1

Part I – Basic Principles of HPLCPart I – Basic Principles of HPLC

Chromatography and HPLCVarious HPLC modes and applications

Normal phaseReversed phaseReversed phase ion pairing Ion exchange (IC)SEC (GPC/GFC)Chiral separation

HPLC system modes Isocratic elution Gradient elution

2

What is chromatography?What is chromatography?

Chromatography is one of the separation technique.

The main purpose of chromatography is

to separateseparate and quantifyquantify the target sample in the matrix.

3

Why mixed sample Why mixed sample can be separated? can be separated?

river bed

direction of flow

4

What is the difference?What is the difference?

Interaction is different by gravity

StrongWeak

5

How can the separation How can the separation be carried out?be carried out?

Separation can be carried out by the column.

packing material, 3-5um

column

6

Interaction between packing Interaction between packing material and samplematerial and sample

Due to difference interaction between packing material and sample, separation can be done.

packing material

sample A

sample B

7

colum

n

mixed sample

Separation can be done Separation can be done by the columnby the column

8

What kind of chromatography?What kind of chromatography?

High Performance Liquid chromatography (HPLC)

Gas Chromatography (GC)Thin-Layer Chromatography (TLC)Capillary Electrophoresis(CE)

9

Advantage of ChromatographyAdvantage of Chromatography

Simultaneous Analysis

High Resolution

High Sensitivity (ppm-ppb)

Small Injection Volume (1-100uL)

10

Advantage of HPLCAdvantage of HPLC

Moderate analysis condition - no need to vaporize the sample like

GCEasy to fractionate the sample

and purifyGood repeatability (C.V. < 1%)

11

Flow Diagram of HPLCFlow Diagram of HPLC

pumpinjector

column

ovendetector

12

Separation modes of HPLCSeparation modes of HPLC

Normal Phase mode (NP)Reversed Phase mode (RP)Reversed Phase Ion Pairing mode (IPC)Ion Exchange mode (IC)SEC mode ( GPC / GFC ) Chiral separation mode

13

Normal Phase ModeNormal Phase Mode

M. Tswett :M. Tswett :first developer of chromatography

Ber. Deut. Botan. Ges., 24, 384 (1906)Adsorptionsanalyse und chromatographischechromatographische Methode. Anwendung auf die Chemie des Chlorophylls.

14

Normal Phase ModeNormal Phase Mode

Petroleum etherPetroleum ether

CaCOCaCO33

Chlorophyll'sChlorophyll's

ChromatoChromatograph

ColorsColors

15

Normal Phase ModeNormal Phase Mode

First developer used CaCO3 as Separation Column

Petroleum ether as Developed Solvent

We defined this combination as

Normal Phase mode Normal Phase mode ColumnColumn :: polar propertypolar property

SolventSolvent : : non polar propertynon polar property

16

Normal Phase HPLC ColumnsNormal Phase HPLC Columns

Silica gel type : general useCyano type : general useAmino type : for sugar analysisDiol type : for protein analysis

Silica gel

SiSi

-Si-CH2CH2CH2CN-Si-CH2CH2CH2NH2

-Si-CH2CH2CH2OCH(OH)-CH2(OH)

Modified Si

17

What is the interaction?What is the interaction?

Silica gel (polar)

Hydrogen bondingHydrogen bondingNon-polar

18

Hydrogen BondingHydrogen Bonding

Compounds contain -COOH : Carboxyl group -NH2 : Amino group -OH : Hydroxyl group

Hydrogen bonding – strong interactionHydrogen bonding – strong interaction

Note: Compounds with bulk group, hydrogen bonding may not form due to stereo hindrance

Compounds do not contain these groups

No hydrogen bonding – weak interactionNo hydrogen bonding – weak interaction

19

Retention Time and Retention Time and Hydrogen bondingHydrogen bonding

OH

HO

SiOH

SiOH

StrongStrong

WeakWeak

or

Very WeakVery Weak

20

Mobile phase solvents for normal Mobile phase solvents for normal phase HPLCphase HPLC

Primary solvents (non-polar) Hydrocarbons (Pentane, Hexane, Heptane, Octane) Aromatic Hydrocarbons (Benzene, Toluene, Xylene) Methylene chloride Chloroform Carbon tetrachloride

Secondary solvents Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF), Dioxane,

Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol

A primary solvent is used as mobile phase. Addition of secondary solvents is to to adjust retention time.

