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한별 방
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1 High Performance Liquid Chromatography Basics

2 Contents About the separation process Modes of HPLC The basics of HPLC instrumentation 에대하여이해하실수있습니다.

3 Chromatography 혼합된시료성분이이동상과고정상사이를흐르면서흡착, 분배, 이온교환 또는분자크기배제작용등에의해각각의단일성분으로분리되는것을 말한다. 분리, 정성, 정량등의분석목적과분리, 정제, 분취목적에이용된다. Injection Interaction Elution abacbc ccbbaa c b a Mobile Phase carries the sample through the stationary phase as it moves through the column Stationary Phase the phase which remains fixed in the column, e.g. C18, Silica

4 Chromatography 의분류 Chromatography Gas Chromatography Liquid Chromatography Supercritical Fluid Chromatography Gas-Solid Gas-Liquid Adsorption Partition Ion exchange Size Exclusion Affinity Gel Filtration Gel Permeation

5 Chronology of Liquid Chromatography 1906 Chromatography 용어의최초사용 1940 s 분배, 종이크로마토그래피 1950 s 박막, 겔 - 여과및기울기용리크로마토그래피 1960 s HPLC 상용화 M.S. Tswett. Ber. Dtsch. Bot. Ges. 24: (1906)

6 Separation Technique I have TWO separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which should I use? HPLC? GC?

7 Comparison of HPLC and GC Hydrophilic Polarity Hydrophobic Amino acids Inorganic ions Volatile Synthetic Sugar Sugars carboxlylic Glyphosatefood dyes alcohols acid Aldehydes Enzymes Ketones PG, OG, DG Sulfonamides Glycols phenols Aflatoxins Fatty acids Antibiotics Nitriles BHT, BHA, Nitrosamine THBQ Flavonoids Organophosphorous PAHs Anabolica Natural food dyes antioxidants Alcohol TMS pesticides Aromatic amines derivativ Fat soluble vitamins e of PCB Polymer monomers Triglycerides Phospho-lipids Epoxides sugars Essential oils C2/C5 hydrocarbons Fatty acid methylester Aromatic esters Volatile Volatility Nonvolatile

8 Comparison of HPLC and GC Sample Volatility Sample Polarity Sample Thermal Lability Sample Molecular Weight Sample Size Sample Preparation HPLC No volatility required Sample must be soluble in mobile phase Separates both polar and nonpolar compounds PAH ~ inorganic ions Analysis can take place at or below room temperature No theoretical upper limit In practicality, solubility is limit Sample size based upon column I.D. Sample must be filtered Sample should be in same solvent as mobile phase GC Sample must be volatile Samples are non-polar and polar Sample must be able to survive high temperature injection port and column Typically < 500 amu Typically 1-5 ul Solvent must be volatile and generally lower boiling than analytes

9 Comparison of HPLC and GC Separation Mechanism Detectors HPLC Both stationary phase and mobile phase take part Most common UV-Vis (Wide range of non-destructive detectors) 3-dimensional detectors Sensitivity to pg GC Mobile phase is a sample carrier only Most common FID, universal to organic compounds Water Polarity of Mobile Phase Hexane Gas Liquid Solute Molecular Weight

10 HPLC Instrumentation & Chromatogram Injector Mixer Pumps Chromatogram Column Detector Solvents

11 Separation Parameter Column stationary phase selection Column length and diameter Mobile phase composition Temperature Flow rate Sample size

12 The Goal of separation Resolution between sample components tr(b) tr(a) t R = t /W = t /W(A) + W(B)/2 = 2 t / W(A) + W(B) t0 t R(B) W(A) W(B) 분리도는두피크의분리된정도를간단히표시해주는값 Baseline 에서분리되는경우 R 값은 1.5

13 Factors Influencing Resolution Efficiency Capacity Selectivity

14 Selectivity Factors : 컬럼에서용출되는각성분의분리의차이 To Change Selectivity Change Mobile Phase Composition Change to Different Mobile Phase Change Mobile Phase ph Change Column Temperature Use Special Chemical Effects Change Stationary Phase

