Search

An analytical method for determining potential endocrine disruptors (bisphenol A, 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol, p-t-butylphenol, p-pentylphenol, p-hexylphenol, pt-octylphenol, p-heptylphenol, nonylphenol) by solid-phase extraction (SPE) and High Perfomance Liquid Chromatography(HPLC) equipped with fluorescence and variable wavelength detector has been developed. The SPE process for sample concentration was performed on a commercially available Oasis HLB cartridge packed with polymeric sorbents. The effect of elution solvent and elution volume on the recoveries of the analytes were investigated with HPLC. Average recovery of >85% was achieved with 60mg sorbents using 5mL of methanol as elution solvent. Phenolic compounds in canned drinks, beverages and water samples were surveyed by this proposed method.

Although it has been well-recognized that microcystin-leucine-arginine (MCLR), the most common form of microcystins, induces neurotoxicity, little is currently known about the underlying mechanism for this neurotoxicity. Here, we found that MCLR (10 ng/μL/mouse, i.c.v.) induces significant neuronal loss in the hippocampus of mice. MCLR-induced neurotoxicity was accompanied by oxidative stress, as shown by a significant increase in the level of 4-hydroxynonenal, protein carbonyl, and reactive oxygen species (ROS). Superoxide dismutase-1 (SOD-1) activity was significantly increased, but glutathione peroxidase (GPx) level was significantly decreased following MCLR insult. In addition, MCLR significantly inhibited GSH/GSSG ratio, and significantly induced NFκB DNA binding activity. Because reduced activity of GPx appeared to be critical for the imbalance between activities of SODs and GPx, we utilized GPx-1 overexpressing transgenic mice to ascertain the role of GPx-1 in this neurotoxicity. Genetic overexpression of GPx-1 or NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly attenuated MCLR-induced hippocampal neuronal loss in mice. However, PDTC did not exert any additive effect on neuroprotection mediated by GPx-1 overexpression, indicating that NFκB is a neurotoxic target of MCLR. Combined, these results suggest that MCLR-induced neurotoxicity requires oxidative stress associated with failure in compensatory induction of GPx, possibly through activation of the transcription factor NFκB.

An analytical method for determining phenol considered priority pollutants of the US EPA andprecursor of toxic phenolic compounds by solid-phase extraction (SPE) and high performance liquidchromatographic systems (HPLC) equipped with fluorescence and mass selective detectors have been developed.The SPE process for sample preconcentration was performed on a commercially available Oasis HLB cartridgepacked with polymeric sorbents. The effect of pH, elution solvent, and elution volume on the recoveries ofthe analytes were investigated with HPLC/FLD. Average recovery of >87.0% was achieved with 60 mg sorbentsusing 5 mL of methanol as an elution solvent at pH=3.요약: Priority pollutant이며 페놀성화합물의 전구체인 페놀을 고체상추출법을 이용하여 추출 정제하여형광검출기와 질량분석기가 각각 장착된 고속액체크로마토그래프를 이용하여 분석하는 방법을 설정하여보았다. 고체상추출은 고분자성물질이 충진된 Oasis HLB cartridge를 이용하여 최적의 조건을 확립하였으며 이때의 평균회수율은 87.0% 이었다. 형광검출기를 이용한 경우가 질량분석기를 이용한 경우보다검출한계가 낮았으나 선택성의 측면에서 질량분석기가 우수하였다. Key words: phenol, hplc, solid phase extraction(spe), hplc/msd

Cyanobacterial toxins, such as microcystins, which exist in Korean lakes, are strongly toxic in fish, cattle, and humans. This study performs a quantitative analysis of cyanobacterial toxins in water by comparing the strip method and the HPLC method. Because the detection ranges of the strip method and the HPLC method are different, the water samples were diluted. The comparison of the strip method and the HPLC method was made using seven samples that contained different concentrations of microcystin. The quantitative results produced by the strip analysis were significantly aligned with the results of the HPLC analysis. The results of correlation analysis were r

In this study, the thermal denaturation mechanism and secondary structures of two types of human insulin nanoparticles produced by a process of solution-enhanced dispersion by supercritical fluids using dimethyl sulfoxide (DMSO) and ethanol (EtOH) solutions of insulin are investigated using spectroscopic approaches and molecular dynamics calculations. First, the temperature-dependent IR spectra of spherical and rod-shaped insulin nanoparticles prepared from DMSO and EtOH solution, respectively, are analyzed using principal component analysis (PCA) and 2D correlation spectroscopy to obtain a deeper understanding of the molecular structures and thermal behavior of the two insulin particle shapes. All-atom molecular dynamics (AAMD) calculations are performed to investigate the influence of the solvent molecules on the production of the insulin nanoparticles and to elucidate the geometric differences between the two types of nanoparticles. The results of the PCA, the 2D correlation spectroscopic analysis, and the AAMD calculations clearly reveal that the thermal denaturation mechanisms and the degrees of hydrogen bonding in the spherical and rod-shaped insulin nanoparticles are different. The polarity of the solvent might not alter the structure or function of the insulin produced, but the solvent polarity does influence the synthesis of different shapes of insulin nanoparticles.

E-mail: pyod@kangwon.ac.krReceived March 20, 2014, Accepted June 16, 2014Key Words : Supercritical fluid extraction, Curry, Curcumin, Carbon dioxideA dried curry powder is well known food additive in manyAsian countries. Curcumins are main coloring substancesand active compounds in curry powder which is the naturalyellow pigment in turmeric isolated from the rhizome of theplant Curcuma longa. Curcumins are consisted of curcumin([(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione) and two related compounds, demethoxy-curcumin (DMC) and bis demethoxycurcumin (BDMC).The chemical structure of curcumin is shown in Figure 1.Curcumins gives specific flavor and yellow color to currypowder.

