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1 HPLC 기초개론

2 Contents Ⅰ. HPLC 기초이론 Ⅱ. HPLC 시스템의구성 Ⅲ. 분리모드에따른컬럼선택 Ⅳ. 이동상용매 ( 용매의선택및준비 )

3 Ⅰ. HPLC 기초이론

4 Chromos (colour) + graphein (write) : in Greek Chromatography System / Instrument Chromatograph Chemical separation technique Chromatogram Electronic results of of chromatographic separation

5 Chromatography 혼합된시료성분이이동상과고정상사이를흐르면서흡착, 분배, 이온교환 또는분자크기배제작용등에의해각각의단일성분으로분리되는것을말한다. 분리, 정성, 정량등의분석목적과분리, 정제, 분취목적에이용된다. Injection Interaction Elution abacbc ccbbaa flow a b c 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

6 History of Chromatography

7 Separations Separation 은고정상과이동상사이에서의이동의차이에의해서이루어진다.

8 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

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

10 Comparison of HPLC and GC

11 Comparison of HPLC and GC HPLC GC Volatility - 휘발성이요구되지않음 - 샘플은이동상에서용해성을가져야함휘발성 Polarity 극성및비극성화합물의분리비극성및극성화합물 ThermalLability 분석은상온에서일어남고온의주입구및컬럼에서열적안정성을가져야함 MolecularWeight Preparation 이론적인한계치없음 ( 실제적으로, 용해성에제한됨 ) 샘플은필터해야하고, 이동상과같은용매에녹아야함 분석가능 Mw 의범위가좁다 : < 500 amu 용매는휘발성을가져야하며, 일반적으로분석대상물질보다 B.P 가낮아야함. Size 샘플량은컬럼의내경에따름일반적으로 1~5 ul Separation Mechanism 고정상과이동상사이에서일어남. - 이동상은단지샘플을이동시키는역할 - 고정상과샘플의친화력 Detectors 일반적으로 UV-VIS 검출기사용일반적으로 FID 를비롯하여, 대부분의유기물분석

12 Modes of High Performance Liquid Chromatography 흡착 (Adsorption) : Normal phase 분배 (Partition) : Reverse phase, Normal phase 이온교환 (Ion exchange) : Ion Exchange 분자체 (Size exclusion) : Gel Permeation Chromatography(GPC) Gel Filtration Chromatography(GFC)

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

14 Normal Phase Liquid Chromatography Functional groups의 polarity에의한분리작용 일반적으로비수용성, 비극성용매를사용하며극성이낮은시료부터용리 고정상으로사용되는고체흡착제의표면과이동상으로사용되는액체에대한친화력의차이가분리를좌우 Si-OH Hexane polar O 극성낮은시료 극성컬럼 Si-OH--OH 극성높은시료

15 Normal Phase Liquid Chromatography Columns Silica(Adsorption) Cyano(Bonded Phases) Amino Diol Mobile Phase Organic Solvents Good For Mixtures of structural isomer compounds not readily soluble in organic/water mobile phases

16 Normal Phase Liquid Chromatography separation principles

17 Reverse Phase Liquid Chromatography 가장일반적인 chromatography technology 고정상으로사용되는고체지지체의표면액상과이동상으로사용되는액체에대한분배력의차이에따라분리되는원리 Bonded phase의개발로고정상에대한이동상의조성변화, 이동상변형제첨가등다양한응용상의변화가가능하게됨으로써널리사용하게됨.

