent acidity to find the conditions at which the surface of a metal oxide powder began to dissolve. The theoretical masses of surface monolayers of metal oxide powders were compared with the experimentally determined masses of dissolved material thought to be from the surface to test whether surface and bulk dissolution phenomena in acidic conditions are separable and quantifiable. A set of methods was tested that could first dissolve a metal oxide sample’s surface, then separate and detect analyte species by chelation ion chromatography. Surface characterization by ion chromatography could be more cost-effective than existing methods, and reveal chemical properties of the sample where existing methods only give physical composition and properties.

Nanoflow gradient generator for capillary high-performance liquid chromatography.

A novel method of generating a nanoflow gradient elution for a capillary high-performance liquid chromatography (HPLC) system has been developed. An important feature of this system is that any gradient (GR) profile generated by a conventional microflow GR pump can be asymptotically traced and converted as a corresponding nanoflow GR profile simply by using a 10-port switching valve with two injection loops installed. Consequently, it has been called an "asymptotic trace 10-port valve" (AT10PV) nanoflow GR generator. Performance of the AT10PV nanoflow GR generator was tested in the range of flow rates from 50 to 500 nL/min. The test demonstrated that the AT10PV nanoflow GR generator can asymptotically trace the original gradient profile with good reproducibility. A capillary HPLC system using the AT10PV nanoflow GR generator provides reasonably good repeatability of peak retention times on the chromatogram of the tryptic digest of a BSA sample, RSD of less than 0.3% at a flow rate of 200 nL/min. It also enables sequential running of a series of sample injections in the same manner as conventional analysis at microflow rates.

A new HPLC method to determine glomerular filtration rate and effective renal plasma flow in conscious dogs by single intravenous administration of iohexol and p-aminohippuric acid.

A high-performance liquid chromatography method to determine iohexol (IOX) and p-aminohippuric acid (PAH) in the plasma of dogs is evaluated according to recovery, reproducibility, and linearity utilizing a gradient pump. The mobile phase consists of 50mM sodium dihydrogen phosphate with 0.5mM tetrabutylammonium chloride, the pH is adjusted to 4.1, methanol is added to the final ratio of 90:10 (v/v), the flow rate is set at 1 mL/min, and separation is achieved with an ODS2 Luna column. The UV detector is set at 254 nm. IOX and PAH are used for evaluation of the effective renal plasma flow (ERPF) and glomerular filtration rate (GFR). The present method tested in three dogs demonstrates the accuracy in the evaluation of ERPF and GFR. Because of its precision and simplicity and low cost, it can be considered a good tool for ERPF and GFR in small animal practice.

Performance and reliability of splitless microliter gradient pumps in a metabolic stability study using cytochrome P450/3A4 and capillary liquid chromatography–mass spectrometry

The performance of two commercially available capillary LC pumps (MicroPro (Eldex, USA), Evolution 200 (ProLab, Switzerland)) generating really splitless gradients in the microliter per minute range was tested in detail concerning their applicability for routine drug discovery. A standard method to study metabolic stability against CYP450 isoform 3A4 was selected. This method was transformed into a fast splitless capillary LC–MS method. Both pumps generated reproducible gradients at flows of 5–10 μl/min within 10–15 min. Although gradient formation of the MicroPro system was very reproducible, its equilibration time was too long for fast gradients around 5 μl/min. The Evolution 200 pump offered a good performance with 180 μm i.d. columns at a flow rate of 6 μl/min. The precision of the retention time of the internal standard (ISTD) varied between 1.4 and 3.4% ( n=131–152, three different columns tested). Up to 800 injections of sufficient performance on one column and a stable enough response of the ISTD for 16 h sequence duration were obtained. Accuracy between 95 and 105% and precision ≤8.4% for 1′-hydroxylated midozolam were reached. The IC 50 values of the miniaturized assay (drug candidate BAL4815 1.7±0.5, itraconazole 0.46±0.06, and ketoconazole 0.12±0.01 μM) agreed well with those of the conventional approach. Details concerning method optimization and limitations in operation are discussed in detail. Still, the overall performance of the capillary LC pumps cannot cope completely with that of conventional HPLC pumps in terms of user-friendliness.