For the quantitative detection of algal toxin, microcystin, a chemiluminescence immunochromatographic assay method was developed. The developed system consists of four parts, chemiluminescence assay strip (nitrocellulose membrane), horse radish peroxidase labeled microcystin monoclonal antibodies, chemiluminescence substrate (luminol and hydrogen peroxide), and luminometer. The performance of the chemiluminescence immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. The detection limit of chemiluminescence immunochromatographic assay system is several orders of magnitude lower than with HPLC. The chemiluminescence immunochromatography and HPLC results correlated very well with the correlation coefficient (r 2 ) of 0.979.

For the production of ethanol from freshwater cyanobacteria, a new pretreatment method using supercritical fluid was introduced. In this study, it was found that the supercritical fluid could penetrate inside the cell wall and help to liberate starch from cyanobacterial cells which resulted in the increase of the efficiency of ethanol production. For Microcystis aeruginosa, supercritical fluid pretreatment increased the amount of ethanol produced from cyanobacteria from 1.53 g/L to 2.66 g/L. For Anabaena variabilis, the amount of ethanol was increased from 1.25 g/L to 2.28 g/L. With use of supercritical fluid pretreatment, the efficiency of the process to obtain higher ethanol yields from freshwater cyanobacteria was improved upto 80%. The optimum temperature and pressure conditions for supercritical fluid pretreatment were determined as the temperature of and the pressure of 120 atm. This study demonstrates the feasibility of using supercritical fluid pretreatment for ethanol production using freshwater cyanobacteria.

Cyanobacterial toxins, microcystins which exist in Korean lakes show strong toxicity to fish, cattles and human. In this study, we tried to analyze cyanobacterial toxin, microcystin in the Microcystis cultivation solution using test strip, although the most common analytical methods for the detection of microcystin are HPLC and ELISA. This new anlytical method used the advantages of high specifisity and rapidness of test strip, high sensitivity of fluorescence reader. Therefore, we could analyze the trace amount of microcystin existed in various water samples without using the microcystin standards.

A new chemiluminescence immunochromatographic analysis system with high sensitivity and high reproducibility was developed for the determination of microcystins (MCs) in water. Horse radish peroxidase (HRP) labeled microcystin monoclonal antibody was used for the sensitive chemiluminescence detection. The chemiluminescence immunochromatographic analysis system was composed of microcystin LR (MCLR)-monoclonal antibody (mAb)-Horse Radish Peroxidase (HRP) conjugate, MCLR-BSA conjugate, luminol, hydrogen peroxide mixture solution, an immunochromatographic assay strip and luminometer. To detect the concentration of microcystins in water, we utilized one spot analysis of the strip instead of flow type analysis. We could detect the microcystins in water at a concentration as low as 9.45 pg/mL with the chemiluminescence (CL) detection.

A quantitative analysis of prostate-specific antigen (PSA) in samples of human blood serum by fluorescence immunochromatography using monoclonal antibodies to PSA was developed. The fluorescence immunochromatographic analysis system is composed of anti-PSA-monoclonal antibody (mAb), fluorescence conjugates in detection solution, a immunochromatographic assay strip, and a laser fluorescence scanner. A fluorescence immunochromatographic analysis system was employed to detect PSA on the basis of the area ratio between the control line and the test line of the strip. Under optimal conditions, the area ratio was proportional to PSA concentration ranging from 0.72 to 46.0 ng/mL with a detection limit of 0.72 ng/mL.

We demonstrate the possible application of the sandwich type surface-enhanced Raman scattering (SERS) immuno-assay using antigen-antibody binding for detection of prostate-specific antigen (PSA) in cancer cells. In this sandwich type of SERS immunoassay, to capture antigens onto the immobilized layer of antibodies on the gold substrate we prepared the monolayer of gold nanoparticles on the APTMS-derivatized surface of a glass slide by using the SAM technique. This sandwich type of SERS immunoassay in which antigens on the substrate specifically capture antibodies on a Raman reporter (DSNB coated gold nanoparticles with R6G) could successfully detect PSA at low levels. A strong SERS spectrum of Raman reporter was observed only with a substrate in which PSA is present.

Supercritical fluid extraction (SFE) with supercritical carbon dioxide and solvent extraction of isoflavones from soybeans were investigated. Extraction efficiencies under several different extraction conditions were examined. For SFE, pressure, temperature, organic modifier content, were optimized. The best extraction conditions were 55°C, 100 bar, and 7.5% ethanol modifier. Extraction recoveries of daidzein (97.3%) and genistein (98.0%) were obtained. For the solvent extraction, with 30 mL of 1 N HCL and 80% ethanol, daidzein of 676.3 µg/g and genistein of 659.9 µg/g were extracted. Data shows that SFE gives a little lower isoflavones recovery compared to conventional extraction with organic solvents. However, the SFE method has several advantages over the solvent extraction method. Sample handling steps are minimized, thus reducing possible losses of analytes and saving analysis time. No cleanup steps are employed, and no organic solvent extractions are involved in this method.