15 Capacity Factor Capacity factor is characteristic of a specific compound at a given mobile phase composition, temperature, and column type K 은시료의머무름을나타내는지표 ( 시료와고정상이얼마나강하게상호작용하는가를나타냄 )

16 Efficiency HETP = L/N N(Theoretical plate) 은이론단수라함. 컬럼의효율을의미함 HETP(the Height Equivalent to a Theoretical Plate) - 이론단해당높이. 시료분자가이동상과고정상사이에서평형에도달하는데필요한컬럼의길이임

17 HPLC Concept Normal and Reversed Phase Mode Stationary Phase Mobile Phase Normal Polar Non-polar Reversed Non-polar Polar Isocratic vs. Gradient Elution Isocratic The mobile phase composition is held constant throughout the run Gradient The mobile phase composition changes over time in a predetermined way

18 Properties of Organic Solvents Commonly Used in HPLC Nonpol ar Polar Solvent Miscible with water UV cutoff Refractive Index (20 ) Solvent Strength, 18ε 0 (silica) Viscosity (20, CP) Hexane No Isooctane No Carbon tetrachloride No Chloroform No Methylene chloride No Tetrahydrofuran Yes Diethyl ether No Acetone Yes Ethyl acetate Poorly Dioxane Yes Acetonitrile Yes Propanol Yes Methanol Yes Water Yes >

19 Modes of High Performance Liquid Chromatography Types of Compounds Separated Naturals Weak Acids Weak Bases Ionics, Bases, Acids Compounds insoluble in Water, Organic Isomers Ionics Inorganic ions Mode Stationary Phase Mobile Phase Reversed-Phase C-18, C-8, C-4, C-2 Water/Organic Phase Ion-Pair C-18, C-8 Water/Organic Ion-Pair Reagent Normal-Phase Silica, Amino, Cyano Diol Organics Ionics Ion Exchange Anion or Cation Aqueous/Buffer Inorganic ions Exchange Counter Ion Resin High MW Compounds Polymers Size Exclusion Silica Styrene Gel Filtration-Aqueous Gel Permeation-Organic Divinylbenzene

20 Useful Solvents for Reversed-phase Chromatography Reverse phase 에서이동상으로사용되는용매 - Water Elution Strength - Methanol - Acetonitrile - Isopropanol - Dioxane - Tetrahydrofuran - Mixed Solvents (from above)

21 Other applications by RPLC Separation of Weak Acids by RPLC Ion-Pair Reversed-Phase HPLC for Strong Acids and Bases

22 Ion Exchange Chromatography 이온친화력에의하여분리하며용매의농도에따라분리도가달라짐. Resin counter ion sample

23 Size Exclusion Chromatography Mechanism: Elution order is a function of molecular size Stationary Phases Cross-linked gels with defined Pore size Must be non-interactive with sample Mobile Phases Aqueous solvents - Gel Filtration Organic solvents - Gel Permeation

24 Instrumentation HPLC 시스템의구성 1. 탈기장치 (Degasser) 2. 펌프 (Pump) 3. 시료주입기 (Injector) 4. 컬럼 (Column) 5. 컬럼온도조절기 (Thermostatted Column Compartment) 6. 검출기 (Detector) Degasser Pump Injector Column Detector Data System

25 Instrumentation 탈기 (Degassing) 1. 목적 : 이동상중의용존산소, 질소, 기포등을제거 2. 방법 : He sparging Vacuum (Filtration) Ultra-sonication On-line Degassing

26 Instrumentation Pump Binary Pump Choice for high-throughput applications Quaternary Pump Maximum versatility, Choice for method development

27 Instrumentation Sample Injector Requirements: Reproducible introduction of the sample volume into the mobile phase flow. Automatic Injectors or Manual Injectors Manual Injector INJECT Position LOAD Position

28 Instrumentation Sample Injector-Automatic Injector Sampling unit Metering device From Pump Waste To Column 6-port valve Mainpass position Bypass position Automatic Injector Sample injection Drawing the sample