18 Reverse Phase Liquid Chromatography Columns C8-Octyl Silyl C18-Octadecyl Silyl Cyanopropyl Aminopropyl Polymer-based Mobile Phases water with: Methanol Acetonitrile Isopropanol Tetrahydrofuran Partitioning mechanism related to analyte solubility in mobile & stationary phase Less water soluble more retention As the number of carbon atoms increases more retention Unsaturation decreases retention 용출순서 Polar Non-polar compound

19 Reverse Phase Liquid Chromatography Bonded compound (C18, C8, CN, Ph) 와 분석물질간의상호작용에의한분리 고정상은다공성지지체에화학적으로결합되어있음 일반적으로극성용매사용하며비극성이큰물질이나중에용리됨 Acetonitrile (CH3CN-H2O) non-polar -Si-O-Si-C18 - OH

20 Reverse Phase Liquid Chromatography Predict the Elution Order 1. n-hexanoic acid 2. propionate 3. n- butyric acid 4. valeric acid benzene 2. benzoic acid 3. toluene 4. phenol

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

22 Increase the Organic Modifier Content to Decreases Retention

23 Ion Exchange Chromatography 이온화도차이에의한분리 이온성화합물및무기이온의분리에사용 Electrostatic force 이용 Polystyren or Silica gel 표면에이온성을가진화합물을화학적으로결합한고정상사용 이동상으로완충액 (Buffer solution) 사용 시료의분리는 ph, 이온세기, 이동상에포함된이온종류의영향을받음

24 Ion Exchange Chromatography 이온친화력에의하여분리하며용매의농도에따라분리도가달라짐. Resin counter ion sample

25 Ion Exchange Chromatography

26 Separation of Weak Acids

27 Ion-Pair Reversed-Phase HPLC for Strong Acids and Bases

28 Buffer Solution 의사용목적 이온성시료일때 이온억제및이온쌍형성 생리활성물질의순수분리, 정제시 ph 조절 이온성물질분리시 이온강도조절

29 Size Exclusion Chromatography Mechanism: Elution order is a function of molecular size Stationary Phases Cross-linked gels with defined pore size Be certainly non-interactive with sample Mobile Phases Aqueous solvents - Gel Filtration Organic solvents - Gel Permeation

30 Size Exclusion Chromatography 충전제 : Styrene, divinylbenzene 일정크기의 pore를가진충전물을사용 분리원리 - 물리적인분리방법 - 경로차에의한분리 - 큰분자가먼저용출되고작은분자가나중에용출됨 응용 : 고분자의분자량및분자량분포도를구함

31 Molecular Weight Determination by Size Exclusion

32 Separation Parameter 컬럼고정상의선택 컬럼길이와직경 이동상조성 온도 유속 샘플량

33 Chromatography parameter t2 R t 2 R K = t 1R t0 t1 R t α = t 2 R t 1 R R = 2 t W1 +W2 t0 t 1 R Inject W1 W2

34 The Goal of separation Resolution between sample components tr(b) t W t = = 2/(W A + W A ) 2 t W A + W A tr(a) t t0 t R(B) WA WB 분리도는두피크의분리된정도를간단히표시해주는값 Baseline 에서분리되는경우 R 값은 1.5 피크간머무름시간차이가클수록, 띠폭의평균값이작을수록 R 값증가

35 Factors Influencing Resolution R = α-1 α (N) K 1+K Selectivity Efficiency Capacity

36 Chromatographic Parameter : Selectivity Factor t2 R t1 R t 2 R α = t t 2 R t 0 t 1 R t 0 = t 2 R t 1 R t0 t 1 R Inject W1 W2

37 Selectivity Factors : 컬럼에서용출되는각성분의분리의차이 t 2 R t 1 R 선택성을바꾸기위해서는.. 이동상조성의변화 다른이동상종류선택 이동상의 ph 변화 컬럼온도변화 이동상첨가제첨가 고정상의변화 a > 1.2 : 분리능이있다. a = 1 : 분리능이없다.

