detection in hplc -...

Detection in HPLC

Dr. Shulamit Levin, 1

Selecting the Right Detector:Selecting the Right Detector:Types of Detectors in HPLCTypes of Detectors in HPLC

Shulamit LevinShulamit Levin

•UV/VIS•Refractive index •Fluorescence•Electrochemical •Conductivity •Mass-spectrometric (LC/MS) •Evaporative light scattering

•Appendix:•Cutoff of solvents UV•Troubleshooting of RI detector as an example

The Detector is the The Detector is the ““EyeEye”” of the HPLC Systemof the HPLC System

HPLC Columnin Oven

Auto Sampler

Pumpflows 50-5000µL/min)

Fraction Collector

Waste

DetectorControl &Data Processing

1. Fucose2. Galactosamine3. Glucosamine4. Galactose5. Glucose6. Mannose

20.00Minutes5.00

mV

0.00

300

12

34

5

6

a bc d

DetectorsDetectors

UV/VISUV/VISRefractive index Refractive index FluorescenceFluorescenceElectrochemical Electrochemical Conductivity Conductivity MassMass--spectrometric (LC/MS) spectrometric (LC/MS) Evaporative light scatteringEvaporative light scattering

Optical Bench of UVOptical Bench of UV--VIS DetectorVIS Detector

DeuteriumArc Lamp

RotatingDiffraction

Grating190 to 600nm

Taper-CellFlowCell

Beam SplitterMirrors

DualPhotodiode

ApertureSlit

Illumination Lens

Beam-DefiningApparatus

Optical Light Path

Sample side

Reference side

Detection in HPLC

Dr. Shulamit Levin2

Beer's LawBeer's Law

Reduce Pathlength Reduce Concentration

Absorbance = Extinction Coefficient x Pathlength x Concentration

UV ChromophoresUV Chromophores

UV ChromophoresUV Chromophores UVUV--Vis chromophoresVis chromophores

λmax Em x 10-3 @ λmax

Adenine 260.5 E = 13.4Guanine 275 E = 8.1Cytosine 267 E = 6.1Thymine 264.5 E = 7.9Uracil 259.5 E = 8.2NADH 340 E = 6.23NAD 260 E = 18

Detection in HPLC

Dr. Shulamit Levin3

UV spectrum of 10 UV spectrum of 10 nMnM mobile phasemobile phase

AU

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

pH 0.94

TFA

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

265.1 295.8

pH 2.0

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

205.2

pH 2.26

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

nm210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

pH 2.78

Sodium PhosphateFormic Acid

Acetic Acid

U.V. CutU.V. Cut--offs for some Common Solventsoffs for some Common Solvents

SolventSolvent UV CutoffUV Cutoff SolventSolvent UV CutoffUV CutoffWaterWater 180180 N-HeptaneN-Heptane 197197MethanolMethanol 205205 CyclohexaneCyclohexane 200200N-PropanolN-Propanol 205205 Carbon tetrachlorideCarbon tetrachloride 265265AcetonitrileAcetonitrile 190190 ChloroformChloroform 245245THFTHF 225225 BenzeneBenzene 280280AcetoneAcetone 330330 TolueneToluene 285285Methyl acetateMethyl acetate 260260 Methylene chlorideMethylene chloride 232232Ethyl AcetateEthyl Acetate 260260 TetrachloroethyleneTetrachloroethylene 280280NitromethaneNitromethane 380380 1,2-Dichloroethane1,2-Dichloroethane 225225

All wavelengths reported in nm.All wavelengths reported in nm.

Remember Solvents chosen can affect detection!!Remember Solvents chosen can affect detection!!

UV Detection of UV Detection of AccQAccQ--Tag Amino Acid DerivativesTag Amino Acid Derivatives

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

AU

Minutes

Aspa

rtic

Acid

Glu

tam

ic A

cid

Hyd

roxy

prol

ine

Ser

ine

Asp

arag

ine

AMQ

Gly

cine

Glu

tam

ine H

istid

ine

NH

3Th

reon

ine

Arg

inin

e

Alan

ine

Pro

line

Alph

a-am

inob

utyr

ic a

cid

Tyro

sine

Cys

teic

Aci

dVa

ine

Mte

hion

ine

Orn

ithin

eLe

ucin

e

Lysi

ne

Phe

nyla

lani

ne

Tryp

toph

anIsol

euci

ne

Diode Array DetectorDiode Array Detector

Detection in HPLC

Dr. Shulamit Levin4

Extraction of 3D DataExtraction of 3D Data

Time

Abso

rban

ce

Chromatogram

λ1

λ2

Wavelength

Abso

rban

ce Spectrum

PDA Spectrum Index PlotPDA Spectrum Index PlotDNPH Derivatives 0.25 ng Each PeakDNPH Derivatives 0.25 ng Each Peak

