Natural Product Isolation PDF

5. Natural Product Isolation Natural product chemistry research 1. Extraction 2. Fractionation 3. Isolation 4. Quantification 5. Structure elucidation 6. Assay 2 1. Extraction: purpose - Unknown bioactive compound - Known compound present in an organism - A group of compounds (structurally rela...

5. Natural Product Isolation Natural product chemistry research 1. Extraction 2. Fractionation 3. Isolation 4. Quantification 5. Structure elucidation 6. Assay 2 1. Extraction: purpose - Unknown bioactive compound - Known compound present in an organism - A group of compounds (structurally related) - ID of all 2nd metabolites for chemical fingerprinting or metabolomics study 1. Extraction (scale: amount for what??) -Extraction for purifying a sufficient amount of a compound to characterize it partially or fully. (ex. MS or NMR) - Level of purity? - Enough material for structure ID of previously isolated co mpound (ex, TLC, HPLC, MS): small quantity - Enough material for further study (clinical trial): large quantity 4 1.Extraction: method - Drying and gliding or homogenizing or maceration - Choice of extraction method a. maceration b. boiling c. soxhlet d. supercritical fluid extraction e. sublimation f. steam distillation 5 1. Extraction: method - Choice a. of solvent Polar extraction: water, ethanol, MeOH b. Medium polarity extraction: EtOAc (ethyl acetate), DCM (dichloromethan) c. Nonpolar: n-hexan pet-ether, chloroform 부표 11 용매의 약자 = Acetic anhydride = Amylacetate 수 BuOH =w-Butanol 다 Z-BuOH = i5t?-Butanol Et2NH = Diethylamine /-BuOH = ?er/-Butanol Et3N = Triethylamine CHC13 = Chloroform HCOOH = Formic acid = Methylenedichioride HOAc = Acetic acid CH2N2 = Diazomethane MeCOEt = Methylethylketone CH3CN = Acetonitrile MeOH = Methanol AC2O AmOAc 우 다 O CH2cI2 우 EtOAc ^EtOH 우 1 引 2（） = Ethylacetate = Ethanol = Ether C5H5N = Pyridine Me2co = Acetone 앙112 = Pentane PhOH = Phenol c6H6 = Benzene PrOH =n-Propanol DMSO = Dimethylsulfoxide i-PrOH = fso-Propanol 7 1. Extraction: solvent jl =—— Physicochemical Properties of O)me Common Solvents Used in Natural Products Extraction Solvent -Hexane Dichloromethane 77-Butanol iso-propanol zz-Propanol Chloroform |Ethyl acetate Acetone Methanol Ethanol Water 乃 Polarity index Boiling point (℃) Viscosity (ePoise) 0.0 3.1 3.9 3.9 4.0 4.1 4.4 5.1 5.1 5.2 9.0 69 . 41 . 1 11； 1 82 92 61 77 56 65 78 100 0.33 0.44 2.98 2.30 2.27 0.57 0.45 0.32 0.60 1.20 1.00 Solubility in water (% w/w) 0.001 1.6 7.81 100 100 _________ 0 R1 즈 8.7 100 100 100 100 Ex : water saturated butanol Partition, CC 8 2. Fractionation J —— - cocktail of compounds - initial separation by similar polarities or molecular size a. two phases of a liquid-liquid extraction b. contiguous elute from chromatography column - vacuum liquid chromatography (VLC) - column chromatography (CC) - size-exclusion chromatography (SEC) - solid-phase extraction (SPE) adsorbent 스sintered glass frit 〜 conical vacuum adaptors 卜 to water aspirator - not to generate too many column filter 9 lask 3. Isolation: protocol -known (ref.) or unknown -unknown case: qualitative tests for the for the type of compounds (phenolic, steroids, alkaloids, flavonoids) a. b. c. d. e. Hydrophobicity or hydrophilicity: solvent partition Acid-base properties: pH 3, 7, 10今extraction Charges: adding various ion exchanger Heat stability: 