Solvent Extraction과 Gel Chromatography를 이용한 Phenoxyethanol Galactoside와 Chlorphenesin Galactoside의 정제

정경환 한국응용과학기술학회 2017 한국응용과학기술학회지 Vol.34 No.4

We investigated the purifications of PE-gal and CPN-gal, synthesized by transgalactosylation reaction using recombinant β-gal. The reaction mixture containing PE and PE-gal was first mixed with EA, and thereafter PE and PE-gal were distributed in two-phase (EA/water) system. In this system, PE and PE-gal was selectively moved into EA and water phase, respectively. Then, the water phase was collected, and silica gel chromatography was carried out using the collected water phase. Finally, we compared two purified PE-gal samples using HPLC and TLC analysis, in which the one was purified only by silica gel chromatography and the other was purified by EA extraction followed by silica gel chromatography. In the latter case, the residual PE was almost completely removed, whereas, in the former case, the residual PE was remained remarkably. Additionally, the purification yield of PE-gal was about 21% on the basis of mole. In the same purification protocol, CPN-gal was able to be purified using EA extraction followed by silica gel chromatography, in which the residual CPN was almost removed when CPN-gal was purified by EA extraction followed by silica gel chromatography. 재조합 β-galactosidase (β-gal) 을 이용하여 transgalactosylation 반응으로 합성된 2-phenoxyethanol galactoside (PE-gal)과 chlorphenesin galactoside (CPN-gal)의 정제를 실시하였다. 먼저 PE와 PE-gal이 포함된 반응물에 ethyl acetate (EA)를 넣고, EA/water 이상계 시스템에서 PE와 PE-gal을 분획하였다. 이 시스템에서 PE는 EA층으로 PE-gal은 water층으로 분획되었다. 그리고, 물층을 모아서 silica gel chromatography를 실시하였다. 최종적으로, silica gel chromatography만 실시하여 PE-gal을 정제한 경우와, EA 처리 후 silica gel chromatography를 실시하여 PE-gal을 정제한 경우의 정제 PE-gal에 대하여 HPLC (High performance liquid chromatography)와 TLC (thin-layer chromatography) 분석을 수행하였다. 그 결과 EA를 처리하여 분획한 후, silica gel chromatography를 수행한 시료에서 잔여 PE가 완전히 제거되는 것을 관찰하였고, silica gel chromatography만 실시하여 PE-gal을 정제한 경우에는 상당량의 잔여 PE가 관찰되었다. 이 때, mole 기준으로 약 21%의 정제 수율을 확인할 수 있었다. 마찬가지로 CPN-gal의 정제에서도 EA 분획 처리 후, silica gel chromatography를 수행하였더니, 잔여 CPN이 거의 없는 순수한 CPN-gal을 얻을 수 있었다.

식품중의 수산분석법에 관한 연구

김복성,이영자,홍기형,이창희,류시생,진희숙,송성실,김용철,이명환 국립보건연구원 1992 국립보건원보 Vol.29 No.2

Environmental Sciences : Development and Comparison of Analytical Methods for Measuring Simazine Herbicide Using Gas Chromatography/Ion Trap, Gas Chromatography/Mass Selective Detector, and High Performance Liquid Chromatography/Triple Quadrupole Mass Spe

( Ill-min Chung ),( In Myoung Park ),( Seung Hyun Kim ) 한국응용생명화학회(구 한국농화학회) 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.5

Simazine is widely used to manage weed growth in the agricultural and industrial fields. Three analytical methods, including gas chromatography (GC)/ion-trap (IT), GC/mass selective detector (MSD), and high-performance liquid chromatography (HPLC)/triple quadrupole mass spectrometers (HPLC/MS/MS), were developed and compared for simazine quantification. The spike recoveries by GC/IT, GC/MSD, and HPLC/MS/MS were 100, 117, and 82.5%, respectively. Different spike recovery depending on analytical methods could be due to matrix effect, different instrumental sensitivity, losses during sample preparation or all of the above. The limit of detection was 0.0361, 0.0263, and 0.0013 mg/kg by GC/IT, GC/MSD, and HPLC/MS/MS, respectively. The HPLC/MS/MS with a positive atmospheric pressure chemical ionization (APCI) ionization mode was the most rapid (7 min), sensitive (limit of detection: 0.0013 mg/kg, limit of quantification: 0.0042 mg/kg), and precise (relative standard deviation: 0.5%) method for simazine quantification.