Insulin was precipitated from solution in dimethylsulfoxide using supercritical carbon dioxide as an antisolvent. Solution enhanced dispersion by supercritical fluids (SEDS) process was applied to produce nano‐size insulin particles. We used dimethylsulfoxide to help the supercritical carbon dioxide to extract water from the aqueous protein solution. Various sizes of insulin nano‐particles were successfully prepared with a narrow particle size distribution from aqueous dimethylsulfoxide solution without using any additive. The theoretical particle sizes were deduced from the calculated droplet sizes based on a modified Jasuja's equation. The calculated mean particle sizes and the experimentally obtained ones were compared and the results showed an excellent correlation coefficient (R2) of 0.9738.

A method for the detection of microcystins (microcystin LR, RR, and YR) in cyanobacteria by supercritical fluid extraction (SFE) and liquid chromatography‐mass spectrometry (LC/MS) has been developed. Supercritical fluids for the analytical extraction of nonvolatile, higher molecular weight compound, and microcystins from cyanobacteria were investigated. The microcystins included in this study are sparsely soluble in neat supercritical fluid CO2. However, the microcystins was successfully extracted with a ternary mixture (90% CO2, 9.5% methanol, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous methanol fluid system gave high extraction efficiency for the extraction of the polar microcystins from cyanobacteria. The microcystins were determined by liquid chromatography‐tandem mass spectrometry (LC/MS/MS).

Fatty acid methyl esters were separated by a packed capillary supercritical fluid chromatographic (SFC) column and detected by a UV detector at the wavelength of 206 nm. The temperature controlled restrictor was designed for UV detection. The temperature controlled restrictor is a 20 cm length of deactivated fused silica of 7 µm i.d., which is held right after UV detector of the packed capillary SFC. The temperature of the restrictor will control the flow rate of the supercritical carbon dioxide mobile phase through the packed capillary column in SFC. Thus, as the pressure in the column is increased during a pressure program, the temperature of 7 µm fused silica tube can be varied to maintain a constant flow rate.

Cyanobacterial toxins, microcystins are very potent hepatotoxins and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, the effects of several environmental factors on production and degradation of toxins produced by cyanobacteria in Lake Soyang have been studied. A new rapid quantification method of microcystins using fluorescence for a detection signal and a lateral-flow-type immunochromatography as a separation system was used. Culture age, temperature, light intensity, pH, N-nutrient concentration, P-nutrient concentration, iron and zinc concentration were the most importantly examined factors. The toxin content was the highest on 17-18 days and at temperatures between 20 °C and 25 °C, and at pH between 8.4 and 8.8.

Supercritical fluid extracts of three Myanmar natural plants, namely, Curcuma aeruginosa, Citrus hystrix, and Azadirachta indica A. Juss were screened for antimicrobial activity against different strains of bacteria and yeast, which are known to cause various types of skin infections. The antimicrobial activity was tested on Bacillus cereus, Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Propionibacterum acnes, and Malassezia furfur. None of the plants tested produced consistent inhibition of the Gram‐negative bacteria E. coli and yeast M. furfur. Three plants consistently inhibited bacterial growth of Bacillus subtilis. The antimicrobial activity profile showed that Bacillus subtilis was the most susceptible bacterial strain. According to the antimicrobial profile, SFE extracts of C. aeruginosa presented no significant difference in inhibitory activity on all Gram‐positive bacterial strains. A. indica leaf extracts showed the highest antibacterial activit...

Microcystins are a family of cyclic polypeptides produced by different species of cyanobacteria (blue green algae), which can form blooms in lakes and water reservoirs. However, it is difficult to detect microcystins directly in the water since the concentration of the toxins in water is usually too low. It is necessary to develop a simple and quick method to detect microcystins. In this paper, different detection characteristics of fluorescence immunochromatography and gold colloidal immunochromatography for analysis of cyanobacterial toxins were studied. These two immunochromatography assays are easy to perform, rapid, sensitive, and their quantitative range is within detectable microcystin concentrations in water samples. The fluorescence immunochromatographic system has the unique advantages of low detection limit, and satisfactory accurate results are obtained. The gold colloidal immunochromatographic system has the strong advantage of direct detection of microcystins at the test site withou...

Solution-enhanced dispersion by supercritical fluids (SEDS) was applied to produce nano-sized recombinant human growth hormone (hGH) particles. Ethanol was used to help the supercritical carbon dioxide to extract water from the aqueous protein solution. Various sizes of hGH nanoparticles were successfully prepared with a narrow particle size distribution from aqueous ethanol solution without using any additive. The theoretical particle sizes were deduced from the calculated droplet sizes based on a modified Jasuja's equation. The calculated mean particle sizes and the experimentally obtained ones were compared and the results showed an excellent correlation coefficient (R (2)) of 0.995.

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is low and the detection method is usually complicated. We developed a rapid analytical detection method of microcystins gold colloidal immunochromatogeraphic strip. The sensitivity of the strip is about 1 ng/mL for microcystin LR; it is able to distinguish visually among different concentrations of microcystin solutions. The developed gold colloidal strip can detect microcystins within 15 min and does not require either a complicated extraction system, or trained or qualified experts.

The use of supercritical fluids for the analytical extraction of the nonvolatile, higher molecular weight compound, microcystin LR, from cyanobacteria was investigated. The microcystin LR included in this study was sparingly soluble in neat supercritical fluid CO2. However, the microcystin LR was successfully extracted with a ternary mixture (90% CO2, 9.5% acetonitrile, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous acetonitrile fluid system gave a high extraction efficiency for the extraction of the polar microcystin LR from cyanobacteria. In our laboratory, in the past, aqueous acetic acid‐modified CO2 was used for the extraction of microcystins from cyanobacteria and aqueous methanol was also used as a modifier. In this study, we tried a new organic solvent, aqueous acetonitrile as a co‐solvent, so that we could obtain a higher extraction efficiency of the microcystin LR from cyanobacteria than in earlier experiments.