90 C for 10 min Size: dialysis tubing for macromolecule 10 4. Quantification - compound yield (recovery): important 三흐-------O - The reason of activity loss during isolation 1. retain in column 2. Unstable 3. active compound precipitated out when loading on to the column 4. spread across a wide range of fractions 5. activity of extract: presence of synergy among a number o f compounds今separated今not active 11 4. Quantification: “Poor-yield” Problem - Poor yield or poor recovery: major problem in natural product isolation -1,900g of Taxol from 6,000 Taxus brevifolia/ 27,300kg bark (NCI 1990) 1. finding a better source: different species, plant part, cultivation condition 2. semisynthesis from abundant precursor 3. total synthesis 4. tissue culture 12 6. Assays - Assays of natural product 1. filtered or centrifuged: remove any insoluble matter 2. readjust original pH 3. Positive and negative control in any assay 4. series of dilutions to determine major target compound 5. sensitive assay 13 Extraction of Plant Secondary Metabolites Selection, Collection and ID of Plant Materials O - Plants selection 1) traditionally used 2) random collection 3) species based 식물성분 추출분리 1 . 신선한 식물조직: hot alcohol or 건조 - 건조 식물시료의 경우 장시간 보존 가능 미생물 오염에 주의 필요 채집시 다른 식물 혼입에 주의 식물시료 감정 후 표본을 공인 표본실에 저장 16 Drying and Grinding O - Drying at < 30C: avoid decomposition by heat - Protected from sunlight: avoid decomposition by UV - For flesh sample study: extract as soon as possible using MeOH or EtOH 今deactivate enzyme in plant - Small quantities: milled using spice mill grinding今increasing surface area今improve extraction efficiency 17 식물성분 추출 예비실험 1. 신선한 식물조직 hot alcohol 처리: 효소에 의한 변화 방지 및 추출용매로 역할 O 1. 계통적 추출법: ether（ 지방, 수지, 정유, chlorophyll, alkaloid, 배당체, 수지, 색소, tannin） 今CHCl3 （ 탄성 gum 추출） 今Me OH （ saponin, 당, 배당체, tannin, alkal oid 추출） 今H2O （ tannin, 배당체 추 출） - 추출 성분이 어느 분획에 완전 분리되는 것이 아니 라, 수개 분획에 걸쳐서 분포됨. 18 대규모 추출분리: 추출 O 1. 건조된 시료 추출시 MeOH 사용今세포 막 파괴今세포내 성분 추출 가능 （ ether, CH2Cl2 또는 CHCl3 같은 비극성 용매로 는 세포내 성분 추출 불가） 2. 지용성성분 추출시 CH2Cl2 로 바로 추출 하는 것 보다 재료를 일단 MeOH에 담가 두었다가 CH2Cl2 와 MeOH의 1:1 혼합용 매로 추출하는 것이 효과적임. 19 Extraction Extraction of Plant Secondary Metabolites 331 Ground Plant Material 1 .Macerate with MeOH 2 .Concentrate under vacuum MeOH Extract--------------------------1. Suspend in 90% MeOH 2. Partition with hexane or petroleum ether 90% MeOH Partition Hexane Extract 1. Concentrate 2. Suspend in H2O 3. Partition with CHCI3 Lipid 제거 Aqueous Extract Crude CHC13 Partition 1. Wash with 1% NaCl in H2O to remove tannins 2. Evaporate to dryness CHCI3 Partition (Essentially Free of Tannins) 1% NaCl Solution (Waste) Fig. 1. General proced나re for preparing extracts representing a range of pola-20 rities, including a virtually tannin-free chloroform extract (19). 대규모 추출분리: 액체분배법（ 상분리） - Ether로 탈지한 재 료를 EtOH로 온침하 고 감압농축 후 물을 가한 후 ether 혹은 CHCl3로 추출하면 극성이 적은 phenol성 물질, terprnoid, alkaloid 같은 물질이 이행 된다. O - 수층을 BuOH 또는 EtOAc로 추출하면 극 성이 큰 flavonoid, 배당체 또는 saponin이 추출된다. 21 Saponin extract Ground Plant Material 1. Defat with hexane or petroleum ether Hexane or Petroleum Ether Extract (Contains fats, waxes, and chlorophylJs) 1. Add diethyl ether to precipitate crude saponin mixture 22 대규모 추출분리: 배양액성분의 분리 O 식물수침액 또는 미생물 배양액 같은 수 용액은 1. ether, CHCl3, EtOAc, BuOH 순으로 분배 추출하 는 방법으로 천연물 분리 2. 