38 Parameters which will affect selectivity 용매세기가센이동상은분석물질을컬럼에서빨리용출시킴 용매세기가약한이동상은분석물질의분리를향상시킬수있으나, 분석물질은컬럼에서오래머무르게됨.

39 Chromatographic Parameter : Capacity Factor t2 R t 2 R t1 R t K = t 1R t0 t0 t 1 R Inject W1 W2

40 Capacity Factor 용량인자 (R) 은이동상조성, 온도및컬럼의종류에따른특정성분의특성임. = t 1 R t0 K 은시료의머무름을나타내는지표 ( 시료와고정상이얼마나강하게상호작용하는가를나타냄 ) 단일용매를이용한분석인경우 K 은 2~10, 보통 2~6 이최적값

41 Capacity Factor 피크의용량인자를변화할수있는가장좋은방법은이동상조성의변화 이동상의세기를증가시키면용리액의 용량인자는감소함. 역상에서, 유기용매의 10% 증가는피크의 용량인자를 2 배혹은 3 배감소시킨다.

42 Chromatographic Parameter : Efficiency

43 Calculating Efficiency N = 16( t R ) 2 Wb = t R W 1/2 HETP = L/N N(Theoretical plate) 은이론단수라함. 컬럼의효율을의미함 HETP(the Height Equivalent to a Theoretical Plate) - 이론단해당높이. 시료분자가이동상과고정상사이에서평형에도달하는데필요한컬럼의길이임. - 낮은값을가질수록컬럼효율높음

44 Ⅱ. HPLC System 의구성

45 HPLC System 의구성 1. 탈기장치 (Degasser) 2. 펌프 (Pump) 3. 시료주입기 (Injector) 4. 컬럼 (Column) 5. 컬럼온도조절기 (TCC: Thermostatted Column Compartment) 6. 검출기 (Detector) Degasser Pump TCC TCC Injector Column Detector Data System

46 탈기 (Degassing) 1. 목적 : 이동상중의용존산소, 질소, 기포등을제거 2. 방법 : He sparging Vacuum (Filtration) Ultra-sonication On-line Degassing Vacuum Chamber A B C D Control Assembly Solenoid Valve Vacuum pump

47 On-line Degasser 의원리 Solvent 가 membrane tube 를지나면서 gas 는제거하고, liquid 는통과시키면서일정한진공상태를유지함

48 Pump 1. 역할 : 이동상을이동상저장용기에서끌어내어시료주입기로연속적으로밀어주는역할 2. 요건 : - 일정한유속과압력을유지할것 - 다양한용매를사용할수있을것 3. Isocratic 및 Gradient -Isocratic: 분석시간동안이동상조성의변화없음 -Gradient: 분석시간동안이동상조성이시간의흐름에따라변함

49 Isocratic pump Damper Outlet valve Purge valve To ALS and Column Active Inlet valve Waste QA/QC 의응용에맞는설계

50 Binary pump Purge valve Mixer Damper Inlet valve Waste Outlet valve Outlet valve Inlet valve High-pressure 혼합원리에의해설계

51 Quaternary pump Vacuum chamber Inlet valve Damper Solvent bottles Purge valve Proportioning valve Waste 용매혼합의최고의유연성을제공

52 Injector 1. 역할 : 분석하고자하는시료를용매의흐름에실어준다. 2. 종류 수동 (Manual Injection Valve) 자동 (Auto Injector)

53 Manual Injector

54 Autosampler Mechanical Design 100 x 2ml vials tray Metering device Sample loop 40 x 2ml vials tray Robotic arm with vial gripper Sample tray Injection valve 15 x 6ml vials tray

55 Auto Injector Standard loop volume : 300 µl Total delay volume : 300 µl + V inj Minimal (bypass) delay volume : 6.2 µl Vial gripper Sampling unit Metering device 6-port valve From pump To column To waste Widest dynamic injection range : 0.1 µl ~ 1.8 ml

56 Auto Injector

57 컬럼온도조절기 (Thermostatted Column Compartment) 역할 : 분리능향상및분석결과의 재현성보장을위해컬럼온도를 적절하게설정, 유지한다.