Solvents which possess UV transparency are often preferred for easy detection.

21

Increase of solvent polarityIncrease of solvent polarity

0 % 2 % 5% / MeOH 0 % 2 % 5% / MeOH

1 : Dioctyl phthalate2 : Dibutyl phthalate3 : Diethyl phthalate4 : Dimethyl phthalate

Solvent : Hexane

22

Column : Non-polar property

Solvent : Polar property

- Normal Phase mode - Column : polar property

Solvent : non polar property

Reversed Phase modeReversed Phase mode

23

Reversed Phase HPLC ColumnsReversed Phase HPLC Columns

C18 (ODS) type C8 (octyl) typeC4 (butyl) typePhenyl type TMS typeCyano type

-Si-C18H37

Si

Non-polar propertyNon-polar property

24

What is the interaction?What is the interaction?

HydrophobicHydrophobic interactioninteraction

Non-polar

polar solvent

25

HydrophobicityHydrophobicity

If the sample has more CH3CH2CH2--- : Carbon chain

: Aromatic group

If the sample has more -COOH : Carboxyl group -NH2 : Amino group

-OH : Hydroxyl group

Hydrophobicity Hydrophobicity is stronger.is stronger.

Hydrophobicity Hydrophobicity is weaker.is weaker.

26

Retention Time and HydrophobiciRetention Time and Hydrophobicityty

OH

OH

C18 (ODS)

Strong

Weak

27

Mobile phase solvents for Mobile phase solvents for reversed phase HPLCreversed phase HPLC

Water (buffer) + Organic solvents When buffer is used, the concentration and pH are imp

ortant factors Methanol (MeOH), acetonitrile (A CN) or THF are co

mmon organic solvents for r.p HPLC.

Optimization of water (buffer) and organic solvents’ ratio is very important.

28

Increase of solvent polarityIncrease of solvent polarity

20 % 30 % 40% / H2O20 % 30 % 40% / H2O

1 : p-Hydoxymethylbenzoate2 : p-Hydoxyethylbenzoate3 : p-Hydoxypropylbenzoate4 : p-Hydoxybutylbenzoate

Solvent : MeOH

29

Effect of stationary phaseEffect of stationary phase

C18 (ODS)

Strong

C8

sample

sample

sample

C4

Medium

Weak

30

Effect of stationary phaseEffect of stationary phase

Analytical Conditions Column : Shim-pack CLC-ODS Mobile phase : MeOH : H2O = 7 :3 Flow rate : 1.0 mL/min Temperature : 40 C Injection volume : 10 uL Detection : UV-254 nm

Peaks

1. Methyl benzoate

2. Ethyl benzoate

3. n-Propyl benzoate

4. n- Butyl benzoate

ODS C8 TMS ODS C8 TMS

31

Normal Phasegood separation for stereo isomers

(Vitamin E etc..)changeable retention time

Reversed Phasegood repeatable retention time rugged stationary phase

Normal vs Reversed PhaseNormal vs Reversed Phase

32

Reversed Phase Reversed Phase Ion-Pair ChromatographyIon-Pair Chromatography

Ion-Pair Reagent

33

Ion-Pair ReagentsIon-Pair Reagents

Anion CompoundsTetra-n-butylammonium hydroxide (TBA)

Cation CompoundsButanesulfonic acid sodium salt (C4)Pentanesulfonic acid sodium salt (C5)Hexanesulfonic acid sodium salt (C6)Heptanesulfonic acid sodium salt (C7)Octanesulfonic acid sodium salt (C8)Decanesulfonic acid sodium salt (C10)