대량의 알코올 또는 acetone을 가하여 침전분리 3. Amberlite XAD-2 또는 XAD-4 resin column을 통과시켜 흡착시킨 후 acetone이나 MeOH로 용출 등의 방법을 많이 사용한다. 23 Fermentation brotH of JVoc：dr^dia sp < 미생물 균체 제거 Filtered Fil trate 너 | Extracted with EtOAc EtOAc extract Concentrated, waskiecl with water and dried on anhydrous Na2so스 Oil 수분제거 Crude spirocardin B Crude spirocardin A Benzene-EtOAc (= 1 -3:1) Benzene-EtOAc | (사: 1 - 3:1) Silica ge! CC Silica gel CC Oil Spirocardin B 0H Fig. 2. Isolation of microbial natural products: spirocardins A and B fr 24 Ncy cci rdid sp>. '------- OH OH Alkaloid extract O Ground Plant Material 1. Defat with hexane or petroleum ether 2. Extract with MeOH, filter, concentrate Defatted Methanolic Extract 1. Suspend in tartaric acid, titrated to pH 5 2. Partition with EtOAc, pre-saturated with H2O Aqueous-Acid Phase Ethyl Acetate Partition 1 1. Make alkaline with으으（&血이 compounds） Na2c（人 solution, titrating to pH 11, or as desired 2. Partition with EtOAc 25 Aqueous Extract Ethyl Acetate 牛- Lipid: Interfering Compounds in Assay - false positive in assay, - broad at 1.2-1.4ppm in 1H-NMR - reverse phase CC or solid phase extraction : remain in column - Plant Pigments Carotenoids (interfere electron capture detection), Vegetable Tannins (false positive in assay) Plasticizers: solvent, filter paper, plastic apparatu s, MeOH extraction in plastic container (ex) phthalate 26 Detection of Phytochemical Groups in Extracts (false positive in many assays) - Alkaloids: Mayer reagent, Dragendorff reagent - Sesquiterpene Lactones and Cardiac Glycosides : Kedde reagent, Baljet reagent - Flavonoids: Shinoda test, Sulfuric test - Other Polyphenols: Ferric chloride, Gelatin-salt - Sterols: Lieberman-Burchad test - Saponins: shaken今foam (>15min) 28 Chromatography Mechanism ■ Adsorption ■ Partition ■ Size exclusion chromatography (SEC) ■ Ion-exchange 29 Older strategies ■ Focus on chemistry of compounds not activity ■ Straightforward isolation and identification of compounds 30 Modern strategies ■ Production of natural products libraries ■ Tissue culture, genetic manipulation, combinatorial biosynthesis ■ Chemical fingerprinting & metabolomics 31 Classical chromatographic techniques ■ ■ ■ ■ Thin-layer chromatography (TLC) Preparative TLC Open-column chromatography (CC) Flash chromatography (FC) : VLC (vacuum Liquid Chromatography) adsorbent sintered glass frit conical vacuum adaptors water aspirator test tube filter flask 32 Modern chromatographic techniques ■ High-performance thin-layer chromatography (HPTLC) ■ Solid-phase extraction (e.g., Sep-Pak) ■ Multiflash chromatography (e.g., SepBox) ■ Countercurrent chromatography (CCC) ■ High-performance liquid chromatography (HPLC) ■ Hyphenated techniques (HPLC-DAD, LC-MS, LC-NMR, LC-MS-NMR) 33 1 Sep-Pak Sample Extraction Products High-performance thin-layer chromatography (HPTLC) mobile pha&e stationary phase Countercurrent chromatography (CCC) 34 Plant material | STEP 1: | Successive Soxhlet extraction with Solvent A, B and C ▼ Small scale Bioassay (7n vitro} Bioactivity guided approaches 구조?, 양? Bioassay (/n vitro) V fclxtract A Kxtract B Kxtract C? 