29 HPLC 컬럼의종류 재질에따른분류 Steel 컬럼 PEEK 컬럼 분리기작에따른분류 순상컬럼 역상컬럼 이온컬럼 크기배제컬럼

30 순상컬럼이란? Functional Group 의 Polarity 에의한분리 일반적으로 Non-aqua, Non-polar Solvent 사용 극성이큰물질이나중에용출됨. CH3 Si-OH Hexane Polar CH3 CH3 Si-OH OH interaction CH3 Silica phase Zorbax Rx-silica, Inertsil SIL-100A, Inertsil SIL-150A, Bonded phase( 극성도를줄여주는역할 ) Zorbax CN, Zorbax NH2, Lichrosorb CN, Lichrosorb NH2, Lichrosorb Diol, Inertsil NH2, Inertsil CN,Inertsil Diol

31 역상컬럼이란? Bonded compounds(c18, C8, CN, Ph) 와분석물질간의상호작용에의한분리 일반적으로 Polar Solvent 사용 비극성이큰물질이나중에용출됨. ACN/H2O Nonpolar CH3 O-Si-C18 CH3 interaction OH

32 Agilent Reverse Phase 컬럼선택 Reversed-Phase Chromatography NEW! 2006 Specialty products StableBond Diisopropyl Diisobutyl bonding Eclipse XDB Dimethyl bonding Double endcapping Eclipse Plus Dimethyl bonding Double endcapping Extend-C18 Bidentate-C18 Double endcapping SB-C18 Diisobutyl-C18 SB-CN Diisopropyl-CN Eclipse XDB-C18 Eclipse Plus C18 Eclipse Plus C8 Rx-C18 Dimethyloctadecylsilane Rx/SB-C8 Diisopropyl-C8 SB-Phenyl Diisopropyl- Phenyl SB-C3 Triisopropyl SB-AQ Diisopropyl Eclipse XDB-C8 Eclipse XDB- Phenyl Eclipse XDB-CN Eclipse Plus- Phenyl-Hexyl Eclipse PAH NEW! 2007 Bonus-RP Embedded amide Diisopropyl-C14 Triple endcapped

33 컬럼선택 1 - 용매에따른고정상선택 분자량크기 분자량 <2000 분자량 >2000 Hexane YES Silica Organic Sol NO NO MeOH YES CN, Ph, C8, C18 Water NO YES YES THF YES GPC column 수용성 GPC NO Ionic YES NR3, COO, SO3

34 컬럼선택 2 - 공극, 고정상, 입자크기, 내경결정 분자량 <2000 분자량 >2000 공극크기결정 (Pore Size) 80~120A 컬럼의 Pore Size는분석하려는분자의크기에따라결정됩니다. 300A 일반적으로크기가작은분자들은 80~120A 인컬럼에서는확산이잘됩니다. 그러나, 펩타이드나단백질의경우 300A 의컬럼을사용하시는것이좋습니다. Eclipse XDB-C18 결합상결정 (Colum Bonded Phase) 중간극성시료나, 비극성시료의머무름시간을최대화하기위해서 C18 컬럼으로시작합니다. 분리능이 C18Phase에서좋지않거나, 큰단백질을분석할경우 C18과같은더짧은탄소길이의컬럼을사용합니다. 300SB-C18 <5um 기본입자크기결정 (Particle Size ) 분자크기에상관없이일반적으로입자크기가 5um 인컬럼을사용합니다. 그러나, 입자크기가작을수록더우수한 efficiency 와 Resolution 을제공하며, 길이가짧은컬럼에서도이용될수있습니다. <5um 4.6mm 컬럼내경결정 (Column Diameter ) 컬럼직경은얼마나많은시료를투입하는가에따라결정됩니다. 직경이작을수록좋은 Peak 형태를유지하면서더적은시료들이투입되고, 이는 Sensitivity 를향상시키게되어,LC/MS 나미량시료분석에가능합니다. 4.6mm