Polyaromatic hydrocarbons were separated by a capillary supercritical fluid chromatographic (SFC) column and detected by a UV detector at the wavelength of 280 m. The temperature-controlled restrictor was designed for UV detection. The temperature-controlled restrictor is a 20 cm length of deactivated fused silica of 7 mm i.d. which is held right after UV detector of the capillary SFC. The temperature of the restrictor will control the flow rate of the supercritical carbon dioxide mobile phase through the capillary column in SFC. Thus as the pressure in the column is increased from 1500 psi to 4000 psi during a pressure program, the temperature of 7 m fused-silica tube can be varied from 100 to 350 to maintain a constant flow rate.

Blooms of toxic microorganisms are commonly found in freshwater in several areas of the world. Freshwater poisonings are mainly caused by species of cyanobacteria, also known as blue-green algae. They develop in eutrophic water wherever proper conditions for their growth are found, including a mild wind, a water temperature between 15 and 30 C, a neutral or alkaline pH and a rather high level of mineral nutrients. As a consequence of the extended use of nitrates and phosphates, blooms occur more and more frequently. There is a risk that these blooms contaminate water supplies that are used as recreational areas or as drinking water reservoirs. This is a serious water quality problem because many of the cyanobaterial species are able to produce potent toxins. The most frequently reported toxins are hepatotoxins. They are classified as microcystins and nodularins, the former representing the largest group. All microcystins have a common cyclic heptapeptide structure consisting of (-DAla-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where MeAsp stands for erythro-β-methylaspartic acid, Mdha for Nmethyldehydroalanine, Adda for 3-amino-9-methoxy-2,6,8trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Z for the variable amino acids that give its name to the molecule. For example, microcystin LR has X = Leusine and Z = Arginine, microcystin RR has X = Arginine and Z = Arginine. Microcystins can be synthesized by various genera of cyanobacteria such as Microcystis, Oscillatoria, Anabaena or Nostoc. They have been responsible for the poisoning of fish, birds, wild and domestic animals in many countries. Adverse effects on human health have also been recognized. Recent researches have actually shown that microcystins act as type 1, 2A and 3 protein phosphatase inhibitors, as well as tumor promoters when present in nanomolar concentrations. Nowadays, WHO guidelines have been established for the monitoring of microcystins in water. Maximal values of 0.1 μg/L in drinking water for a long term exposure or 1 μg/L for a short term exposure have been proposed on the basis of laboratory experiments of toxicity on mice and pigs. Blooms of blue-green algae, which have been increasing in recent years, have the potential to cause problems for the water supply industry. Recent occurrences of toxic algal blooms have highlighted the potential risk of toxins entering drinking water supplies, and posing a threat to public health. This necessitates a need for research to determine the effectiveness of different water treatment processes for removing algal toxins. At present, slow sand filtration has shown some potential to remove toxins by biodegradation. However, not all types of toxin were successfully removed. The limited amount of work on ozonation has shown that it is an effective process for removing toxins, even with relatively low ozone doses. Ozonation does produce by-products, but these have been shown to be non-toxic. Activated carbon adsorption has been shown to be capable of effectively removing different toxins. With powdered activated carbon (PAC), high PAC doses were generally required. However, most of the tests were conducted with higher toxin concentrations than would probably be found in raw waters. Granular activated carbon (GAC) can effectively remove toxins, but the studies have not fully assessed the most suitable GAC. In addition, the PAC doses required may be impractical to dose at a treatment works. For GAC adsorption to be effective, contact times of more than 15 minutes should be considered, assuming that microcystin does not biodegrade on the GAC. Activated Carbon Fiber (ACF) is a recently developed adsorbent which has more superior adsorbing capacity to active carbons. Recently, Chungbuk National University (Chungju, South Korea) has developed a manufacturing process of ACF by using various precursors of fabrics, cottons, viscous rayons and mixed fabrics etc. ACF made by fabrics and cottons has never been used to remove microcystins in water sample. There is no published data available that has investigated water treatment processes using ACF made by fabrics and cottons, In this study we aimed to obtain basic data on the adsorption of the toxic peptide, microcystin LR to the ACF that was prepared in Chungbuk National University. Our future goal would be to identify and develop ACF treatment processes suitable for removal of algal toxins and to provide a basis for estimating potential water treatment usage.

For the detection of cyanobacterial toxin, an Enzyme-linked immunosorbent assay (ELISA) was integrated into a PDMS microchip. The conjugates of microcystin-LR (MCLR) and keyhole limpet hemocyanin (KLH) were adsorbed on the surface of polystyrene beads and these MCLR-KLH polystyrene beads were introduced into a microchamber. MCLR on the surface of polystyrene beads reacted with horseradish peroxides (HRP) conjugated anti-MCLR monoclonal antibody (mAb) which had a competitive reaction with MCLR in water sample. After the enzyme substrate 3,3,5,5-tetramethyl benzidine (TMB) was injected into the chamber and catalyzed by HRP, the color change was detected with a liquid-cord waveguide. This integration shortened the conventional ELISA analysis time from several hours to about 30 min with only 4.2 L MCLR sample consuming which was useful for the environmental analysis. More over, troublesome operations required for ELISA could be replaced by simple operations. The microchip based detection system showed a good sensitivity of 0.05 g/L and maintained good reliability through its quantitative range with low coefficients of variation (2.5-10.5%).

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.