시간 + 경제성 Plant material Successive Soxhlet extraction with Solvent A, B and C Extract A ACTIVE extract (s) Bioassay {in vitreh Preliminary fractionation by Flash Chromatography/VLC/COSepPak ACTIVE fraction (s) Different chromatographic techniques guided by in vitro bioassay isolation and purification of compound (s) ACTIVE compound (s) Structure determinat ion (UV, IR. MS, NMR, X-Ray etc) 13ioassay of active compound (s) using different concent rat ions to determine the potency 7" vivo assay and ---------------------------- 느 Clinical trials toxicological studies 수 Application for approval------------------------ Patent application Pharmaceutical production and marketing soxhlet 35 Supercritical Fluid Extraction(초임계추출법): process of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent - Environmental improvement and reduced product contami nation - Selectivity - Speed Ex : caffeine, Volatile oil, hops 36 Table 3 Various Supercritical Solvents and Their Critical Conditions ■日^■■山■■■*■■■■페느■■■■■■日■■게■■■日그^^^^^^퍼■■드^ Fluid Carbon dioxide Ethane Ethylene Propane Propylene Trifluoromethane • Chlorotrifluoromethane T richlorofluoromethane Ammonia Water Cyclohexane 77-Pentane Toluene Critical temperature (K) Critical pressure (bar) 304.1 31 C 305.4 282.4 369.8 364.9 299.3 302.0 471.2 405.5 647.3 553.5 469.7 591.8 73.81 48.8 50.4 42.5 46.0 48.6 38.7 44.1 113.5 221.2 40.7 33.7 41.0 37 Planar Chromatography Thin-layer chromatography (TLC) 흡착제를 균일한 박층으로 만들어 사용 ■ 일반적으로 silica gel 박층 사용하며 지용 성 물질 분리에 적합 ■ 분석 시간이 짧음 ■ 용매, 발색 시약의 제한이 없음으로 여러 종류의 물질 분석에 이용 가능 ■ 38 Planar Chromatography Thin-layer chromatography (TLC) 39 Mechanisms of TLC Separation ■ Adsorption Chromatography ■ Partition Chromatography ■ Size-inclusion/exclusion Chromatography ■ Ion Exchange Chromatography 41 Adsorption and hydrogen bonding between compound and sorbent face (normal phases) Adsorbed compound Hydrogen bond normal phases / H Sorbent surface ? (Silanols) Si、 OH 42 Common reverse phases for partition chromatography -Si-CH2(CH2)6CH3 -Si-CH2(CH2)16CH3 ODSC18 RP-8 C8 -Si-CH2(CH2)2NH2 Phenyl “해 —Si-CH2cH20cH2cHeH2 ' Amino -Si-CH2(CH2)3 Diol OH -Si-CH2CH2CH2CN Cyano 43 Table 2 Simple Systems for TLC Solvent system Sorbent Notes Hexane:ethyl acetate (fctOAc) Petrol:diethyl ether (Et2O) Silica gel Petrol :chloroform (CHC13) Silica gel T oluene:ethyl acetate:acetic acid CTEAJ CHCI3： acetone Silica gel Universal system)——can substitute hexane for petroleum spirit or pentane A universal system for relatively nonpolar metabolites. Excellent for terpenes and fatty acids. Care should be taken with Et2O as explosive mixtures are formed in air Considerably useful for the separation of cinnamic acid derivatives and in particular the coumarins Vary the composition, i.e., 80:18:2 or 60:38:2—excellent for acidic metabolites Benzene: acetone Silica gel | Butanolracetic acidrwater Butanol: water:pyridine: toluene | Methanol:water Silica gel Silica gel Silica gel Silica gel ＜三18 A general system for medium polarity oroducts Useful for the separation of aromatic products. Care should be taken as benzene is a highly carcinogenic solvent. Substitute toluene for benzene A polar system for flavonoids and glycosides Sugar analysis system. Try 10:6:6:1. Development may take 4 h Start with 100% MeOH to determine wnetner metaooiites will move irom me origin. Increase water concentration to ‘slow’ down products. The addition of small amounts of acid or base may improve chromatography | Acetonitrile:water Methanol: water Methanol: water C18 /c2 Polyamide Cellulose A universal simple reverse-phase system Universal Used for the separation of highly