Cheanyeh Cheng

Analysis of the enzymatic racemization of D-aspartic acid to L-aspartic acid by the on-line coupling of a solid-phase extraction column and a ligand-exchange high-performance liquid chromatography column.

D-Aspartic acid can be enzymatically biotransformed with D-amino acid oxidase and aminotransferase to L-aspartic acid. The reaction was surveyed at three temperatures and a period of 3 days, however, L-aspartic acid can be produced only at the reaction temperature 90 degrees C. However, the separation of D-aspartic acid and L-aspartic acid by ligand-exchange chromatography showed matrix interference. Therefore, the column-switching technique by coupling a solid-phase extraction (SPE) column to the analytical ligand-exchange HPLC column was used to eliminate the matrix effect. The pretreatment of reaction samples with the SPE column was considered as a combination of size-exclusion chromatography and ion-pair chromatography. The ion-pair reagent was 0.005 M sodium 1-octanesulfonate aqueous solution adjusted to pH 2.2. Part of the first eluted peak from the SPE column was then switched through the ligand-exchange column and analyzed with a 0.25 mM Cu2+ aqueous mobile phase of pH 3.6. The quantitative analysis of D- and L-aspartic acids was performed by the standard addition method. Overall, the separation and analysis of D- and L-aspartic acids in the enzymic solution was convenient, fast, and successful with the developed on-line LC-LC column-coupling and column-switching system.

On-line desalting and carbohydrate analysis for immobilized enzyme hydrolysis of waste cellulosic biomass by column-switching high-performance liquid chromatography

An innovative green column-switching high-performance liquid chromatographic (HPLC) technique was developed by coupling traditional and Pb(2+) ion-exclusion columns to study enzyme hydrolysis components of waste cellulosic biomass. Pure water was used as the mobile phase to separate neutral polar analytes in high salt content solution. The column-switching HPLC-RI was connected on-line to the immobilized enzyme reactor for successive on-line desalting and simultaneous analysis of six carbohydrates (cellobiose, glucose, xylose, galactose, mannose, and arabinose) in the hydrolysate of waste paper and waste tree branch by incorporating the heart-cut and the elution-time-difference techniques. Six internal standard calibration curves in the linear concentration range of 0-2,000 microg mL(-1) were prepared. Xylitol was used as the internal standard to give excellent linear correlation coefficients (0.9984-0.9999). The limits of detection and quantification for cellobiose, glucose, xylose, galactose, mannose, and arabinose varied between 0.12-4.88 and 0.40-16.3 microg mL(-1), respectively, with an accuracy of 90-102% and a precision of 0.1-7.8%. Cellulose and hemicellulose contents were higher in waste paper than in waste tree branch.

Simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero by on-line postcolumn derivation fluorescence detection and ultraviolet detection coupled two-dimensional high-performance liquid chromatography

An innovative two-dimensional high-performance liquid chromatography system was developed for the simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero. A C8 reversed-phase chromatographic column with ultraviolet detection was used as the first dimension for the determination of aspartame, and a ligand-exchange chromatographic column with on-line postcolumn derivation fluorescence detection was employed as the second dimension for the analysis of amino acid enantiomers. The fluorimetric derivative reagent of amino acid enantiomers was o-phthaldialdehyde. The hydrolysis of aspartame in Coca-Cola Zero was induced by electric-heating or microwave heating. Aspartame was quantified by the matrix matched external standard calibration curve with a linear concentration range of 0-50 μg mL(-1) (r(2)=0.9984). The limit of detection (LOD) and the limit of quantification (LOQ) were 1.3 μg mL(-1) and 4.3 μg mL(-1), respectively. The amino acid enantiomers was analyzed by the matrix matched internal standard calibration method (D-leucine as the internal standard) with a linear concentration range of 0-10 μg mL(-1) (r(2)=0.9988-0.9997). The LODs and LOQs for L- and D-aspartic acid and L- and D-phenylalanine were 0.16-0.17 μg mL(-1) and 0.52-0.55 μg mL(-1), respectively, that was 12-13 times more sensitive than ultraviolet detection. The overall analysis accuracy for aspartame and amino acid enantiomers was 90.2-99.2% and 90.4-96.2%, respectively. The overall analysis precision for aspartame and amino acid enantiomers was 0.1-1.7% and 0.5-6.7%, respectively. Generally, the extent of aspartame hydrolysis increases with the increase of electro-thermal temperature, microwave power, and the duration of hydrolysis time. D-aspartic acid and D-phenylalanine can be observed with the electro-thermal racemization at the hydrolysis temperature 120°C for 1 day and only D-aspartic acid can be observed at the hydrolysis temperature 90°C for 2 and 3 days. For the microwave induced hydrolysis, only L-aspartic acid was detected at the power 560 W for 1 min and 320 W for 3 min.