35 컬럼선택 3 주입량에따른내경설정 Column Type 직경 (ID mm) 주입량 (Sample Load) 유속 (Typical Flow rate) Sensitivity 증가율 Applications Analytical ~1.5mg 0.5~3 ml/min + Standard separations Solvent Saver ~500ug 0.3~1.5mL/min ++ Narrow Bore ~120ug 0.1~0.5mL/min ++++ Save solvent, uses standard HPLC High sensitivity:limited sample solvent Micro Bore ~50ug 10~100 ul/min high sensitivity LC/MS Capillary 0.5/0.3 1~10ug 1~15 ul/min Nano 0.1/ ~200ng 200~500nL/min Semi-Prep 9.4 1~10mg 5~10 ml/min Preparative ~250mg 20~60 ml/min Very high sensitivity LC/MS peptides&protein Very high sensitivity LC/MS peptides&protein mg preparative separations Hundreds of milligrams up to 1gram

36 Instrumentation Thermosttated Column Compartment 역할 : 분리능향상및분석결과의재현성보장을위해 컬럼온도를적절하게설정, 유지한다.

37 Instrumentation Detector Detector Type Sensitivity Selectivity Useful % Advantages of compounds VWD ng/pg - 80 Low cost DAD ng/pg Peak confirmation Fluorescence pg/fg High sens. Electro- pg/fg + >20 High sens. chemical Conductivity ng/pg - 10 Ion Chrom. Refractive Index Mass Spectrometer ug/ng Universal response ng/pg ++ <100% MW,structural information

38 Instrumentation UV Detector Detector Cell I 0 I A = εbc I 0 = 입사광의세기 ε = 몰흡광도 I = 투과된빛의세기 b = 광로의길이 A= 흡광도 c = 시료농도 Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer s Law.

39 Instrumentation UV Detector Variable Wavelength type - 190~600 nm 사이의모든파장중에서원하는파장을지정하여사용한다 Deuterium lamp Cut-off filter Holmium oxide filter Automated wavelength verification Sample diode Flow cell Easily exchangeable Lens Grating Beam splitter Reference diode Slit Mirror 2 Mirror 1 Programmable wavelength switching

Instrumentation UV Detector Diode Array Type - 190-950 nm 의전파장을동시에 scan 한다.

40 Instrumentation UV Detector Diode Array Type nm 의전파장을동시에 scan 한다. - RT/Wavelength/Absorbance 에대한 3D spectrum 분석가능 More Automated uptimewavelength verification & Calibration Excellent wavelength resolution Extended Wavelength Range Long life deuterium lamp Programmable slit width control

41 Instrumentation Fluorescence Detector 제약 / 임상 : 혈액중 Quinidine 약과그대사물 생명과학 : 유도체화된아미노산 환경 : Polynuclear aromatic hydrocarbons(pah), carbamates 식품 : 비타민, Mycotoxin - Aflatoxin

$Instrumentation Refractive Index Detector 일반화합물의비선택적검출$

42 Instrumentation Refractive Index Detector 일반화합물의비선택적검출 Food analysis (e.g., sugars and lipids) Polymer Analysis (GPC)

43 HPLC in NOW!!! Customers want Conventional LC Ultra-fast LC Faster! More Information! & More Secure Analysis! 비용감소및생산성증가 분석 QC 와프로세스통제속도증가 LC/MS 와 LC/MS/MS 사용량증가 Combinational Chemistry 분야의요구 빠른분석방법개발 용매사용량감소 Category Conventional LC Fast LC Ultrafast LC Cycle times > 5min < 5min < 1 min Key Point! Maximizing Productivity and Assets with RRLC

Agilent 1200 Series RRLC System 5 Major Benefits 1. Speed 20X faster than conventional HPLC 2. Resolution 60% higher resolution than conventional HPLC 3.