polar 44 Detection of Natural Products in TLC ■J------------------------------------------- -■ Ultraviolet Detection • • • non-destructive method UV-active 254nm or 366nm indicators UV-lamp: eye, skin (mutagenic) ■ Spray detection • destructive 45 Ten Simple Spray Reagents for Natural Product TLC Visualization Detection spray Recipe Treatment (1) Vamllin) Sulfuric acid Dissolve vanillin (1 g) in concentrated H2sO4 (2) Phosphomolybdic acid (PMA) Dissolve PMA in ethanol to make a 5% w/v solution (3) Ammonium molybdate (VI) Dissolve ammonium molybdate(VI) (10g) in concentrated H2sO4 (100 mL) Dissolve antimony(III) chloride in a mixture of glacial acetic acid (20 mL) and chloroform (60 mL) Spray onto plate and heat at A universal spray. Many terpenes give 100℃ until coloration red and blue colors. Natural appears products with little functionality may give poor coloration—try spray (2). Spray and heat in a fume cupboard Spray onto plate and heat at Useful to detect many terpenes as 100℃ until coloration blue spots on a yellow background, appears Spray and heat in a fume cupboard Spray onto plate and heat at A universal spray. Many diterpenes 100℃ until coloration give a blue color. Spray and heat in appears a fume cupboard (4) Antimony(III) (5) Tin(lV) chloride Add tin(IV) chloride (10mL) to a mixture of chloroform (80 mL) and glacial acetic acid (80 mL). Notes Spray onto plate and heat at Di- and triterpenes give a red to blue 100℃ for 2-5 min or until coloration. Care should be taken coloration appears when handling this spray as antimony compounds are highly poisonous. Spray and heat in a fume cupboard Spray onto plate and heat for Useful for the detection of flavanoids 5 min at 100℃ or until and terpenes. Tin(IV) chloride is coloration appears poisonous and a lachrymator. Spray and heat in a fume cupboard 46 (6) Dragendorff、 reagent Add 10 mL of a 40% aqueous solution of KI to 10mL of a solution of 0.85 g of basic bismuth subnitrate in acetic acid (10 mL) and distilled water (50 mL). Dilute the resulting solution with acetic acid and water in the ratio 1:2:10 (7) 2f4 Dinitro-phenyl- Dissolve 2,4-dinitrohydrazine phenylhydrazine (0.2 g) in 2-N HCI (50 mL) (8) Perchloric acid (9) Borntrager reagent (10) Ninhydrin A 20% (w/v) aqueous perchloric acid solution A 10% (w/v) ethanolic solution of KOH Add ninhydrin (0.3 g) to a mixture of butanol (100 mL) and acetic acid (3 mL) Generally, no heat is required—— if reaction is not spontaneous, heat until coloration appears Generally, no heat is required — but if reaction is not spontaneous, heat until coloration appears Heat at 100℃ until coloration Heat until color detection Heat at 100℃ until coloration This is the traditional method for alkaloid detection, although care should be taken as some nonalkaloids such as iridoids and some flavonoids give a positive reaction. Alkaloids give a dark orange to red coloration Detects aldehydes and ketones with a yellow to red coloration A universal spray but is useful for steroids and triterpenes For the detection of coumarins and anthraquinones Especially useful for amino acids, amines, and as a general alkaloid spray. Alkaloids appear as a red coloration Not for ID but only for functional group detection! 47 ■ P(Preparative)-TLC ■ CP(centrifugal preparative)-TLC Fig. 6. Circular band separation through CPTLC development. 