58 컬럼온도조절기 (Thermostatted Column Compartment)

59 Detector 의역할 역할 : 컬럼에서분리된시료가일정한간격으로 검출기를통과할때시료의존재및양을일정한규칙에의해인식하여전기적인신호로바꾸어준다.

60 Detector 의종류 UV/vis Detector 다파장검출기 (Multi Wavelength Detector) 가변파장검출기 (Variable Wavelength Detector) 다이오드어레이검출기 (Diode-Array Detector) 형광검출기 (Fluorescence Detector) 굴절률검출기 (Refractive Detector) 전기화학검출기 (Electrochemical Detector) 전도도검출기 (Conductivity Detector) Agilent HPLC Detector

61 HPLC Detector 의특징 Useful % Detector Type SensitivitySelectivity of compounds Advantages VWD ng/pg - 80 Low cost DAD ng/pg Fluorescence pg/fg Peak confirmation High sens. Electrochemical pg/fg + >20 High sens. Conductivity Refractive Index Mass Spectrometer ng/pg - 10 ug/ng ng/pg ++ <100% Ion Chrom. Universal response MW,structural information

62 UV/VIS Detector (VWD, MWD, DAD) 검출원리 Detector Cell I 0 I I 0 = 입사광의세기 ε = 몰흡광도 I = 투과된빛의세기 b = 광로의길이 A= 흡광도 c = 시료농도 photodiode 에도달하는특정파장의빛의양과시료의농도사이의 관계를나타내면, Log I 0 /I = A = εbc

63 대표적인발색단 Chromophore Structure λmax(nm) Amine -NH Ethylene -C=C- 190 Ketone =C=O 195 Ester -COOR 205 Aldehyde -CHO 210 Carboxyl -COOH Nitro -NO Phenyl 202, 255 Naphthyl 220, 275

64 UV/VIS Detector 종류와구조 Variable Wavelength Detector (VWD) Multi Wavelength Detector (MWD) Diode Array Detector (DAD)

65 UV/VIS Detector 종류와구조 Variable Wavelength Detection(VWD) type - 190~600 nm 사이의모든파장중에서원하는파장을지정하여사용한다.

66 UV/VIS Detector 종류와구조 Diode Array Detection(DAD) type - 190~950 nm 의전파장을동시에 scan 한다. More uptime More Uptime Automated wavelength verification & Calibration Excellent wavelength resolution Extended Wavelength Range Long life deuterium lamp Fast Optimization of sensitivity and resolution

67 Optical Design: Conventional vs. Diode-array Detector Detector Source Entrance slit DAD Entrance slit Exit slit Source Concave grating Flow cell Flow cell Concave grating Conventional Scanning Detector Diode-array Detector

68 DAD 장점 신속한전파장스펙트럼확인 다파장검출 탁월한파장재현성 기기의신뢰성 Fast scan

69 UV/VIS Detector 응용분야 Drugs : Amide(Quinidine) Foods : Fatty Acid Flavor : Flavone Peptide & Protein Vitamins : Retinol

70 Fluorescence Detector(FLD) 검출원리 원리 - Excitation filter를통과한빛은 sample cell에존재하는시료분자를들뜬상태로만들어주고바닥상태로떨어지면서흡수한파장보다긴파장의빛을방출함 - 빛의발광량 : 시료농도에비례 - 시료 : 분자구조가형광을띠거나형광유도체를만들었을때검출가능 형광물질 - Benzene 고리치환물중작용기가 -OH, -OCH 3, -H 2, -F, -NH(CH 3 ), -N(CH 3 ) 2, -CO 등인화합물

71 Fluorescence Detector 구조

72 Fluorescence Detector 응용분야 제약 / 임상 : 혈액중 Quinidine과그대사물 생명과학 : 유도체화된아미노산 환경 : Polynuclear aromatic hydrocarbons(pah), carbamates 식품 : 비타민, Mycotoxin - Aflatoxin