Glucose metabolism and bioreduction of 2-butanone byCandida utilis studied by means of ion-exchange chromatography

Abstract The metabolism of glucose during cell growth was important for the production yield of the yeast mediated bioreduction of ketones. Thus, the choice of the very simple acyclic ketone 2-butatone as the substrate forCandida utilis can easily explain the effect of glucose metabolism on the enantioselective bioreduction mechanism of ketone. The glucose metabolism was extended by adding the sorbitol (polyol) pathway to the Embden-Meyerhoff pathway due to the existence of a NADP-specific polyol dehydrogenase inC. utilis. The metabolite, glycerol, was produced via the sorbitol pathway which could suppress the production of ethanol by the Embden-Meyerhoff pathway. The reaction mechanism of bioreduction showed that ethanol, an energy source of the bioreduction, must be oxidized to acetic acid to activate the oxidoreductase. The formation of acetic acid, therefore, can serve as a mark for the success of bioreduction. On the other hand, the unconsumed glucose and the less added 2-butanone indicated a negative effect for the bioreduction. Overall, the microbial biotransformation of 2-butanone toS-form 2-butanol studied by ion-exchange chromatography was successful.

Online eluent-switching technique coupled anion-exchange liquid chromatography–ion trap tandem mass spectrometry for analysis of non-steroidal anti-inflammatory drugs in pig serum.

A novel method for online extraction, pH-gradient separation, and analysis of nine non-steroidal anti-inflammatory drugs (NSAIDs) was developed by coupling online eluent-switching technique to single anion-exchange chromatographic column/ion trap mass spectrometer (MS) and used for monitoring NSAIDs residues in pig serum. A neutral eluent and a pH-gradient eluent were used for extraction and separation of NSAIDs, respectively. Each of nine NSAIDs has an MS precursor ion of either [M−H]− or [M−Na]−. The extracted ion chromatogram for a specific product ion of each NSAID was used for its quantitative analysis. The dynamic linear ranges of calibration curves were all 0–200 ng mL−1 (R2 > 0.9950). The analysis accuracies estimated by spiking standard concentrations at 20, 100, and 200 ng mL−1 were 80.5–99.9%. The corresponding intra-day and inter-day precisions (RSD%) were 2.5–14.5% and 2.9–15.2%, respectively. The limit of detection/limit of quantitation of NSAIDs were 1.3/4.3, 0.5/1.6, 0.2/0.5, 2.5/8.2, 1.5/4.9, 0.6/2.1, 0.6/2.0, 0.5/1.7, and 0.6/2.1 ng mL−1 for carprofen, diclofenac, flunixin, ibuprofen, ketoprofen, meclofenamic acid sodium, mefenamic acid, niflumic acid, and tolfenamic acid, respectively. After 1 h injection of a dose containing 2 mg kg−1 weight pig of flunixin and tolfenamic acid to the pigs, a residue amount of 3480 ± 36 ng mL−1 and 431 ± 13 ng mL−1, respectively, was reached for the incurred pig serum specimens and both residues were reduced to about 20 ng mL−1 at the time of 24 h.

An Electronic Tongue System Design Using Ion Sensitive Field Effect Transistors and Their Interfacing Circuit Techniques

This paper proposes an electronic tongue system design using ion sensitive field effect transistors (ISFETs), extended-gate FET (EGFET) and their interfacing circuit techniques. Bridge-type constant voltage, constant current, and temperature compensation circuitries have all been developed for ISFET to sense hydrogen and chloride ions for water quality monitoring applications. This design offers a sensitivity of over 54 mV/pH and an improved temperature coefficient (T.C.) of 0.02 mV/degC; in addition, a sensitivity of 43 mV/pCl can be achieved by using a proposed extended-gate FET with a mixed polyvinyl chloride (PVC), chloride ionophore III (ETH9033) and lipophylic salt on the indium-tin-oxide (ITO)/glass substrate.

Application of ligand-exchange chromatography to the assay of l-alanine from dl-aspartic acid by Pseudomonas dacunhae

A direct chiral ligand-exchange chromatographic method was developed to monitor L-alanine production by fermentation. A mobile phase containing aqueous 0.25 mM Zn2+ solution is utilized to separate amino acids in the fermentation medium. The detection limit for L-alanine is 0.5 ppm and the analysis time for one sample is about 8 min. As sample preparation is simple and the matrix effects are minimal, the assay is fast and convenient. The results indicate that the method has potential for the analysis of complicated fermentation media.