48 Two-dimensional-TLC 2nd development (2nd dimension) Fig. 9. Two-dimensional TLC plate after two developments. 49 Adsorption QOQOO O C그 <Z> O O <=u 4 J A V H U -/Knalyte mixture 그 = Strongly adsorbed to ttie stationary phase and less soluh>le in. the mot>ile phrase ◊ = Loosely t>ovxnd to the stationary phase and more soluble in tbie mobile phase Mig. 1 • lirustratcd t>asic principle of a<ds<z>rp>tion chromatograi^tiy. 50 Partition 51 Size exclusion chromatography (SEC) 52 Commonly Used B이ided-Phase Silica Phase Description Cl TMS, trimethyl Reversed-phase material C2 RP-2, dimethyl Reversed-phase material Structure Application Ideal for polar and multifunctional compound separation Si—CH3 Ideal for polar and multifunctional compound separation _；i-C H 2 5 C4 Butyl Reversed-phase material Ideal for HIC and ion-pairing chromatography of large proteins and macromolecules --J!i—C H 4 g C5 C6 C8 Pentyl Reversed-phase material Hexyl Reversed-phase material MOS, RP-8, LC8, Octyl Reversed-phase material Useful for hydrophobic proteins and oligonucleotide ■J •i-C H 5 li 느C6Hl3 1 —s CH 1 느 8 l7 , Useful for ion-pairing chromatography Useful for various types of nonpolar and moderately polar natural product isolation 53 C12 C18 CN NH2 OH Phenyl Dodecyl Reversed-phase material ——< 거-C12H25 ODS, RP-18, LC18, octadecyl Reversed-phase material CPS, PCN, cyano, cyanopropyl, Nitrile Reversed-phase or normal-phase material Amino, amino propyl silyl (APS) Reversed-phase, normal­ phase or weak ion­ exchange material Diol, Glycerol Reversed-phase or normakphase material C6H5 Reversed-phase material J서;_厂 TJ 서 2；>1 으 18 니 37 （리厂t 么）저 라니 Useful for various types of nonpolar and moderately polar natural product isolation Most retentive for nonpolar compounds. Ideal for various types of moderately polar and polar natural product isolation unique seiecuvity for polar natural products in both reversed and normal-phase modes. Ideal for carbohydrate separation -I -- 1 4；_ （리-以:丁시러2 1 ______________ U— 1 2;. （아1之사計1 Used for various medium polar and polar natural product separation Useful for analyzing aromatic natural products 1 1 Phenyl ether C6H5(C3H6O linker) Reversed-phase material 1 1 ___________ < Si­—(CH2)3Ph ! Ideal for extremely polar aromatic compound separation Table 2 Commo이y Used Sephadex G-series Sephadex type G-10 G-15 G-25 G-50 G-75 G-100 G-150 G-200 Sephadex LH-20 Bead size (jim) 40-120 40-120 20-50 20-80 50-150 100-300 20-50 20-80 50-150 100-300 20-50 40-120 20-50 40-120 40-120 40-120 Size Fractionation range (molecular weight) <700 <1500 100-5000 100-5000 100-5000 100-5000 500-10,000 500-10,000 500-10,000 500-10,000 1000-50,000 1000-50,000 1000-100,000 1000-100,000 >100,000 >100,000 Pumping system Sep-Pak column I LI IJJ Upper frit assembly Packing material Solvent reservoir Packing material To vacuum 츠 Lower frit assembly To fraction collector Fig. 6. Solid-phase extraction (Sep-Pak). Fig. 7. Application of pump in LPLC. 56 57 Gas chromatography 휘발성물질 분리 및 분석에 적합 ■ 분리능, 분석 감도 가장 우수 Moniterperpene, sesquiterpene 등의 휘발성 ■ 천연물 분석 ■ 휘발되기 힘든 물질의 경우 ester 유도체화 또는 trimethylsilylether 화합물로 유도하여 휘발성화하면 GC로 분석 가능함 ■ 58 ■ HPLC 이동상으로 액체를 사용하고 압력은 가하 여 강제로 고정상을 통과 ■ 모든 비휘발성 물질 분석 가능 ■ 59 Normal Phase chromatography 유고정상의 고체 흡착제 표면과 시료간의 친화력 크기에 따라 분리. 셔시료의 분자 입체 구조 및 극성 도는 고정상과의 상호작용에 큰 역할 여정지상 (stationary phase) : Silica gel, Alumina 3이동상 (mobile phase) : 비 극성 유기 용매. 