34

Ion PairingIon PairingSeparation of Carboxylic AcidsSeparation of Carboxylic Acids

Column: Bonded C18

Mobile Phase: H2O/MeOH1:1 with Tetrabutyl-Ammonium Hydroxide

35

Ion PairingIon PairingSeparation of Amino CompoundsSeparation of Amino Compounds

Analytical Conditions Column : Shim-pack CLC-ODS Mobile phase : [A] : [B] = 9 : 1 [A]

100 mM phosphate buffer (pH=2.1)

0.8 mM sodium octane sulfonate [B] acetonitrile Flow rate : 1.5 mL/min Temperature : 40 C Injection volume : 10 uL

Peaks1. Nicotinic acid 6. Thiamine

2. Nicotinamide 7. Caffeine

3. Pantothenate 8. Folic acid

4. Pyridoxine 9. Biotin

5. Riboflavin 10. Riboflavin

Phosphate

36

Ion Paring Ion Paring Important ConsiderationsImportant Considerations

Type of Ion-Pair reagentsConcentration of Ion-Pair reagentspH of solvent

R-COOH RCOO- + H+

(pKa=4.5)

R-NH2 + H+ R-NH3+

(pKa=6.0)

37

Type of Ion-Pair ReagentsType of Ion-Pair Reagents

Mobile Phase: H2O/MeOH1:1,with 0.005M ion pairingreagent and 1% HOAc

1 Maleic Acid2 Phenylephrine3 Phenylpropanolamine4 Naphazoline5 Phenacetin6 Pyrilamine

Hexane Sulfonate Pentane Sulfonate

38

Concentration of Concentration of Ion-Pair ReagentsIon-Pair Reagents

39

Washing column in case of TBAWashing column in case of TBA

If amine modifier or TBA ion pair reagent is used as a mobile phase, the column must be washed when analysis is finished.

In order to remove these amine modifier or TBA ion pair reagent, sodium perchlorate is effectively working. Because sodium perchlorate has strong affinity to amine compounds.

Following solution may be recommended for column washing 0.1% phosphoric acid including 100 mM sodium perchl

orate : methanol = 1 : 1

40

Ion Exchange ModeIon Exchange Mode

N+

R

R

R

Sample

SO3- Sample

+

++

++

++

+

++++

+

Ion Attraction Force

41

Ion Exchange ModeIon Exchange Mode

Biological Fields (protein, peptide, amino acid analysis)Ion chromatography

Cation ExchangersStrong Cation Exchange (SCX) (R-SO3

-)Weak Cation Exchange (WCX) (R-COO-)

Anion ExchangersStrong Anion Exchange (SAX) (R4N

+)Weak Anion Exchange (WAX) (DEAE)

42

Protein Analysis Protein Analysis using WCX columnusing WCX column

Analytical ConditionsColumn : Shim-pack WCX-1Mobile phase :

[A] 20 mM phosphate buffer (pH=6.0)

[B] A+0.25M sodium sulfate

[A] - [B] 30 min linear gradient Flow rate : 1.0 mL/minTemperature : ambientDetector : UV-280 nmInjection volume : 10 uL

Peaks

1. albumin

2. myoglobin

3. -chymotrypsinogen A

4. liponuclease A

5. lisozyme

43

Ion Exchange modeIon Exchange modeImportant ConsiderationsImportant Considerations

pH of Buffer solutionConcentration of Buffer solutionElution Method

Isocratic elutionpH gradient elution increasing Ionic strength gradient

44

What is SEC ?What is SEC ?

Size Exclusion Chromatography (SEC)Size Exclusion Chromatography (SEC)GPC (Gel Permeation chromatography)

mainly polymer science fieldGFC (Gel Filtration Chromatography)

mainly biological science field

45

Principle of SECPrinciple of SEC

No Interaction ForceNo Interaction ForceDifference of Traveling TimeDifference of Traveling Time

46

Elution orderElution order

SECColumn

47

Purpose of GPC / GFCPurpose of GPC / GFC

GPCMolecular WeightMolecular Weight measurement of

polymerGFC

SeparationSeparation of protein

48

Relationship between Relationship between MW and RTMW and RT

Mol

ecul

ar W

eigh

t (L

ogM

W)

Exclusion limit

Permeation limit

Time

49

Calibration CurveCalibration Curve

Inject the standard polymer sample one by one to know the relationship between molecular weight and retention time.