A simple HPLC method with pulsed EC detection for the analysis of creatine

The objective of this study was to develop a simple and sensitive LC method for the determination of creatine in aqueous solutions as well as in rat plasma using electrochemical detection. The chromatographic system consisted of a GP50 gradient pump, an ED40 pulsed electrochemical detector, and an AI-450 chromatography automation system (Dionex). The mobile phase consisted of a mixture of water, acetonitrile, 0.01 M sodium acetate, and 1.0 M sodium hydroxide (2.5:2.5:90:5, V/V/V/V) at a flow rate of 1.0 ml/min. The chromatographic separation was achieved at 45 °C on a column with a polyhydroxylated glucose and sulfonated stationary phase. The retention times of creatine and creatinine was 3.50 and 4.73 min, respectively, with creatine fully resolved from its major degradation product, creatinine. The standard curves were linear over the concentration range of 0–20 μg/ml. Within-day and day-to-day relative standard deviations (R.S.D.) were less than 10%. This method was used to study dissolution characteristics of various creatine salts in water.

EFFECT OF MAGNESIUM ON THE GROWTH AND ALKALOID PRODUCTION OF HAIRY ROOT CULTURES

EFFECT OF MAGNESIUM ON THE GROWTH AND ALKALOID PRODUCTION OF HAIRY ROOT CULTURES

COMPREHENSIVE EVALUATION OF DIFFERENT SOLIDAGINIS HERBA EXTRACTS

COMPREHENSIVE EVALUATION OF DIFFERENT SOLIDAGINIS HERBA EXTRACTS

Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification.

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).

On-line pH modification of carbonate eluents using an electrolytic potassium hydroxide generator for ion chromatography

Classical gradient elution, based on the application of a gradient pump used for mixing two or more prepared eluent components in pre-determined concentrations, was replaced by a chromatography system equipped with an isocratic pump and an electrolytic KOH generator. The isocratic pump delivered a constant concentration eluent composed of pure hydrogencarbonate solution. Carbonate ions, the main component of carbonate/hydrogencarbonate-based eluents, were formed by titration of hydrogencarbonate with KOH formed on-line in the electrolytic KOH generator. By changing the concentration of electrolytically-generated KOH, the eluent composition could be changed from pure hydrogencarbonate to a carbonate/hydrogencarbonate buffer, and finally to a carbonate/hydroxide-based eluent. The described system was tested to achieve pH-based changes of retention behavior of phosphate under constant inflow eluent composition conditions.

Two-pump at-column-dilution configuration for preparative liquid chromatography-mass spectrometry.

Preparative liquid chromatography-mass spectrometry (LC-MS) is widely used in parallel synthesis schemes to expedite purification. Recently, an alternative sample loading scheme, at column dilution, has been shown to dramatically increase the mass loading capacity of LC-MS purification methods. The prototype system utilized separate sample loading and binary gradient pumps. We report here a configuration for effecting at-column dilution using only the two pumps that provide the binary gradient flow. The advantages of a two-pump configuration are reduced cost, reduced space requirements, simplified control, and reduced service and maintenance issues. The two-pump at-column-dilution configuration is demonstrated for large- and small-scale LC-MS purifications. Purification on scales appropriate for high-throughput parallel synthesis can be achieved with small-scale chromatography using at-column dilution; purification of 20 mg of material is demonstrated using a 4.6 mm x 150 mm column and a flow rate of 3 mL/m. Reducing the scale of chromatography required for LC-MS purification has significant benefits, including the following. It requires less expensive columns, consumes less solvent, generates smaller-volume fractions (shorter dry-down time and the ability to collect into small-volume collector formats, such as 96-well plates), and has the potential for faster separations.

Determination of REEs distribution in monazite and xenotime minerals by ion chromatography and ICP-AES.