호분리 가능한 시료 : 약한 극성의 시료 분리 상 유호작용 힘 : 수소결합(hydrogen bonding), Silica gel 60 1 Reverse Phase chromatography POROUS PARTICLE ―“——-- __ BONDED PHASE •시료가 두 상（고정상과 이동상）에 대한 용해도 차이에 의해 분배 정도가 달라져 분리가 이뤄짐. 62 해 Diol phase : –OH 작용기가 있어 시료와 hydrogen bond 형성. • Amino phase : Hydrogen bond + Van der Waals Carbohydrate & more polar sample. 63 수 C8 phase : Reverse-phase Chromatography에 이용 . R hydrophobic & Hydrophilic 상호작용에 기초. O- Si - (CH2)7- CH3 R 하 C18 phase : Reverse-phase Chromatography 에 이용. ODS R O- Si - (CH2)17 - CH3 R 64 Stationary Phases Commonly Used in HPLC SOLVENT STRENGTH NORMAL PHASE hexane is o-octane t oluene chloroform methylene chloride tetrahydrofuran et hyl ather ethyl acetate acetone acet onitrile iso-propyl alcohol methanol REVERSED PHASE water methanol Iso-propyl alcohol acetonitrile acetone ethyl acetate ethyl ether tetrahydrofuran methylene chloride chloroform toluene iso-octane hexane 66 Define separation goal Increase Polarity 心Polar 수None- polar Increase Molecular weight Water insoluble Adsorpt 10n Partition Gel Permeation Water soluble Exchange Gel Filteration 67 Detectors of HPLC 부 UV/Vis Detector Refractive In dex- Detector Fluorescence Detector Evaporation Lighter Scat tering Detec tor 68 UV/VIS – Detector ► Beer-Lambert Law A=£*b*c Signal is limited by £ : Compound's molar absorptivity Light source : D2 lamp (190~380nm) Tungs ten lamp (380~800nm) b : Flowcell’s optical pathlength C Concentration of sample in optical path duterium lamp Application : amplifier Aromatic ring, Double bond, Triple bond HPLC 검출기 중 가장 많이 사용함. grating 69 Refractive Index Detector 31 6 stainloss steel tubing = ------- 仁＞ ■ =너 1 2 3 4 5 TefIon tubing Connection by female nut Connection by flango Heat exohanger Flow cell Sam pie ceil Reference cell Heat exchanger Joint block 3 - way solenoid valve 6 7 From column To waste liduid reoept크cle Reference cell과 sample cell에 포함된 시료와 용매의 굴절률 차이에 의하여 검출. Universal : carbohydrate, sugar Fluorescence Detector absorption Energy level 2 hv Energy level 1 luminescence Energy level 2 hv Energy level 1 71 ELSD (Evaporation Lighter Scattering Detector) 후원리 Nebulizer 를 통해서 샘플과 이동상이 질소에 의해서 뿜어져 나오면 Heated Zone 에서 이 동상이 증발하여 없어지고 샘플은 입자화 되 어서 광을 통과. 샘플은 일정양의 광을 흡수하고 난 후 샘플 자체에서 산란今검출 느사용범위 모든 물질에 감응함으로 선택성 의 폭이 넓다. 72 Comparative Table for HPLC Detectors UV Fluorescence RI ELSD Sensitivity ng pg ug ng Detection selectivity selective highly selective universal universal Temperature influence small small large large Gradient elution possible possible impossible Possible 73 Isocratic Gradient 74 Optimize separation conditions What is resolution ? Rs =1/4 (α -1/α)(√N)(k’/1+k’) 1 > > > Rs : Resolution α : Column selectivity N : Column efficiency K’ : Capacity factor 77 Capacity factor k 시료가 컬럼 고정상에 충분히 머무르게 하 는 효과 이동상 의 용매 강도를 의미 VR - V0 = t R - t 0 t’ R V0 t0t0 VR = 머무름 용량 V0 = 용매의 머무름 용량 tR = 머무름 시간 t0 = 용매의 머무름 시간 t’ R = 실제 머무름 시간 k’ = 0 : 시료 Vo에서 용출 2- 6이 적당 78 Selectivity α (separation factor) 누 Selectivity : α 수각 성분의 분리 차이 노1 : 두 시료가 전혀 분리 안됨 α t’ R(B) -t 0 t’ R(A) -t 0 t’ t’ R(B) R(A) 78 Efficiency N （ Theoretical plate number） 거!------- - N = 16（tR/W）2 최소 3,000 이상이어야 （10,000 이상일 때 신뢰） 컬럼의 효율을 의미함 （길이, 충진제입자, 유속, 온도…） 동일조건 컬럼에서 시료에 따라 N이 달라짐 （지용성＞ 약산성＞산성＞ 염기성. 당, 이온성 물질） 79 Efficiency N (Theoretical plate number) 거！