No. time(min) mol. wet.1 22.0 55000002 22.6 18000003 23.4 8600004 25.0 4000005 27.4 1600006 31.0 500007 33.8 200008 38.4 40009 39.8 2000

10 42.8 60011 46.8 80

50

Actual Sample InjectionActual Sample Injection

To calculate the MW,

GPC software is required.

51

Protein Separation Protein Separation using GFC columnusing GFC column

Analytical ConditionsColumn : Asahipak GFA-50Mobile phase :

0.1 M sodium phosphate

0.1 M NaCl (pH=7.0)Flow rate : 0.5 mL/minTemperature : ambientDetector : UV-280 nmInjection volume : 10 uL

Peaks

1. glutamate dehydrogenase

2. lactate dehydrogenase

3. enolase

4. adenylate kinase

5. cytochrome C

52

Chiral Separation ModeChiral Separation Mode

C*

H

COOHCOOH

NH2

RR HOOCHOOC*C

NH2

RR

HM

irror

(L) form (D) form

Physical properties are all the same except optical rotation.

*Chiral Center

53

Separation of Chiral CompoundSeparation of Chiral Compound

Direct separation using Chiral ColumnDiastereomeric derivatization

(L) + (R) (L)-(R)

(R) + (R) (R)-(R)Enantiomer Diastereomer

Diastereomer can be separated Diastereomer can be separated by reverse / normal phase column.by reverse / normal phase column.

54

Direct separation Direct separation using Chiral Columnusing Chiral Column

Stationary phase has chiral moiety.

(R)

(R)(L)

(R)

Strong interaction

Weak interaction

55

Gradient Elution in HPLCGradient Elution in HPLC

Isocratic elution modeOne mobile phase with a constant

composition Gradient elution mode

Multi mobile phases with changing composition

56

Isocratic Elution SystemIsocratic Elution System

pump injector

column

ovendetector

Single SolventSingle Solvent

57

Gradient Elution SystemGradient Elution System

pumpinjector

column

ovendetector

pump

A

B

Time

B

con

cen

trat

ion

58

Isocratic Elution ModeIsocratic Elution Mode

Long Time AnalysisLong Time Analysis

Bad SeparationBad Separation

MeOH / H2O = 6 / 4

MeOH / H2O = 8 / 2

( column : ODS type )

59

Gradient Elution ModeGradient Elution Mode

95%

30% MeO

H c

once

ntr

atio

n

60

Part II Instrumentation of HPLCPart II Instrumentation of HPLC

Solvent delivery pump

Sample injector

Column oven

Detector

61

Flow Diagram of HPLCFlow Diagram of HPLC

pumpinjector

column

ovendetector

62

Desirable Pump PerformanceDesirable Pump Performance

High Pressure Resistance

Precise Flow Rate

Low Pressure Fluctuation

63

Pump pressure fluctuationPump pressure fluctuation

P=0.5

P=1.5

Methanol 1.0mL/min 40 kgf/cm2

Low pressure-fluctuation pump

High pressure-fluctuation pump

This pressure fluctuation will affect detector noise.

64

Plunger reciprocating pumpPlunger reciprocating pump

motor and cam

plungerplunger seal

check valve

pump head

10 -100uL

65

AdvantageLow pressure fluctuationVery easy to replace the another

solvent

DisadvantageChange the plunger seal

Plunger reciprocating pumpPlunger reciprocating pump

66

Dual plunger Dual plunger with parallel flow line with parallel flow line

check valve

plunger head

Very low pressure fluctuation Refractive index detector Conductivity detectorElectrochemical detectorMS detector

The number of maintenance parts is more.