Different detection characteristics of fluorescence immunochromatography method and high performance liquid chromatography (HPLC) method for the analysis of cyanobacterial toxins were studied. In particular, low and high limits of detection, detection time and reproducibility and detectable microcystin species were compared when fluorescence immunochromatography method and high performance liquid chromatography method were applied for the detection of microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa. A Fluorescence immunochromatography assay system has the unique advantages of short detection time and low detection limit, and high performance liquid chromatography detection method has the strong advantage of individual quantifications of several species of microcystins.

An efficient method for the simultaneous separation and purification of microcystins (LR), (RR), and (YR) from the cyanobacterium is described. The method involves supercritical fluid extraction for the fast extraction and column chromatography for the purification of the microcystins LR, RR, and YR. A new method for the simultaneous extraction of microcystins LR, RR, and YR was developed by using supercritical CO2. Three kinds of microcystins were successfully extracted with a ternary mixture (90% CO2, 9.4% acetic acid, 0.6% water). The separation and purification procedure results in a purity of up to 95% for microcystins RR and YR without the need for a preparative high performance liquid chromatography (HPLC). Only for the purification of microcystin LR, we used a further preparative HPLC purification step. The advantage of the method developed here is that the sample handling steps were minimized, thus reducing possible losses of microcystins and saving extraction and purification time.

The need for the identification of compounds present in complex gaseous mixture or after separation by gas chromatography (GC) using vapor-phase infrared (VPIR) spectrometry is becoming increasingly important. This is primarily due to advances in Fourier transform infrared (FTIR) spectrometry allowing the acquisition of complete spectra in a few tenths of a second with detection limits of less than 1 ppb in long path infrared gas cells and below 10 nanogarms in the GC/FT-IR interface. With a GC/FT-IR system hundreds of spectra can be generated per chromatogram. Clearly, the limiting processes are the spectral searching and, in cases where the search results are ambiguous, spectral interpretation steps. A powerful digital computer is needed to control the interferometer and perform the Fourier transformation from the time domain to the frequency domain in order to obtain the spectrum. This makes the incorporation of automatic spectral interpretation in the same computer an obvious and sensible capability. It should be kept in mind that the ultimate goal of the chemist is not simply to produce a collection of spectra but rather to determine what chemical compounds are present in the unknown samples and what their significance is to the problem at hand. The laborious task of spectral identification has been greatly simplified by the use of computer-based library searching routines. Two general classes of algorithms has been developed: procedures that seek to make an exact identification of an unknown by direct comparison with spectra of known compounds (which require the spectrum of the unknown to be in the reference databases), and more general approaches striving to identify all the functional groups in the unknown. A wide range of encoding schemes has been proposed 1 to represent spectra in a digital form suitable for library searching and interpretation. The ideal storage format retains the minimum amount of information necessary for correct identification of unknown compounds in a form permitting rapid numerical comparison. The optimum instrumental parameters for acquiring spectra for spectral searching and identification are a function of the sample, so that practical considerations require some degree of standardization in sampling and measurement. In addition, specific computer characteristics such as word size, amount of semiconductor memory, and types of mass storage devices must also be taken into account. Buechi et al. 2 have discussed the compilation of spectral

New monoclonal antibodies (mAbs) against microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an immunochromatographic assay system was designed for the detection of cyanobacterial hepatotoxins, MCs in water. The immunochromatographic assay system is composed of anti‐MCs‐mAb, fluorescence conjugates in detection solution, an immunochromatographic assay strip, and a laser fluorescence scanner. The performance of the immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. As the detection limit of HPLC is several orders of magnitude higher than with immunochromatographic assay systems, attention was also given to preconcentration of samples with solid phase extraction cartridges.

A rapid and efficient method for extraction and isolation of microcystin-LR from the cyanobacterium is described. The method involves supercritical fluid extraction (SFE) for the fast extraction and silica gel flash chromatography for the purification of the microcystin-LR. The microcystin-LR can be successfully extracted with aqueous methanol modified supercritical carbon dioxide fluid. The procedure results in a purity of up to 95% microcystin-LR with the following preparative HPLC purification steps. The advantage of the method developed here is that the sample handling steps are minimized, thus reducing possible losses of microcystin-LR and saving extraction and purification time.

Analysis of lysophosphatidic acids (LPAs) is of clinical importance as they can serve a potential marker for ovarian and other gynecological cancers and obesity. It is critically important to develop a highly sensitive and specific method for the early detection of gynecological cancers to improve the overall outcome of this disease. We have established a novel quantification method of LPAs in human plasma by negative ionization tandem mass spectrometry (MS-MS) using multiple reaction monitoring (MRM) mode without the conventional TLC step. Protein-bound lipids, LPAs in plasma were extracted with methanol : chloroform (2:1) containing LPA C 14:0 as an internal standard under acidic condition. Following back extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol. The reconstituted solution was directly injected into electrospray source of MS/MS. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H] - of C16:0, C18:2, C18:1, C18:0 and C20:4 LPA, respectively. Q2 ions selected for MRM were m/z 79, phosphoryl product. Using MS/MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interferences. This method allowed simultaneous detection and quantification of different species of LPAs in a plasma over a linear dynamic range of 0.01-25 μmolL - 1 . The detection limit of the method was 0.3 pmol/mL, with a correlation coefficient of 0.9983 in most LPAs analyzed. When applied to the plasmas of normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.

The optimum flow rates for the nitrate ion were measured experimentally using 1 m×50 μm i.d. Latex particle coated silica capillary. The Van Deemter curves for nitrate at 75, 110 and 150°C were also obtained. Increasing the column temperature resulted in an increase of the optimum flow rate for the nitrate ion. The theoretically calculated optimum flow rates in 50 μm i.d. open tubular ion chromatography matches fairly well with actual experimental data.