Chromatography용 Paper, $\mu$RPC Column 및 Superose Column을 이용한 정자의 이동을 자극하는 난포액 성분의 분리

박영식 한국수정란이식학회 1998 한국동물생명공학회지 Vol.13 No.3

난포액내 함유되어 있는 단백질성분 중에서 sucrose 층으로부터 정자의 swim-up 이동을 자 극하는 성분을 분리하기 위하여 paper chromatography (PC) 및 reverse phase column (RPC) 과 superose column (SC)를 이용한 액체 chromatography의 분리효과를 조사하였던 바 결과는 다음과 같다. 1. Chromatography용 paper로 분리한 각 band 의 성분은 첨가농도가 증가할수록 정자의 이동과 운동을 자극하였으며, 특히 band 1 성분은 정자의 이동을 유의하게 증가시켰다. 그러나, 동일 첨가수준에서 bands 성분의 정자 이동과 운동자극효과는 난포액의 효과에 비하여 유의하게 낮았다. 2. $\mu$RPC를 이용 2~5mm 난포로 부터 분리한 성분중 RT3.33, RT7.00, RTl3.87 및 RTl6.6A 성분은 정자의 이동을 자극하였으나, 자극효과는 매우 적었다. 3. $\mu$RPC를 이용 10mm 난포로 부터 분리한 성분은 정자의 이동과 활력을 자극하지 않았다. 4. SC를 이용 2-5mm 난포로 부터 분리한 성분 중 RVI.35 성분과 RV0.82 성분은 정자의 이동과 운동을 유의하게 자극하였다. 결론적으로 난포액내 정자의 이동과 운동을 자극하는 단백질 성분은 superose column을 이용하여 효과적으로 분리할 수 있으며, 분리된 RVI.35 성분과 RV0.82 성분은 정자의 swim-up 분리를 자극하였다. To efficiently separate a protein stimulating sperm swim-up migration and movement from follicular proteins, the effect of paper chromatography and liquid chromatography with reverse phase column and superose column on protein separation was examined. And the results obtained were as follows; 1. The band component that was separated with paper chromatography stimulated sperm migration and movement depending on its additional levels. Especially, band I component significantly increased sperm migration. But, all components of bands 1, 2 and 3 showed lower sperm migration and movement, compared to follicular fluid at the same additional level. 2. Among the components separated from follicular protein of 2~5mm follicles with reverse phase column ($\mu$RPC), components at retention time (RT) of 3.33, 7.00, 13.87, and 16.6A minutes stimulated sperm migration within a limited range. 3. All components separated from follicular protein of 10mm follicles with $\mu$RPC column didn't stimulate sperm migration and movement. 4. Among the components separated from follicular protein of 2~5m follicles with superose column, components at retention volume (RV) of 1.35 and 0.82 ml significantly stimulated sperm migration and movement. In conclusion, protein components stimulating sperm migration and movement were efficiently separated with superose column in Smart system. Especially, components of RV 1.35 and RV0.82 stimulated sperm swim-up separation.

Size-exclusion chromatography법에 의한 식품 중 알긴산프로필렌글리콜 분석법 확립

정은정,최유정,이근영,윤상순,임호수,김용석,Jeong, Eun-Jeong,Choi, Yoo-Jeong,Lee, Gunyoung,Yun, Sang Soon,Lim, Ho Soo,Kim, Yong-Suk 한국식품위생안전성학회 2017 한국식품위생안전성학회지 Vol.32 No.5