67

Dual plunger Dual plunger with tandem flow linewith tandem flow line

check valve

Main plunger Sub plunger

Low pressure fluctuation UV / PDA detectorFluorescence detector

The number of maintenance parts is less. So this design is suitable for routine analysis.

68

Single plunger Single plunger

check valve

High pressure-fluctuation Low sensitivity analysis using UV / PDA and Fluorescence detector

The number of maintenance parts is minimized.

69

HPLC InjectorsHPLC Injectors

Manual injector

Auto injector

70

6 port valve system6 port valve system

column

load position inject position

pump pump column

sample loop

71

Manual InjectorManual Injector

Rheodyne Manual InjectorRheodyne Manual Injector

72

Manual InjectorManual Injector

[LOAD][LOAD] [INJECT][INJECT]

Column

Pump

Column

Pump

73

Relationship between injection Relationship between injection volume and detector's responsevolume and detector's response

Volume of Injection

Res

pon

se o

f D

etec

tor

Half volumeof sample loop

3 times volumeof sample loop

Loop injection

Partialinjection

74

Injection MethodInjection Method

Partial Injection MethodPartial Injection Methodbetter to inject less than half volume of

sample loop Loop Injection MethodLoop Injection Method

better to inject more than 3 times volume of sample loop

75

Dispersion and Dilution EffectDispersion and Dilution Effect

loop

pump

column

drain

drainpump

column

loop

drain

drain

Sample ZoneDilution ZoneDispersion Zone

Less than half volume Less than half volume More than half volume More than half volume

76

Dispersion and Dilution EffectDispersion and Dilution Effect

drain

drain

pump

column

loop

Sample ZoneDilution Zone

Less than 3 timesLess than 3 times More than 3 timesMore than 3 times

drain

drain

pump

column

loop

77

How to inject the sampleHow to inject the sample- Rheodyne Manual Injector -- Rheodyne Manual Injector -

Insert a syringe at INJECT position.Turn knob to the LOAD position.Inject a sample.Turn a knob to the INJECT position.Remove the syringe.Wash an injection port.

78

INJECT / LOAD positionINJECT / LOAD position

[INJECT ][INJECT ] position position

[LOAD][LOAD] position position

79

End Position of Drain PipeEnd Position of Drain Pipe

Injection portInjection port

Must put the end of drain pipe at higher than Injection port

80

Washing for Injection PortWashing for Injection Port

Use of Needle Port Cleaner

81

CautionCaution

Do not use pointed or beveled needle tip. Must use square end type.

Do not use more than pH 10 solution.Must change rotor seal.

82

Column Temperature Column Temperature Control DevicesControl Devices

Column Oven (heat /or cool) Heated Column Jacket

Aluminum block

Insulated Column Jacket Water bath

Column temperature control devices are functioning to keep the column temperature constant. The temperature fluctuation of column will influence retention time reproducibility.

83

Detectors for DetectorsDetectors for Detectors

Ultraviolet / Visible detector (UV/VIS)Photodiode Array detector (PDA)Fluorescence detector (RF)Conductivity detector (CDD)Refractive Index detector (RID)Electrochemical detector (ECD)Mass spectrometer detector (MS)

84

Ultraviolet / Visible detectorUltraviolet / Visible detector

Ein Eout

ACl= - log (Eout / Ein)

l

C : concentration

Lambert-Beer's law

(A : absorbance)

CellCell

85

Ultraviolet / Visible detectorUltraviolet / Visible detector

Sample Cell

Reference Cell

Photodiode

Photodiode

Ein

EinEin

Grating

D2 / W lamp

Eout

86

Lambert-Beer's lawLambert-Beer's law

ACl= - log (Eout / Ein)

Ab

sorb

anc

e

Concentration

linear range2.5

87

SpectrumSpectrum

275 nm275 nm

208 nm208 nm275 nm275 nm

208 nm208 nm

88

Additional FunctionsAdditional Functions

Dual Wavelength modeRatio Plot modeWavelength Time Program modeWavelength Scan mode

89

Dual Wavelength ModeDual Wavelength Mode

90

Ratio Plot ModeRatio Plot Mode

91

Wavelength Time Wavelength Time Program ModeProgram Mode

92

Wavelength Scan ModeWavelength Scan Mode

93

Sample Cell

512 Elements Photodiode Array

Grating

D2 / W lamp

Photodiode Array detectorPhotodiode Array detector

One element can detect one absorbance at one wavelength.