Design of a multi-frequency Bio-Impedance Spectroscopy system Analog Front-End and Digital Back-End with system on-chip implementation

In this paper, the design of a Bio-Impedance Spectroscopy system is presented. The system consists of a Direct Digital Synthesis Sine-Wave Generator (DDS-SWG), an Analog Front-End (AFE), an Analog-Digital Converter (ADC), and a Digital Back-End implemented using a Field Programmable Gate Array (FPGA). The FPGA also drives the ADC, the DDS-SWG, and the LCD. The AFE consists of a high output impedance Load-In-Loop Current Source (LL-CS) and a set of High Common-Mode Rejection Ratio (CMRR) Instrumentation Amplifiers (IA). It uses a simple and straightforward impedance extraction algorithm using just division and constant multiplication. The CMOS implementation of the AFE using the TSMC 0.35um 2P4M is also presented. The system forms part of a platform that aims to predict a urolithiasis (urinary stone) event.

Online capillary solid-phase microextraction coupled liquid chromatography-mass spectrometry for analysis of chiral secondary alcohol products in yeast catalyzed stereoselective reduction cell culture

An online solid-phase microextraction coupled liquid chromatography-electrospray ionization-ion trap mass spectrometry was developed for the analysis of trace R- and S-4-phenyl-2-butanol (R- and S-pbol) in salt rich cell culture of Saccharomyces cerevisiae catalyzed stereoselective reduction of 4-pheny-2-butanone (pbone). A Supel-Q PLOT capillary column was used for the extraction and deionized distilled water was used as the extraction mobile phase. The extraction flow rate and extraction time were at 0.1 mL min(-1) and 0.95 min, respectively. The three target analytes, pbone, R-pbol, and S-4-pbol, were desorbed and eluted by the mobile phase of water/methanol/isopropanol (55/25/20, v/v/v) with a flow rate of 0.5 mL min(-1) and analyzed by a chiral column. The mass spectrometric detection of the three target analytes was in positive ion mode with the signal [M+Na](+). The matrix-matched external standard calibration curves with linear concentration range between 0 and 50 μg mL(-1) were used for quantitative analysis. The linear regression correlation coefficients (r(2)) of the standard calibration curves were between 0.9950 and 0.9961. The yeast mediated reduction was performed with a recation culture of yeast incubation culture/glycerol (70/30, v/v) for 4 days. This biotransformation possessed 82.3% yield and 92.9% S-enantomeric excess. The limit of detection (LOD)/limit of quantification (LOQ) for pbone, R-pbol, and S-pbol was 0.02/0.067, 0.01/0.033, and 0.01/0.033 μg mL(-1), respectively. The intra-day and inter-day precisions from repeated measurements were 10.8-21.1% and 11.6-18.7%, respectively. The analysis accuracy from spike recovery was 84-91%.

Design of a multi-sensor readout chip for amperometric, potentiometric, impedometric, and colorimetric bio-sensor applications

Point-of-Care Testing (POCT) devices are being deployed to provide personalized healthcare monitoring or for remote monitoring whenever there is no accessible medical facility. In line with POCT, Lab-on-a Paper or Lab-on-a-Chip devices are taking flight. These include the hybrid of the sensor and readout circuit fabricated into a chip. To date, there is no single chip solution that harmonizes the four electrochemical sensing modalities which is the contribution of this work. The design of a multi-sensor readout chip is presented; this is composed of amperometric, potentiometric, impedometric, and colorimetric readout circuits implemented on TSMC 0.18 µm 1P6M technology. The chip can be used to implement a POCT to assist a patient in assessing the condition of his urinary system through urine quality indicators such as: pH value and ionic concentration of calcium (Ca2+) via potentiometric sensing; uric acid, citric acid, and calcium oxalate concentrations via amperometric sensing; osmolality or total dissolved salts through impedometric sensing; and urine turbidity, color and crystallization through its optical properties. The readout circuit designs for diversified sensing mechanisms were developed and investigated in this paper. In addition, a power savings of ∼60dB can be achieved when all the circuits of the proposed chip are disabled. The sub-circuit enable was implemented using PMOS pass transistors. Non-simultaneous sub-circuit enable both reduces the power budget and prevents the inter-sensor interference or cross-talk.