------Plate Number for Well-packed HPLC Column Particle Diameter 10㎛ 10㎛ 5㎛ 5㎛ 5㎛ 3㎛ 3㎛ 3㎛ 3㎛ Column length 15 ㎝ 25 ㎝ 10 ㎝ 15 ㎝ 25 ㎝ 5㎝ 7.5 ㎝ 10 ㎝ 15 ㎝ Plate Number N 6,000-7,000 8,000-10,000 7,000-9,000 10,000-12,000 17,000-20,000 6,000-7,000 9,000-11,000 12,000-14,000 17,000-20,000 8 Sample pretreatment Solid-Phase Extraction (ex. sep-pak) & Filtration 81 jl 휴 후 셔 石 사 수 Separation Goals Resolution : Rs be greater than 1.5 Separation time : less than 90min P eak height : over signal/noise ratio Pressure : 750 psi ~ 1300 psi (60 bar~120 bar) Peak Asymmetry : 0.9~1.5 Efficiency N : to be greater than 10,000 소 Capacity factor k’: 0.5 ~ 20 (2 – 6) 82 Derelipication Dereplication and Partial Identification of Compounds - To identify novel pharmaceuticals and agrochemical lead compounds rapidly and effectively from complex natural matrices - To avoid spending time and money on the re­ isolation and re-identification - Sensitive, rapid, reproducible 84 Dereplication ■ Analysis without isolation ■ DB 활용 ■ Increase the number of novel compounds identified ■ Make more efficient use of limited resources ■ 경제성 (US $50,000, 3 month: isolation and identification of an natural compound) ■ Taxonomic information: chemotaxonomy 85 Understand Problem (disease, pest etc.) Exclude previously tested organisms Inc rease the number of novel compound V Hit Optimization Selection of Best Leads Development of a Commercial Product 86 Partial Identification of Compounds 303 Plant Extracts ▼ B|| Bioassay/ecdysteroid-specific RIAs Bioassay RIA r1 -ve or cytotoxic -ve Gradient Of 3 RP-HPLC/Bioassay/RIA 30%-100% MeOH in H2O over 30 mins collect 1 min fractions for bioassav/RIA_________________ cf. Rts of active fractions with known ecdysteroids agonist +ve +ve rphytoecdysteroids' Diol NP-HPLC/ Bioassay/RIA 4% MeOH in CH2CI2 collect 30 sec fractions for bio石公公ay/RIA cf. Rts of active fractions with known ecdysteroids agonist +ve -ve "nonsteroidal agonists' antagonist +ve +ve/-ve ’antagonists' CIQ-SPE .—— Q% 25% 60% 1OO% MeOH in H2O ▼ Bioassay (agonist or antagonist) to identify active fraction(s) Gradient Cis RP나너PLC/Bioassay 30%-100% MeOH in H2O over 30 mins collect 1 min fractions for bioassay cf. Rts of active fractions with known (ant)agonists (c니curbitacins, withanolides, stilbenoids) Fig. 2. Dereplication strategy for ecdysteroid agonists and antagonists from 87 at sources. Chromatography for Dereplication ■ ■ ■ ■ ■ ■ Countercurrent chromatography (CC) Centrifugal partition chromatography (CPC): CPCPDAD Solid-Phase extraction (SPE) Thin-layer chromatography (TLC) “ simple, complete polarity range” GC-MS : TMS-derivative HPLC, CE, UV/Vis, MS, LC-MS, IR, LC-MS/NMR ■ Database 88 Hyphenated technique 89 Hyphenated technique ▪Marriage of a separation technique （ 분리） and spectroscopic detection technique （ 분석） ▪LC-MS, LC-NMR ▪LC-PDA-MS, LC-MS-MS, LC-NMR-MS, LC- PDA-NMR-MS 90 Hyphenated technique for chemical fingerprinting and quality control of herbal medicine Fingerprinting ▪Herbal medicines: geographic origins, sources, harvest times ▪Quality of raw material: GAP (good agricultural practice, GMP (good manufacturing practice) ▪TLC今GC-MS or LC-MS, LC-NMR-MS …… Chemotaxonomy Metabolomics 91 Hyphenated technique Separation technique Further detection technique(s) Optional analyte enrichment Optional multiple hyphenation Fig. 1. Hyphenated technique. 92 93