73 Refractive Index Detector(RID) 검출원리 원리 - 시료의농도변화에따른굴절률을측정 - Cell은 sample cell과 reference cell로구성되며 reference cell 은이동상으로채워지고분석시간동안이동상은 sample cell만을통과하면서흐름 응용범위 - 굴절률에의하여분석하므로거의모든물질에사용할수있으나주로고분자, 당등의분석에이용 - 일반적으로 UV/ Visible, Conductivity 검출이어려운시료에이용

$Refractive Index Detector 구조 1. Flow in 2. Heater 3. Heater exchanger 4.$

74 Refractive Index Detector 구조 1. Flow in 2. Heater 3. Heater exchanger 4. Sample cell 5. Purge valve 6. Recycle valve 7. Waste container 8. Reference cell 9. Solvent bottle

75 Optical Unit Design : RID Tungsten lamp Diodes Zero glass Heat exchangers Flow cells Mirror

76 Refractive Index Detector 응용분야 일반화합물의비선택적검출 식품분석 ( 예 : 당, 지방등 ) 폴리머분석 (GPC)

77 Ⅲ. 분리모드에따른컬럼선택

78 Columns 구성 : - 관모양의용기에충전제를채워서사용 - 분석하고자하는시료의종류에따라컬럼의크기및충전제의종류를선택하여사용 역할 : 혼합상태의시료를화학적또는물리적특성에따른 머무름정도의차이에의해분리

79 HPLC Column 은? 여러가지물질을각각의성분으로분리시키는곳 입자크기 재질 1.8 μm, 3.5 μm, 4 μm, 5 μm, 6 μm, 7 μm, 10 μm, 37 μm, 55 μm, 75 μm Silica, Alumina, Silica-Bonded, Polymer(resin)- Bonded 형태 : Spherical, irregular Pore 크기 : 50 A ~ 300 A

80 컬럼선택시고려사항 화학적요소 ( 선택인자 (a), 용량인자 (k)) 충전제의표면성질 이동상의조성 물리적요소 ( 이론단수 (N)) 충전용기의내경, 길이, 재질 충전제의입자크기 충전제의분포상태

81 HPLC 에사용되는컬럼의내경 Type 컬럼내경 (mm) 길이 (cm) 유속 (ml/min) 일반컬럼 (Conventional) 고속분리용 (High Speed) Microbore Microbore Capillary (Packed)

82 컬럼충전제의종류 Base Material Examples/Varieties 사용비율 Silica gel Polymer Alumina Others High Surface area, wide pore Polymethacrylates PS-DVB Polyethyleneglycol Polydextrans Neutral Acidic Graphitized Carbon, Zirconia 75% 20% 2% 3%

83 Silica Column 의구조

84 Column

85 역상크로마토그래피 고정상 ( 비극성 ) 이동상 ( 극성 ) 대상시료 C18, C8, C4, phenyl -물 -물과섞이는이동상, 변형제 중성, 약산및염기성용질등 ZORBAX C18(ODS) ZORBAX C8 ZORBAX phenyl

86 역상크로마토그래피의장점 이동상에완충염 (Buffer Salts), 이온쌍시약과같은다양한첨가제를적절히추가함으로써비이온성, 이온성, 또는이온화가능한시료를분리할수있음 소수성결합상컬럼 (Hydrophobic Bonded Phase Column) 은재현성이좋으며, 안정함 대부분의유기화합물분석이가능 수용성용매에녹아있는시료는직접주입가능 분리하고자하는시료분자의소수성정도에따라용리순서의예측이용이

87 순상크로마토그래피 고정상 ( 극성 ) 이동상 ( 비극성 ) 대상시료 -흡착 : Silica Gel, Alumina - 결합상 : Amino, Cyano, Diol - Hexane, Methylene chloride, Organic -Steroid -Aflatoxin - Vitamin, fat-soluble ZORBAX Rx-SIL ZORBAX CN ZORBAX NH 2

