• 제목/요약/키워드: 이온크로마토그래피

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토양균에서 항생물질 및 효소억제제의 분리와 구조 연구

  • 구양모;이윤영;김경자;최응칠;김범태;주정호;이창훈
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1994.04a
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    • pp.179-179
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    • 1994
  • 새로운 항생물질을 개발하기 위하여 토양으로부터 분리한 균주를 액체 및 고체배지에서 배양하여 여러 검정균에 대하여 종이디스크법으로 항균효력을 조사하였다. 그 결과 (+), G(-), fungi 등에 강한 항균 효력을 보인 토양균 SNUS 8810-43과 Mycobacterium, fungi에 항균력을 보인 토양균 SNUS 8810-129를 선택하여 각각의 배양액에서 항생물질을 분리하고, 분리한 항생물질의 구조를 규명하고자 하였다. 토양균 SNUS 8810-43의 배양액으로부터 항생물질을 분리하기 위하여 양이온 교환 수지 관 크로마토그래피와 셀룰로오스 관 크로마토그래피를 수행하여 시료 JJH-II-46-43을 얻었다. 시료 JJH-II-46-43의 IR, $^1$H-NMR, $^{13}$C-NMR, $^1$H-$^1$H COSY, $^1$H-$^{13}$C COSY, FAB-MS 스펙트럼을 얻어 분리한 항생물질의 구조를 분석하여 이 항생물질의 구조가 N-methylstreptothricin과 동일하다는 것을 확인하였다. Mycobacterium smegmatis에 강한 활성을 나타내는 물질을 토양균 SNUS 8810-129로 부터 분리하였다. 토양균 SNUS 8810-129를 배양한 V-8 아가판을 메탄올로 추출하여 이를 실리카겔 관 크로마토그래피와 preparative TLC로 시료 LCH-IV-17B, LCH-III-387을 얻었다. 시료LCH-IV-l7B, LCH-III-387의 $^1$H-NMR, $^{13}$C-NMR, FAB-MS, CI-MS, IR등의 스펙트럼을 얻어 분리한 항생물질의 구조를 분석하여 이 항생물질이 glycolipid계 항생물질이라는 것을 알았다. $^{13}$C-NMR 상의 자료와 화학적인 방법으로 구성당을 조사한 결과 이 항생물질을 이루고있는 당은 rhamnose 임을 알았다. 또 이 항생물질을 구성하는 지방산은 화학적인 방법과 MS 스펙트럼, $^{13}$C-NMR 스펙트럼으로부터 hydroxydecanoic acid인 것으로 확인되었다. 항생물질 LCH-III-387와 항생물질 LCH-IV-l7B는 각각 rhamnose를 1, 2개 포함하고 있는 것으로 확인되었다. 그리고 동일한 탄소수의 지방산을 가지고 있는 것으로 생각되었다. 이들 항생물질을 이루는 구성당과 지방산간의 정확한 연결및 구조, 생리활성에 관한 연구는 계속 수행중에 있다.

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Characterization of Extracellular Peroxidase from Pleurotus ostreatus (Pleurotus ostreatus에서 분비되는 Peroxidase의 특성)

  • 배성호;신광수;강사욱;하영칠;최선진;김규중;최형태
    • Korean Journal of Microbiology
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    • v.27 no.4
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    • pp.348-356
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    • 1989
  • An extracellular peroxidase found in culture broth of Pleurotus ostreatus was induced by syringic acid. This enzyme was fractionated by DEAE Sephadex A-50 ion exchange chromatography and gel filtration chromatogrphy on Sephadex G-150. The enzyme is a glycoprotein containing 35.7% carbohydrate. The results of SDS-linear polyacrylamide gradient gel electrophoresis and gel filtration indicate that the enzyme is a dimer consisted of identical subunits (Mr=72,400). The absorption spectrum of the enzyme indicates the presence of one mole of iron protoporphyrin IX per one mole of subunit. Isoelectric point of the enzyme is 4.26 and $K_m$ values for $H_2O_2$ is $7.2{\mu}M$. The enzyme showed its optimal activity at pH 3.5-4.0 and at $40^{\circ}C$. The Km values of this enzyme for ferulic acid and sinapic acid are 2.4 and 12.3 times higher than those of horseradish peroxidase, respectively.