A rapid and efficient method for extraction and isolation ofmicrocystin RR from the cyanobacrerium is described. The method involves supercritical fluid extraction (SFE) for the fast extraction and silica gel column chromatography for the isolation of the microcystin RR. The procedure results in a purity of up to 96–98% microcystin RR without the need for a preparative HPLC. The advantage of the method developed here is that the sample handling steps are minimized, thus reducing possible losses of microcystin RR and saving extraction and isolation time.

A new device to accurately deliver a small amount of methanol into supercritical carbon dioxide fluid is described. Carbon dioxide, the most widely used mobile phase in supercritical fluid chromatography, is a relatively non-polar fluid, and hence the addition of a small amount of methanol could change the solvent strength of the mobile phase. In this work, supercritical CO2 and methanol are delivered from the pump to a 100-μl mixing chamber in which a small magnetic bar is rotating. After passing through the mixing chamber, supercritical CO2 is changed to a new mobile phase with different polarity. The modified mobile phase was successfully used for the separations of polar compounds and polyaromatic hydrocarbons (PAHs).

m tend to fall out of the gas stream and deposit in the mucosa of the upper airways before reaching the alveoli of the lungs but incompletely dissolved protein particles can cause undesired side effects such as inflammation or immune response against the drug protein itself. Pulmonary delivery of a protein drug can avoid the hepatic first-pass effect of and the digestive process typical for an oral admini-stration as well as the discomfort of the needle injection associated with a parenteral one. A good example where such pulmonary delivery can significantly improve the patient’s comfort is the insulin inhalation therapy for diabetics, an issue which has already been investigated in clinical studies.

A new method for the fast extraction of mucrocystins RR and LR in cyanobacterium was developed using supercritical CO2. The microcystins were successfully extracted with a ternary mixture (90% CO2, 9.5% acetic acid, 0.5% water). The method developed here has several advantages over solid-phase extraction sample preparation for the analysis of microcystins. Sample handling steps are minimized, thus reducing possible losses of analytes and saving analysis time. No organic solvent extractions are involved in this method and no clean-up steps are employed.

During its relativelyshort history, supercritical fluid chromatography (SFC) hasbecome an attractive alternative to GC and LC in certainindustrially important applications. SFC gives the advantageof high efficiency and allows the analysis of non-volatile orthermally labile mixtures.The density of the mobile phase in SFC is about 200-500times that in gas chromatography. The effect of shorterintermolecular distances and the resulting increase inmolecular interactions is an enhanced solubilizing capabilityof the solvent towards various solutes. Compounds withmuch higher molecular weights than gas chromatographynormally allows can therefore be chromatographed. How-ever, the most commonly used mobile phases in SFC are allrelatively non-polar fluids. Carbon dioxide, the most widelyused fluid, is no more polar than hexane

Flow/hyperlayer field flow fractionation (Fl/Hy FFF) has been applied for the rapid separation and size characterization of core-shell type poly(l-lactide) (PLLA) microspheres which are widely studied as a potential drug carrier due to its biodegradability. Examined microspheres are prepared by varying the entrapped amount of the retinoic acid as an anti-cancer therapeutic and varying PEG-PLLA copolymers. It is demonstrated that the separation of PLLA microspheres can be achieved within 5 min. by Fl/Hy FFF and the particle size distribution (PSD) is readily obtained from the experimental fractograms. From the size distribution of PLLA particles obtained by Fl/Fl FFF, it is shown that PSD increases with the amount of drug loaded but it does not seriously change with the addition of PEG-PLLA copolymers.

Reversed-phase liquid chromatography (RPLC) separation factors for racemic 2,4-dinitrophenyl (DNP) amino acids (Phe and Trp) having various substituents at different positions were measured on native β-CD and heptakis(3-O-methyl)-β-CD as the chiral stationary phase in order to compare enantioselectivity of these phases. Both native β-CD and heptakis(3-O-methyl)-β-CD showed good enantioselectivity for the DNP–amino acids investigated. Enantioselectivities of the two CD phases for the DNP–amino acids vary with the type and position of the substituent on the DNP moiety of the amino acids. Heptakis(3-O-methyl)-β-CD, the cavity of which is more electron-rich than that of native β-CD, showed in general much better enantioselectivity for the amino acid derivatives studied.

Recently, the solvatochromic comparison method was successfully applied to determine the effects of adding a modifier to the mobile phase in packed capillary columns, and we have extended this method to understand the modifier effect in open tubular capillary column supercritical fluid chromatography (SFC). SFC retention data for a wide variety of compounds at different modifier concentrations were obtained using a 10 m×50 μm I.D. SB-methyl-100 polydimethylsiloxane capillary column. Supercritical carbon dioxide at a pressure of 260 atm was used as mobile phase and methanol as modifier. The LSER regression equation used was log k ′=SP 0 + l log L 16 + s π 2 H + a α 2 H + b β 2 H + r R 2 , where k ′ represents the capacity factor and L 16 represents the partition coefficient of the solute between the gas phase and n -hexadecane at 298 K, and l , s , a , b and r are the regression coefficients. For open tubular column SFC, variation of the l and s coefficients showed a similar pattern to the packed capillary column SFC. However, the b coefficient showed a different pattern from packed capillary SFC, i.e., a gradual decrease with increasing modifier concentration. Based on these observations, the effects of a small amount of a modifier in the supercritical mobile phase and its effects on solute retention mechanisms have been discussed. For open tubular column SFC, the swelling of the chromatographic stationary phase by a modifier and carbon dioxide and the increase of dipolarity of the mobile phase are the most important contributing factors in substantiating the modifier effect on solute retention.