94

Photodiode Array detectorPhotodiode Array detector

TimeW

avel

engt

h

Ab

sorb

anc

e

Chromatogram

Spectrum

95

Photodiode Array detectorPhotodiode Array detector

UV spectra of peaks are obtained, which are supportive for identification.

By checking peak purity, one can know if any impurity is present.

Use Spectrum When Determining Separation Parameters

2

3

1 1: Azelaic acid

2: Benzoic acid

3: Nitrobenzoic acid

30%

15%Acetonitrile concentration

min

Identification by SpectrumIdentification by SpectrumElution sequence

Peak 1

Peak 2

Peak 3

Acetonitrile30% 15%

Azelaic acidAzelaic acid

Benzoic acidBenzoic acid

Nitrobenzoic acidNitrobenzoic acid

98

Comparison by 3 Point SpectrumComparison by 3 Point Spectrum

Acetyl Salicylic Acid

down slope

up slope, peak top

99

Fluorescence detectorFluorescence detector

+ h1

h2+

*

A

A*

A

h1 h2

Excitation Wavelength

Emission Wavelength

FluorescenceFluorescence

*

100

Cell design Cell design of Fluorescence detectorof Fluorescence detector

101

Derivatization ReagentsDerivatization Reagents

OPA reagent for primary aminesOPA reagent for primary aminesCHO

CHOo-phthalaldhyde(OPA)

+ R-NH2 N-R

A

A

ADAM reagent for fatty acidsADAM reagent for fatty acids

CHN29-anthryldiazomethane(ADAM)

+ R-COOH

CH2OCOR

102

Refractive Index detectorRefractive Index detector

Optical System

103

Refractive Index detectorRefractive Index detector

104

Chromatogram of sugarsChromatogram of sugars

Analytical Conditions Column : Shim-pack CLC-NH2 Mobile phase : Acetonitrile / water

= 7 / 3 Flow rate : 1.0 mL/min Temperature : Ambient

Peaks 1. Glycerol

2. Xylose

3. Fructose

4. Glucose

5. Sucrose

6. Manose

7. Lactose

105

Conductivity detectorConductivity detector

I

V

L

A A

K (conductivity) = I [A] / E [V] =A [cm2] / L [cm] * k (k : specific conductivity)

k= (I/E)*(L/A)k= (I/E)*(L/A)

electrode

106

Conductivity detectorConductivity detector

Conductivity is very affected to temperature.Must keep the cell in the temperature control

devise.

107

Type of Ion ChromatographyType of Ion Chromatography

Non-Suppresser typesimple system (easy maintenance)low conductivity mobile phase

Suppresser Typelow back grand currentdifficult maintenance

108

Non-Suppresser TypeNon-Suppresser Type

extremely low ion exchanger

Low conductivity mobile phase

109

Suppresser TypeSuppresser Type

110

Conductivity and Peak ResponseConductivity and Peak Response

111

Analytical Conditions Column : Shim-pack IC-A3 Mobile phase :

8.0 mM p-hydroxybenzoic acid

3.2 mM Bis-Tris * Flow rate : 1.5 mL/min Temperature : 40 C Injection Volume : 100 uL

Peaks 1. F (1.4 ppm) 2. Cl (10200 ppm) 3. NO2 (10 ppm) 4. Br (43 ppm) 5. NO3 (44 ppm) 6. SO4 (431 ppm)

Chromatogram of Anion Chromatogram of Anion in the brinein the brine

Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane

112

Chromatogram of Cations Chromatogram of Cations in Tap waterin Tap water

Analytical Conditions Column : Shim-pack IC-C3 Mobile phase : 2.0 mM Oxalic Acid Flow rate : 1.0 mL/min Temperature : 40 C Injection volume : 100 uL