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Analytical Determination of Alkylphenols, Chlorophenols and Bisphenol A in Biota Samples using Gas Chromatography/Mass Spectrometry (기체 크로마토그래피/질량분석기를 이용한 생물시료 중 알킬 페놀류, 클로로페놀류 및 비스페놀 A의 분석법)

  • Hong, Jongki;Kim, Hyup;Baek, In-Girl;Kim, Do-Gyun;Seo, Jung-Joo;Seo, Jong-Bok;Chung, Bong-Chul;Pyo, Hee-Soo;Kim, Kyung-Rae;Kim, Yong-Hwa
    • Analytical Science and Technology
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    • v.13 no.4
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    • pp.484-493
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    • 2000
  • The simultaneous analysis of alkylphenols, chlorophenols and bisphenol A in biota samples was performed by gas chromatography-mass spectrometry-selected ion monitoring mode. The phenols were extracted from sample with organic solvent and Forisil and Silica columns for clean-up procedure were compared. Recovery studies were performed at 1-ppm level of phenols added to each biota sample. Their recoveries ranged between 83 and 116% with coefficient of variations of 2.4-11.9%. To improve the detection limits of phenols, trimethylsilyl (TMS) derivatization was applied. The gas chromatographic properties of free phenols and TMS derivatized phenols were also investigated.

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Isolation and Characterization of L-Ascorbic Acid-Producing Enzyme in Neurospora crassa (Neurospora crassa의 L-Ascorbic Acid 생산효소의 순수 분리 및 이의 특성에 관한 연구)

  • Kim, In-Sil;Lee, Yeon-Hee
    • Korean Journal of Microbiology
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    • v.32 no.2
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    • pp.132-138
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    • 1994
  • L-Ascorbic acid-producing enzyme in Neurospora crassa was found to exist in mitochondria and the activity of this enzyme was increased by the addition of D-fluconno-${\gamma}$-lactone or L-gulono-${\gamma}$-lactone in the media. L-Ascorbic acid-producin enzyme in N. crassa has been purified with ammonium sulfate precipitation. DEAE Sepharose CL-6B ion exchange chromatography. Sephacryl S-200 gel filtration chromatography and Reactive yellow 3-agarose dye affinity column chromatography. The specific activity of this enzyme was increased to 239.6 fold and the yield was 2.1%. The molecular weight of the native enzyme was 150.000 dalton when it was estimated with Sephacryl S-200 gel filtration chromatography. Its molecular weight appeared as 75.000 dalton by SDS-polyacrylamide gel electrophoresis. which suggested that this enzyme was consisted with two identical subunits. The optimal pH for this enzyme was 9.0 and the $K_m$ value for D-galactono-${\gamma}$-lactone was 0.073 mM.

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Analysis of Imidazoline Type Cationic Surfactants (Imidazoline계 양이온 계면활성제 분석)

  • Bak, Hong-Soon;Choi, Kyu-Yeol;Lee, Jae-Duk;Kim, Yeo-Kyung;Ahn, Ho-Jeong
    • Applied Chemistry for Engineering
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    • v.9 no.3
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    • pp.404-406
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    • 1998
  • Analysis for the imidazoline type cationic surfactants was performed by the gas chromatography(GC) and high performance liquid chromatography(HPLC). The composition of the alkyl chain distribution was investigated by GC after base/Acid hydrolysis of the imidazoline ring. The distribution of total alkyl chain was separated clearly by a Bondclone C18/NOVA-Pak C18 HPLC column using 50% acetonitrile in methanol containing 0.1M sodium perchlorate as a mobile phase. Alkyl chain distribution and average molecular weight of imidazoline type cationic surfactants were obtained based on these analytical methods. The agreement of results from GC and HPLC was good. The detection limit of imidazoline by HPLC method was 10ng without pretreatment.

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Determination of Trace Iodide in Sodium Bisulfite Aqueous Solution by Ion Chromatography with UV Detection (이온크로마토그래피를 이용한 Sodium bisulfite 수용액 중의 미량 요오드 정량)

  • Park, Yang-Soon;Kim, Do-Yang;Choi, Kwang-Soon;Park, Soon-Dal;Han, Sun-Ho
    • Analytical Science and Technology
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    • v.13 no.3
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    • pp.304-308
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    • 2000
  • The iodide was recovered from a simulated spent fuel to the sodium bisulfite aqueous solution. It was discussed that the trace iodide (below 1 ppm) was determined without the matrix effect of 0.1 M sodium bisulfite and 1 mM $HNO_3$ in aqueous solution by ion chromatography with UV detection. AS4A-SC (DIONEX) column and UV-absorption spectrophotometer were used. The UV-absorption spectra of sodium bisulfite, nitric acid and iodide were obtained, and then 230 nm was selected as an absorption wavelength for iodide determination. 0.1 M NaCl eluent was optimum condition. In this condition the calibration curve of iodide was obtained on the range of about 0-1,000 ppb. The linear coefficient was 0.99993 and the detection limit was 5 ppb. The relative standard deviation was 1.26%.