The advantages of supercritical fluid chromatography (SFC) have recently been realized in particular its rapidity, flexibility, and ability to allow the analysis of substances which cannot be analysed by gas chromatography (GC). The separating power of open-tubular column GC is unparalleled, but its applicability is restricted by the limited volatility and thermal stability of many organic compounds; less volatile compounds can be analysed by high performance liquid chromatography (HPLC), but long analysis times and very small column diameters are required for efficient separations because of the limitations of solute diffusion in the mobile phase. SFC overcomes these difficulties and permits high resolution at low temperatures with short analysis times. Above its critical point a substance such as carbon dioxide has properties which make its use as a chromatographic mobile phase very favourable. The ability of supercritical fluids to dissolve substances was first recorded in 1879, when Hannay and Hogarth studied the solubility of cobalt and iron chlorides in supercritical ethanol. Numerous applications of extraction with supercritical solvents have been described, and the underlying theory fully understood. Following a suggestion by Lovelock in 1958 that a supercritical fluid might be used as a mobile phase in chromatography, Klesper et al. first deminstrated SFC by the separation of nickel porphyrins using supercritical chlorofluoromethanes as mobile phases. Sie and Rijnders and Giddings developed the technique further, both practically and theoretically, and many applications have been reported. Supercritical fluids may be defined as fluids that are at temperatures and pressures above their critical temperatures and critical pressures. Supercritical fluids are now widely used in extraction, fraction, and chromatography. Supercritical fluid separations of the triglycerides in mink oils have not been reported yet, although other triglycerides in fish oil, rapeseed oil, soybean oil, and butterfat were separated and those of butterfat were identified using massspectrometry. In addition to their critical temperatures and pressures, the chromatographically important properties of supercritical fluid are the density, viscosity, and the diffusion coefficients of solutes. Above its critical point, a substance has density and solvating power approaching that of a liquid, but viscosity similar to that of a gas, and diffusivity intermediate between those of a gas and liquid. Hence, the supercritical fluids have properties which make their use as chromatographic phase very favourable. As long as intermolecular interactions are sufficiently strong, supercritical fluids are able to dissolve a variety of solutes, even those with high molecular mass and low volatility. The density of supercritical fluids, and hence the solubility and chromatographic retention of solutes, can easily be changed by changing the applied pressure. This paper deals with the supercritical fluid separations of long-chain hydrocarbons and mink oils that are very difficult to be separated by either GC or HPLC, using pressure programming technique.

In supercritical fluid chromatography (SFC), the role of the flow restrictor is not only to maintain supercritical conditions throughout the column but also to provide the necessary interface between the column and the detector. In the previous paper,1 a new type of flow restrictor called parallel flow restrictor was introduced. At that time, the parallel flow restrictor was used only for the flow control. In this paper, the parallel flow restrictor was used not only for the flow control but also for the interface to the second detector. A supercritical fluid chromatographic system with a combination of two detectors based on physically different principles, flame ionization and UV absorption, was used.

Construction and preliminary characterization of open tubular capillary column ion chromatography is described. Increasing the column temperature resulted in a dramatic increase of the column efficiency. Two different kinds of columns were developed and investigated for capillary ion chromatography. One was a 50μm i.d. fused silica capillary tubing, and another was a 50μm i.d. silica capillary coated with Latex particles (diameter: about 360nm). Both columns showed good separations of inorganic anions, although the capillary coated with Latex particles showed much larger retention than a bare silica capillary.

The use of compressed (dense) gases and supercritical fluids as chromatographic mobile phases in conjunction with liquid chromatographic (LC)-type packed columns was first reported by Klesper et al. in 1962. During its relatively short history, supercritical fluid chromatography (SFC) has become an attractive alternative to GC and LC in certain industrially important applications. SFC gives the advantage of high efficiency and allows the analysis of nonvolatile or thermally labile mixtures. The density of the mobile phase in SFC is about 200-500 times that in gas chromatography. The effect of shorter intermolecular distances and the resulting increase in molecular interactions is an enhanced solubilizing capability of the solvent towards various solutes. Compounds with much higher molecular weights than gas chromatography normally allows can therefore be chromatographed. However, the most commonly used mobile phases in SFC are all relatively non-polar fluids. Carbon dioxide, the most widely used fluid, is no more polar than hexane, even at high densities. Solute polarity should be between that of the stationary phase and the mobile phase in order to have a well behaved separation. Few real samples contain only nonpolar solutes, so a major objective of research in SFC has been directed toward increasing the range of solute polarity that can be handled by the technique. To bring the SFC technique into routine use, mobile phases that are more polar than the commonly used carbon dioxide are necessary. The solvent strength of supercritical CO2, even at high density, is not sufficient for the elution of polar solutes. Polar mobile phase such as NH3 exhibit useful properties, but a more practical way to extend the range of compounds separable by SFC is to use a mixed mobile phase. The solubility of the solute in the supercritical phase can be influenced considerably by adding modifiers to the mobile phase. The use of modifiers has been reported by Jentoft and Gouw and by Novotny et al. The latter group showed that adding 0.1% 2-propanol to pentane as the mobile phase decreases the observed partition coefficient (K) values for many polynuclear aromatic hydrocarbons by 20-35%. Thus, the addition of modifiers (generally organic solvents) to a supercritical mobile phase changes the polarity of the mobile phase and also leads to deactivation of the column packing material. In capillary SFC, most separations are made with pure CO2 because of its compatibility with an flame ionization detection (FID); except for formic acid and water, the addition of any common modifier precludes the use of FID. Modifiers are essential in packed-column SFC for the elution of polar compounds and are extensively used. Several workers have reported the influence of modifiers on peak shape, selectivity and retention time in capillary and packed-column SFC. A simple and effective way to add modifiers to a supercritical fluid mobile phase is to use a saturator column, which is usually a silica column saturated with polar alcohols. In this work, water was used as a polar modifier and a μporasil column as a saturator column. The μ-porasil column was inserted between the pump outlet and the injection valve. During the passage of the supercritical fluid mobile phase through the silica column, a polar modifier (water) can be dissolved in the pressurized supercritical fluid. Dimethylpolysiloxane polymer has been known as more polar polymer than polystyrene polymer. Dimethylpolysiloxane polymer has never been separated using water modified mobile phase. In this paper, using a μ-porasil column as a saturator column, excellent supercritical fluid chromatograms of dimethylpolysiloxane oligomers were obtained.