Ion chromatographic techniques were investigated for the separation and the quantitative determination of some rare earth elements (REEs) in monazite and xenotime minerals. The influences of selected eluents containing complexing acids including oxalic and alpha-hydroxy isobutyric acid (alpha-HIBA) on the retention and hence the separation efficiency of REEs was studied. Different variables affecting the separation of different REEs such as pH, type, and concentration of the mobile phase were investigated. Gradient elution, using an advanced gradient pump, was controlled automatically by the Dionex AI-450 computer software. Separation of REEs was carried out using an Ion Pac CS5A column followed by a post column derivatization reaction with 4-(2-pyridylazo)resorcinol (PAR) and UV-VIS spectrophotometric detection. Mineral dissolution was carried out using sulfuric acid. A comparative evaluation of REE distribution in monazite and xenotime minerals using both ion chromatography (IC) and inductively coupled plasma atomic emission spectrometric (ICP-AES) techniques was carried out.

Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples

This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.

Determination of tripolyphosphate in frozen cod and scallop adductor by ion chromatography

We introduce a method for the determination of tripolyphosphate in frozen cod and scallop adductor by using ion chromatography. The tripolyphosphate was extracted from minced cod and scallop adductor with deionized water by ultrasonic leaching, and then the proteins soluble in water were precipitated with trichloroacetic acid and removed by filtering. An ion chromatograph with an ionpac AS11-HC anion-exchange column, an ASRS (Anion Self Regenerating Suppression), a conductivity detector and a gradient pump (sodium hydroxide gradient) was used. The detection limit was below 5 mg tripolyphosphate/kg cod or scallop adductor. This method is applicable to the determination of polyphosphates in aquatic products and the procedures are easy to implement.

Determination of glutamate and aspartate in microdialysis samples by reversed-phase column liquid chromatography with fluorescence and electrochemical detection

Five different systems for fast determination of aspartate and glutamate in microdialysis samples are described: (I) a high-speed HPLC using a gradient pump with a sharp elution profile, (II) a column switching technique, (III) an isocratic pump with a low-pressure switching valve for one-step gradients, (IV) microbore chromatography using injections of acetonitrile as a wash-out step, (V) on-line connection of microdialysis and HPLC/derivatization. In all cases, automated precolumn derivatization with o-phthalaldehyde-2-mercaptoethanol reagent were used. Both fluorescence and electrochemical detection techniques were evaluated in terms of reproducibility, sensitivity, interference, maintenance and troubleshooting. The electrochemical detection method required a second derivatization step with 0.2 M iodoacetamide to remove excess of a thiol moiety and regular recalibrations after each six to ten injections. Under these conditions the correlation coefficients for electrochemical vs. fluorescence detectors were 0.918 for Asp and 0.988 for Glu for 65 microdialysis samples. Coefficients of variation for six analyses between calibrations were below 3% for both detectors. The limits of detection for both amino acids were about 0.4 pmol for electrochemical detection with a thiol scavenger step, 50 fmol for fluorescence detection using conventional columns and about 20–30 fmol for the microbore system. All systems are suitable for detecting basal levels of Asp and Glu in 5–10 μl microdialysis samples from a rat brain where typical concentrations lie around 1–10 pmol or more. It is concluded that a microbore setup with one isocratic pump and an autosampler optimized for injections of washing solvent between samples is the most practical and economical. The system allows analysis of minute sample volumes down to 1–2 μl.

Electro-elution ion chromatography: controlling sample retention by electrochemically generating or modifying the mobile phase

A new ion chromatographic technique called electro-elution ion chromatography is described. An electrochemical process is used to generate or moderate the mobile phase composition inside the column. The technique offers several advantages over traditional ion chromatography. For example, only water is required as the mobile phase for anion and cation analysis. This reduces cost and chemical waste. Since the concentration of the electrolysis products is directly proportional to the amount of current applied, traditional gradient separation can be accomplished by time-based programming of the current or voltage applied across the column. This eliminates the need for a gradient pump. This technique is self-suppressing, eliminating the need for a suppressor system.

Purification and Characterization of 2-Hydroxybiphenyl 3-Monooxygenase, a Novel NADH-dependent, FAD-containing Aromatic Hydroxylase from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from a recombinant of Escherichia coli JM109, which efficiently expressed the hbpA gene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparent Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM for oxygen. The enzyme was inactivated by p-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the betaalphabeta-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.