88 이온교환크로마토그래피 고정상 ( 극성 ) 이동상 ( 비극성 ) 대상시료 - 음이온또는양이온 교환수지 - Aqueous/ Buffer Counter Ion - 이온성화합물및 무기이온류 ZORBAX SAX ZORBAX SCX ZORBAX WAX

89 크기배제크로마토그래피 겔침투 (Gel Permeation) 겔여과 (Gel Filtration) 고정상 : 다공성 Polystyrene, Silica Gel 이동상 : 지용성 응용 : 고분자화학 Alkyds Epoxies Natural Rubber Polystyrenes Silicones Polyvinylchlorides 이동상 : 수용성 응용 : 생물약품화학 Peptides Structural proteins Enzymes Lipids Nucleic acids

90 HPLC Column Selection

91 컬럼의취급과보관법 Wash the column after use with selected solvents; Flush highly retained sample components from the column. Do not store the column in 100% water. Microbes may grow and clog the column. Don t open the column and repack the material if you want to maintain performance. Use the column at its optimal flow rate - avoid high flow rate. Do not operate silica or bonded phases for extended periods at high temperature. Keep the ph of the mobile phase in an appropriate range for the column.

92 Ⅳ. 이동상용매 ( 용매의선택및준비 )

93 용매조건 모든이동상용매는 HPLC grade를사용 초순수 (18 MΩ이상 ) 을사용 측정하고자하는파장보다낮은 UV cutoff를가진용매사용 용매간섞임성 (miscibility No. 차 15미만 ) 분리하고자하는시료를녹일수있어야함 모든용매는반드시여과및탈기하여사용 낮은점도 이동상은고정상을변화시키면안됨

94 UV Cutoff of Common Solvents Solvent UV Cutoff(nm) Solvent UV Cutoff(nm) Water 180 N-Heptane 197 Methanol 210 Cyclohexane 200 N-Propanol 205 CCl Acetonitrile 190 Chloroform 245 THF 230 Benzene 280 Acetone 330 Toluene 235 Methyl acetate 260 Methylene chloride 232 Ethyl acetate 260 Tetrachloro-ethylene 280 Nitromethane 380 1,2-Dichloroethane 225

95 Solvent Miscibility Acetic acid Acetone Acetonitrile Benzene t-butylmethyl ether Carbon tetrachloride Chloroform Cyclohexane Cyclopentane 1,2-dichloromethane Dichloromethane Diethyl ether Diisopropyl ether Dimethyl sulfoxide dioxane Ethyl acetate ethanol hexane Isooctane Isopropanol Methanol Pentane Tetrahydrofuran Toluene Water Xylene Acetic acid Acetone Acetonitrile Benzene t-butylmethyl ether Carbon tetrachloride Chloroform Cyclohexane Cyclopentane 1,2-dichloromethane Dichloromethane Diethyl ether Diisopropyl ether Dimethyl sulfoxide dioxane Ethyl acetate immiscible immisible ethanol hexane Isooctane Isopropanol Methanol Pentane Tetrahydrofuran Toluene Water Xylene

96 이동상제조 (I) 용매준비 - HPLC grade의용매 - 초순수 ( 비저항값 ;18 MΩ ) -계량시부피비로계량후혼합 - 용매성질에따라흡열, 발열반응가능 - 시일경과시혼합용매의부피비가달라질수있다.

97 이동상제조 (II) Filter 수용성필터 (PVDF 재질 ) 물로활성화한후사용 지용성필터 (PTFE 재질 ) MeOH/ 적합한용매로활성화한후사용

98 이동상제조 (III) 탈기이동상에녹아있는공기, 산소, 기포제거 Vacuum Degassing( 진공탈기 ) 여과 + 탈기 Helium Sparging 직접이동상에 sparging Ultrasonication( 초음파파쇄 ) Online Degasser

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