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The Effect of Temperature and Flow Rate of Eluent on the Separation of Adjacent Lanthanides (La : Ce, Ce : Pr, Pr : Nd) with Displacement Chromatography (치환크로마토그래피에서 온도와 용리액의 흐름속도가 란탄족 원소들 (La : Ce, Ce : Pr, Pr : Nd) 의 분리에 미치는 영향)

  • Ha, Yeong Gu;Song, Gi Hun
    • Journal of the Korean Chemical Society
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    • v.38 no.9
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    • pp.660-666
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    • 1994
  • The effects of temperature and flow rate of eluent on the separation of adjacent lighter lanthanide pairs (La : Ce, Ce : Pr, Pr: Nd) have been studied with displacement chromatography. Two serial columns packed with Amberlite 120 cation exchange resin are used for loading and separation. The retaining ion is $H^+$ ion and the eluent is 0.012M and 0.015M of EDTA solution. The columns and the eluent are maintained at the temperature of 90$^{\circ}C$ and pressurized to reduce vaporizing in the ion-exchange resin column. The eluated solution is analyzed directly with ICP-AES. The separation factors of the lanthanide pairs, La: Ce, Ce :Pr, and Pr: Nd, are 4.6, 2.8, and 1.9, respectively and are higher than that from theoretical calculation at 25$^{\circ}C$. When the flow rate is reduced from 2.5 ml/min to 1.5 ml/min, the HETP is reduced from 1.60 cm to 0.88 cm. The separation efficency can be improved at lower flow rate of eluent and higher operating temperature. The recoveries of pure lanthanides than 99.9% are 49∼77% from this separation.

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Comparative Studies on the Enzymatic Properties of Trypsins from Cat-shark and Mackerel -1. Purifications and Reaction Conditions of the Trypsins- (복상어와 고등어의 Trypsin에 관한 비교 효소학적 연구 -1. Trypsin의 정제와 반응조건-)

  • PYEUN Jae-Hyeung;CHO Deuk-Moon;HEU Min-Soo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.24 no.5
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    • pp.273-288
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    • 1991
  • To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

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Uncertainties of ionic species in snowpit samples determined with ion chromatography system (이온크로마토그래피 시스템을 이용한 눈 시료의 이온성분 측정자료의 불확도 산출)

  • Hong, Sang-Bum;Hur, Soon-Do;Kim, Sun-Mee;Hong, Sungmin;Chung, Ji-Woong;Kang, Namgoo;Kang, Chang-Hee
    • Analytical Science and Technology
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    • v.25 no.6
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    • pp.350-363
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    • 2012
  • To determine ionic species in snowpit samples using ion chromatography system, we described the performance of ion chromatography(IC) system, cleaning method of bottle, and interference by filtering procedure. The limit of detection, reproducibilities, and accuracies determined with BCR$^{(R)}$-408 were 0.01-0.26 ${\mu}g$/L, 0.4-17.4%, 4.5-12.0% for cations and 0.02-0.26 ${\mu}g/L$, 0.1-27.6%, 1.3-5.6% for anions, respectively. Lab blank test for sample bottle indicated that $CH_3CO_2{^-}$, $HCO_2{^-}$, and $NH_4{^+}$ can be easily contaminated in the lab environment. The positive interferences of $NO_3{^-}$ were partly attributed to the cleaning method of bottle. The filtering of melted snow sample should be carefully applied because it can positively affect the concentration levels of some ionic species. Finally, this method was applied to measure ionic species in snowpit samples from the upward area near NEEM camp and the uncertainties of measurement data of $F^-$ were also estimated.

Determination of Phthalates in Biota Samples Using Gas Chromatography/Mass Spectrometry (기체크로마토그래피/질량분석법을 이용한 생체시료 중 프탈산 에스텔류의 분석법)

  • Seo, Jung-Ju;Na, Yuncheol;Hong, Jongki
    • Analytical Science and Technology
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    • v.14 no.5
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    • pp.400-409
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    • 2001
  • A method for the analysis of most common phthalate acid esters (9 secies) in biota samples by gas chromatography-mass spectrometry-selected ion monitoring mode is described. Phthalates in biota samples are extracted by organic solvent and purified by Florisil column. Phthalates are easily contaminated during extraction prodedure. Since the extraction and cleanup steps for biota samples generally are more complicate than those for water or sediment samples, we compared with contamination state of each sample work-up step. By applying this developed method, the overall recoveries ranged between 79 - 117% in biota sample which was spiked with standards. For phthalates used in this study, the quantitaive accuracy, elution pattern on Florisil column, and detection limits were also investigated.

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