44 Agilent 1200 Series RRLC System 5 Major Benefits 1. Speed 20X faster than conventional HPLC 2. Resolution 60% higher resolution than conventional HPLC 3. Highest Flexibility RRLC & standard methods, standard & narrow bore columns Broad range of RRHT columns Fast method transfer: HPLC to RRLC in 5 minutes 4. Highest throughput 2000 Samples/Day on HT-configuration 5. Best usability on proven industry design More uptime, serviceability LC diagnostics, Compliance services New level of instrument control and data handling

45 Agilent 1200 Series RRLC System 1. ZORBAX RRHT 1.8um Columns High Resolution analysis High Speed analysis High Temperture & Speed analysis high resolution, high speed 2. Rapid Resolution Hardware Capabilities Lower dispersion High Pressure limit Configurable delay volume Conventional & U-HPLC High Temperature Column Compartment High sensitive detector (Higher data rate, lower noise)

46 Van Deemter Curve : HETP vs. Volumetric Flow Rate HET TP (cm) Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mm Eluent: 85:15 ACN:Water Flow Rates: ml/min Temp: 20 C Sample: 1.0µL Octanophenone in Eluent 5.0µm 3.5µm 1.8µm H = A + B/u + Cu Volumetric Flow Rate (ml/min) Smaller particle sizes yield flatter curves, minima shift to higher flow rates

47 Van Deemter The Theory Behind For small molecules, smaller particles result in faster diffusion Result: faster chromatography Plate height H Theoretical The smaller the theoretical plate height H, the better the Resolution! H = A + B/u + C x u large particle small particle Resulting Van-Deemter curve Resistance to Mass Transfer H min Longitudinal Diffusion u opt linear flow u Multipath Term

48 When Ultra High Resolution is Needed: ZORBAX Rapid Resolution HT Columns Column Length (mm) Resolving Power N(5 µm) Resolving Power N(3.5 µm) Resolving Power N(1.8 µm) ,500 21,000 32, ,500 14,000 24, ,500 17, ,200 7,000 12, N.A. 4,200 6, N.A. 2,100 2,500 Efficiency (N) Pressure Analysis Time Peak Volume Solvent Usage Analysis Time (Rel. to 150mm) -33% -50% -67% -80% -90% Rapid Resolution HT columns provide the efficiency of longer columns used for high resolution.

49 RRLC for Ultra Fast Analysis

50 RRLC for Ultra Fast Analysis (cont d) 1/15 reduced

51 RRLC for High Resolution Analysis

52 RRLC for High Sensitivity Analysis mau Comparing Same Injection Volume: 4.6 x 150 mm, 5 µm vs. 4.6 x 50 mm, 1.8 µm RRHT A=406 Eclipse XDB-C x 150 mm, 5 µm 6 µl inj. A=761 A=1578 A= mau A=406 A=1580 A=758 A=1385 Eclipse XDB-C18 RRHT 4.6 x 50 mm, 1.8 µm 6 µl inj. Mobile phase: (85:15) MeOH: water Flow =1.0 ml/min. Temp. : ambient Sunscreens: 1. 2-hydroxy-4-methoxybenzophenone 2. Padimate-O 3. 2-ethylhexyl trans-4-methoxycinnamate 4. 2-ethylhexyl salicylate min min

53 1200 Series Rapid Resolution System - Efficient use of pressure mau 700 Better performance at lower pressure than other U-HPLC designs Column 1200 Rapid Resolution RRHT, SB-C18, 2.1 x 50mm, 1.8um Suppler B A BEH, 2.1 x 50mm,1.7um Anal. Time min min Resolution Precision %RSD %RSD Pressure 440 bar 700 bar (+60%) min Sample: Phenones Test Mix Flow Rate: 1 ml/min Gradient: 35-95% ACN in 0.9 min Temperature: 50 Injection volume: 1 µl Injection Technique: ADVR, OI, MCO WL: 245 nm Data Rate: 80 Hz

54 High Speed and High Resolution Liquid Chromatography Important aspects Flow rate (linear velocity) Pressure limits Delay/dwell volumes Extra column volumes Detector data rates

Microsoft PowerPoint - d ppt [호환 모드]

Microsoft PowerPoint - d ppt [호환 모드] HPLC BASICS Chromatography 란? 혼합된시료성분이이동상과고정상사이를흐르면서흡착, 분배, 이온교환또는분자크기배재작용등에의해각각의단일성분으로분리되는것을말한다. 분리, 정성, 정량등의분석목적과분리, 정제, 분취목적에이용된다. Injection Interaction Elution a abacbc aabcbc HPLC System Mobile Phase