HPLC-size-exclusion chromatography에 의해 가공식품에서 알긴산프로필렌글리콜의 함량을 분석하는 방법이 개발되었다. 알긴산프로필렌글리콜을 분석하기 위해 GF-7M HQ column과 LT-ELSD detector가 선정되었다. 알긴산프로필렌글리콜 분석을 위한 전처리 조건으로는 $20^{\circ}C$에서 150 rpm으로 3시간 동안 추출하는 방법이 선정되었다. 알긴산프로필렌글리콜을 5 농도(300, 500, 700, 1,000, and 1,500 mg/kg) 범위에서 검량선을 작성한 결과 직선성($R^2$)은 0.9873으로 측정되었다. HPLC system에 의한 알긴산프로필렌글리콜 분석시 검출한계(LOD) 및 정량한계(LOQ)는 각각 171.43 mg/kg 및 519.50 mg/kg이었다. Size-exclusion chromatography에 의해 얻은 회수율 및 변동 계수(coefficient of variation)는 각각 86.1~110.4% 및 4.1~13.5%이었다. 본 연구에서 개발된 HPLC-size-exclusion chromatography system을 적용하여 134 품목의 가공식품에서 알긴산프로필렌글리콜 함량을 분석하였다. 이 결과들은 이 방법이 가공식품에서 알긴산프로필렌글리콜 함량을 분석하는데 적용할 수 있는 방법이라는 것을 나타낸다. An analytical method for determination of propylene glycol alginate (PGA) in food products was developed by HPLC-size-exclusion chromatography. The GF-7M HQ column and LT-ELSD detector were determined by considering the instrumental analysis conditions for PGA analysis. The pretreatment method for the analysis of PGA was suitable for 3 hr extraction at $20^{\circ}C$ and 150 rpm according to the extraction temperature. Linearity ($R^2$) for the analysis of PGA was 0.9873 at calibration curve range of 300, 500, 700, 1,000, and 1,500 mg/kg (5 points). The limit of detection and limit of quantification of PGA on HPLC system was 171.43 and 519.50 mg/kg, respectively. The accuracy and coefficient of variation obtained by size-exclusion chromatography were 86.1~110.4% and 4.1~13.5%, respectively. By applying the HPLC-size-exclusion chromatography system, it was possible to analyze the contents of PGA in 134 different types of food products.

Analytical method validation for terbutryn using gas chromatography/ion trap, gas chromatography/mass selective detector, and liquid chromatography/triple quadrupole mass spectrometers

장혜원,이장호,최현욱,남태규,김승현,이광근 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.5

Analytical methods including solvent extraction followed by gas chromatography/ion-trap (GC/IT) with scan and MS/MS mode, a GC/mass selective detector (GC/ MSD), and liquid chromatography/triple quadrupole mass spectrometers (LC/MS/MS) were optimized to identify and quantify terbutryn. The spike recovery was 96.5% using GC/IT with scan mode and 103.5% with MS/MS mode, 90.3% by GC/MSD, and 92.5% by LC/MS/MS. The limit of detection (LOD) was 0.0015 mg/kg by GC/IT with scan, 0.026 mg/kg with MS/MS mode, 0.015 mg/kg with GC/ MSD, and 0.026 mg/kg by LC/MS/MS. Of the four methods, GC/IT with scan mode was determined to be the most sensitive (with LOD: 0.0015 mg/kg and limit of quantitation (LOQ): 0.0047 mg/kg), rapid (retention time: 9.6 min) and the most precise method (relative standard deviation: 17%) for the quantification of terbutryn. GC/IT with scan mode proved to be the more sensitive analytical method for terbutryn than other methods in this study, showing better accuracy and rapid analysis.

Gel filtration chromatography와 propionic acid/urea polyacrylamide gel electrophoresis를 이용한 봉독 성분의 분리

최영권,최석호,권기록,Choi, Young-Chon,Choi, Suk-Ho,Kwon, Ki-Rok 대한약침학회 2006 Journal of pharmacopuncture Vol.9 No.2

Objectives : This study was conducted to carry out Purification of Melittin and other peptide components from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis Methods : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. Results : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. The fractions obtained from gel filtration chromatography was analyzed by using SDS-PAGE and propionic acid/urea polyacrylamide gel electrophoresis. The melittin obtained from the gel filtration contained residual amount of phospholipase $A_2$ and a protein with molecular weight of 6,000. The contaminating proteins were removed by the second gel filtration chromatography. Conclusion : Gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis are useful to separate peptide components including melittin from bee venom.