Linear solvation energy relationships (LSERs) were used to verify previously proposed retention mechanisms when an organic modifier is used with an octyldecylsilane bonded phase in packed capillary column supercritical fluid chromatography. Supercritical fluid chromatographic retention data for a wide variety of compounds at different modifier concentrations were obtained using a 60 cm×200 μm I.D. ODS packed capillary column. Supercritical carbon dioxide at a pressure of 260 atm was used as mobile phase and methanol as modifier. The LSER regression equation used was log k′ = SP0 + llog L16 + s π2H + αα2H + bβ2H + rR2, where k′ represents the capacity factor and L16 represents the partition coefficient of the solute between the gas phase and n-hexadecane at 298 K, and l, s, a, b and r are the regression coefficients. By increasing the percentage of modifier added to the mobile phase, the l coefficient decreased gradually. The b and s coefficients were greatly decreased on moving from neat carbon dioxide mobile phase to 2% methanol modifier, thereafter showing a gradual decrease with increasing modifier concentration. From these observations, we substantiate the conclusion that when a small amount of modifier (up to 2%) is added, the predominent modifier effect on the ODS bonded phase is dynamic coating of free silanols on the surface of the stationary phase, and when more modifier is added, another effect, i.e. an increase in the density of the mobile phase leading to an increase in elution strength of the eluent, becomes more important.

The syntheses, structures, and spectroscopic properties of 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]-substituted beta-cyclodextrins have been investigated. Selective activation of the 6(A),6(B)-hydroxy groups was carried out by treating heptakis(2,3-di-O-methyl)-beta-cyclodextrin (1) with 2,4-dimethoxybenzene-1,5-disulfonyl chloride to give 6(A),6(B)-bissulfonate ester 2 in a yield of only 3%. This material was treated with sodium p-(allyloxy)phenoxide in DMF to form 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]-heptakis(2,3-di-O-methyl)-beta-cyclodextrin (3), which had two isomers. One (3A) has the two p-(allyloxy)phenyl arms directed away from the cyclodextrin cavity, and the other (3B) has one of the p-(allyloxy)phenyl groups through the cavity to form a self-inclusion complex. When either 3A or 3B was treated with methyl iodide and sodium hydride, the resulting permethylated 6(A),6(B)-bis-O-[p-(allyloxy)phenyl]heptakis(2,3-di-O-methyl)-6(C),6(D),6(E),6(F),6(G)-penta-O-methyl-beta-cyclodextrin (4) was composed of two isomers, in which 4B is a self-inclusion complex. 3A and 3B also can be converted into a mixture of 3A and 3B in strong base but not when melted in the absence of base. 4A and 4B do not isomerize. Detailed 1D and 2D NMR spectroscopic studies were carried out to characterize the structures of these new compounds, and molecular mechanics techniques were used to explain the experimental facts.

Department of Chemistry, Kwangwoon University, Seoul 139-701, KoreaReceived September 30, 2002Key Words : Microcystin, Monoclonal antibody, CyanobacteriaMicrocystins are a family of cyclic polypeptides producedby different species of cyanobacteria (blue-green algae),which can form blooms in lakes and water reservoirs.

During the last decade, immunochromatographic techniques, in particular fluorescence labeled immunochromatography, have become more popular for the determination of low concentrations of analytes. It offers several potential advantages including highly sensitive detection and wide applications in clinical chemistry, bioanalysis, and environmental analysis. Currently, fluorescence labeled immunochromatography is not the exclusive preserve for a few specialists who are well experienced with antigen-antibody analysis, since the recent developments in fluorescence labeled immunochromatography have become widespread, from simple screening application to quantitative analysis of analytes. In this review, we assembled recent advances in the development and applications of fluorescence labeled immunochromatography techniques, considering their potential benefits in the future.

Any coupling of a spectroscopic detection technique to chromatography for the analysis of complex mixtures inevitably leads to a tremendous bulk of collected data and here gas chromatography/infrared spectroscopy is no exception. To deal with the large amount of data produced, we already demonstrated computer methods that select certain spectra form the data set as being more interesting for the analysis at hand, and focussed attention on the interpretation of those spectra. In this paper, we report an extension of this work that includes automatic determination of proper ranges of frequencies within which characteristic patterns of absorbances for a given functional group will be found. Patterns of absorbances in those correlated regions are used to describe a functionality for selective reduction of infrared data. The method was successfully applied to substituted benzene compounds.