Peaks 1. Na (8.25 ppm) 2. NH4 (0.01 ppm) 3. K (1.66 ppm) 4. Mg (2.22 ppm) 5. Ca (11.85 ppm)

113

Electrochemical detectorElectrochemical detector

Working electrode

AUX electrode

Reference electrode

114

Principal of ECD detectionPrincipal of ECD detection

e-

A

R O + H+

ElectrodeGlassy Carbon (GC)Pt, Ag, Au

[ Applications ]

GC : phenol compounds general usePt : H2O2

Ag : halogen ionAu : sugar analysis

115

Chromatogram of CatecolaminesChromatogram of Catecolamines

Analytical Conditions Column : Shim-pack CLC-ODS Mobile phase :

80 mM phosphate buffer (pH=2.7)

100 mM NaNO3, 200 mg/l SOS

5 mg/l EDTA, 4 % acetonitrile Flow rate : 1.0 mL/min Applied Potential : + 0.8 V Temperature : 40 C Injection volume : 10 uL

Peaks 1. Noradrenalin ( 5 ppb) 2. Adrenalin (5 ppb) 3. Dopamine (5 ppb)

116

LCMSLCMSAtmospheric Pressure IonizationAtmospheric Pressure Ionization

LCMSLCMSAtmospheric Pressure IonizationAtmospheric Pressure Ionization

Pneumatically Assisted ElectroSpray(ESI)

Atmospheric Pressure Chemical Ionization(APCI)

High Voltage

3) Coulon Exclus ion

Ion Evaporation

2) Evaporation

of Solvent

Liquid Samples

+ - + -+-

+-

+ -+

+

+

-

-+-+++-+-

+-+++-+-

++

+

++

+

Liquid Samples HeaterC orona D ischarge

Nee dle

Ion molecular reaction

Nebulizing gas

Nebulizing gas

117

Design of LCMSDesign of LCMS

API Probe

Atmosphere High Vacuum

RP TMP1 TMP2(Vacuum pump system)

MSdetector

118

What kind of compounds What kind of compounds can be analyzed ?can be analyzed ?

ESI drugs and their metabolites peptides proteins many kinds of natural product (-OH, -NH2,-COOH, SO2, PO3 etc.)

APCI pesticides steroids drugs

119

What kind of benefits What kind of benefits LC/MS users can get ?LC/MS users can get ?

Powerful qualitative capabilityPowerful qualitative capability Selective quantitative capability

M/Z

M/Z

M/Z

120

What kind of benefits What kind of benefits LC/MS users can get ?LC/MS users can get ?

Powerful qualitative capability

Selective quantitative capabilitySelective quantitative capability

A:100

D:150B:100C:150

m/z=150m/z=150

TIC TIC

m/z=100m/z=100A

B

C D

121

Analysis of Pesticide Analysis of Pesticide

isoxation oxon

EPN oxon

iprofenfos

303

298

289

577

TIC

122

Analysis of Pesticide 1Analysis of Pesticide 1(Mass Spectrum)(Mass Spectrum)

P

O

O

OC 2H5

NO2

NO

OP

O

OC 2H5

OC 2H5

SP

O

OCH(CH 3)2OCH(CH 3)2

EPN oxon

isoxation oxon

iprofenfos

123

Mass SeparationMass Separation

Maltose

TIC

387

225

195

Glucose

Xylose

OOO

HOH 2C

HOHO

OH HOH 2C

HOOH

OH

OHOH 2C

HO

HOOH

OH

OH3C

HO

HOOH

OH

124

Comparison of each detectorComparison of each detector

LOD : Limit of detection (S/N=3)GC : Gradient Compatibility

PDA sensitivity is slightly less sensitive than normal UV detector.

UV/ VIS RF RID CDD ECD MS

LOD 1 ppb 10 ppt 100 ppb 1 ppb 10 ppt 1 ppb

GC Yes Yes No No No Yes