Alumina Column Chromatography와 HPLC에 의한 토마토의 Dehydrotomatine 및 α-Tomatine 단리방법 연구

최석현 ( Suk Hyun Choi ),김현룡 ( Hyen Ryung Kim ),이진식 ( Jin Shik Lee ) 한국식품영양학회 2010 韓國食品營養學會誌 Vol.23 No.4

Tomato fruits(Lycoperisicon esculentum) synthesize the glycoalkaloids dehydrotomatine and α-tomatine, possibly as defense against bacteria, fungi and insects. We developed a new effective method to prepare and purify dehydrotomatine and α-tomatine that exists in tomato fruits using alumina column chromatography and high performance liquid chromatography (HPLC). The tomato glycoalkaloids(TGA) in tomato was extracted with 2% acetic acid, and then precipitated with ammonium hydroxide(pH=10.5). The dry precipitate substance was applied on alumina column, and then fractionated with water saturated n-butylalcohol. The TGA(Fr. No. 26~36) were collected and dried under reduced pressure. The TGA was performed on a reverse phase HPLC(Inertsil ODS-2, 5 ㎛), eluted with acetonitrile/20mM KH2PO4(24:76, v/v) at 208 ㎚. Two peaks were detected on HPLC, and individual peak was collected by repeating HPLC. Furthermore, to confirm the identity dehydrotomatine and α-tomatine, each peak isolated was hydrolyzed with 1N HCl into sugar and aglycone tomatidine. The sugars were converted to trimethylsilyl ester derivatives. The nature and molar ratios of sugars were identified by gas-liquid chromatography(GLC) and the aglycone by high-performance liquid chromatography(HPLC). The first peak (Rt=17.5 min) eluted from HPLC was identified as dehydrotomatine, and second peak(Rt=21.0 min) was as α-tomatine. This technique has been used effectively to prepare and isolate dehydrotomatine and α-tomatine from tomato fruits.

Separation of Sr, Cs and Ba Using Gas Pressurized Extraction Chromatography With Cation Exchange Resin

Sojin Jeong,Hanul Cho,Byungman Kang,Jihye Kim,Sang Ho Lim 한국방사성폐기물학회 2023 한국방사성폐기물학회 학술논문요약집 Vol.21 No.2

During the initial cooling period of spent nuclear fuel, Cs-137 and Sr-90 constitute a large portion of the total decay heat. Therefore, separating cesium and strontium from spent nuclear fuel can significantly decrease decay heat and facilitate disposition. This study presents analytical technique based on the gas pressurized extraction chromatography (GPEC) system with cation exchange resin for the separation of Sr, Cs, and Ba. GPEC is a micro-scaled column chromatography system that allows for faster separation and reduction volume of elution solvent compared to conventional column chromatography by utilizing pressurized nitrogen gas. Here, we demonstrate the comparative study of the conventional column chromatography and the GPEC method. Cation exchange resin AG 50W-X12 (200~400 mesh size) was used. The sample was prepared at a 0.8 M hydrochloric acid solution and gradient elution was applied. In this case, we used the natural isotopes 88Sr, 133Cs, and 138Ba instead of radioactive isotopes for the preliminary test. Usually, cesium is difficult to measure with ICP-OES, because its wavelengths (455.531 nm and 459.320 nm) are less sensitive. So, we used ICP-MS to determine the identification and the recovery of eluate. In this study, optimized experimental conditions and analytical result including reproducibility of the recovery, total analysis time and volume of eluents will be discussed by comparing GPEC and conventional column chromatography.

Development and Comparison of Analytical Methods for Measuring Simazine Herbicide Using Gas Chromatography/Ion Trap, Gas Chromatography/Mass Selective Detector, and High Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometers

Chung, Ill-Min,Park, In-Myoung,Kim, Seung-Hyun The Korean Society for Applied Biological Chemistr 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.5

Simazine is widely used to manage weed growth in the agricultural and industrial fields. Three analytical methods, including gas chromatography (GC)/ion-trap (IT), GC/mass selective detector (MSD), and high-performance liquid chromatography (HPLC)/triple quadrupole mass spectrometers (HPLC/MS/MS), were developed and compared for simazine quantification. The spike recoveries by GC/IT, GC/MSD, and HPLC/MS/MS were 100, 117, and 82.5%, respectively. Different spike recovery depending on analytical methods could be due to matrix effect, different instrumental sensitivity, losses during sample preparation or all of the above. The limit of detection was 0.0361, 0.0263, and 0.0013 mg/kg by GC/IT, GC/MSD, and HPLC/MS/MS, respectively. The HPLC/MS/MS with a positive atmospheric pressure chemical ionization (APCI) ionization mode was the most rapid (7 min), sensitive (limit of detection: 0.0013 mg/kg, limit of quantification: 0.0042 mg/kg), and precise (relative standard deviation: 0.5%) method for simazine quantification.