260.00 260.00280.00 280.00300.00 300.00320.00 320.00340.00 340.00360.00 360.00380.00 380.00400.00 400.00420.00 420.00440.00 440.00

nm

0.00000.0000

0.00020.0002

0.00040.0004

0.00060.0006

AUAU

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm

CoelutionCoelution of 2 Peaksof 2 Peaks

AU

A

B

Coelution detection at a single wavelength

Coelution is the sum of absorbance of 2 peaks A and B

Peak Purity MeasurementPeak Purity Measurement

2.20 2.40 2.60 2.80 3.00

200.00

250.00

300.00

0.00

0.20

0.40

MaximumImpurity

Spectra at apex and inflection points are displayed

Spectrum at maximum impurity is different

AU

nm

Detection in HPLC

Dr. Shulamit Levin5

Maximum Impurity DetectionMaximum Impurity Detection

260.00 260.00280.00 280.00300.00 300.00320.00 320.00340.00 340.00360.00 360.00380.00 380.00400.00 400.00420.00 420.00440.00 440.00

nm

Minutes

nm

-0.00010 0.00010

0.00000 0.00000

0.00010 0.00010

0.00020 0.00020

0.00030 0.00030

18.40 18.60 18.80 19.00 19.20

AU AU

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm

Hexaldehyde 2,5-Dimethylbenzaldehyde

Coelution of DNPH Hexaldehyde and

2,5-Dimethylbenzaldehyde

Determination of Peak PurityDetermination of Peak Purity

Abs

orba

nce

Time

S t a n d a r d

T im e

U n k n o w n

Peak Purity analyzes all spectra (minimum 15) within a peak against the apex spectrum of the peak itself.

Peak PurityPeak Purity Spectral MatchingSpectral Matching

Spectral match of apex spectrum of the unknown against the apex spectrum of a standard, stored in a user’s library.

Different Spectra Different Spectra –– 53 deg53 deg

200.00 240.00 280.00 320.00nm

EthylPabaEthylparaben

Abs

orba

nce

10 deg of Spectral Contrast10 deg of Spectral Contrast

Similar spectra for structurally related compounds

230.00 250.00 270.00 290.00 310.00nm

TheophyllineDyphylline

Abs

orba

nce

Detection in HPLC

Dr. Shulamit Levin6

Spectral Contrast 0.5 DegreesSpectral Contrast 0.5 Degrees

200.00 240.00 280.00 320.00nm

MethylparabenEthylparaben

Abs

orba

nce

Very similar spectra, CH2 difference

Spectral Contrast can differentiate these spectra

Spectra of nonSpectra of non--UV Active CompoundsUV Active Compounds

210.00 230.00 250.00 270.00 290.00nm

Abs

orba

nce

Analyte and 2 Impurities

DetectorsDetectors


Refractive Index DetectorRefractive Index Detector

Detection in HPLC

Dr. Shulamit Levin7

Refractive Index DetectorRefractive Index DetectorDifferential Refractive Index DetectorDifferential Refractive Index Detector

LAMP

SLED

R

RS

To Amplifier

No sample = n

With sample = n+∆ν

∆X = Const x ∆n

Sugar Analysis Sugar Analysis

-160.00

-140.00

-120.00

-100.00

-80.00

-60.00

-40.00

-20.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00

5.00 6.00 7.00 8.00 9.00 10.00 11.00

mV

Minutes

Fructose

Dextrose Sucrose

Maltose Lactose

SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard

Bagel Extract

Polymer AnalysisPolymer Analysis

0.0050.00

100.00150.00200.00250.00300.00350.00400.00450.00500.00550.00600.00650.00700.00750.00800.00

18.00 20.00 22.00 24.00 26.00 28.00 30.00

MV

Minutes

1260

000

9640

0

5570

SampleName: GPC STDS

Dow 1683

2890

000

1900

00

1030

0

192300

Detection in HPLC

Dr. Shulamit Levin8

LipidsLipids

Del

RIU

5.0 6.0 7.0 8.0Minutes

1 2

250 ng on column 1=Tristearin2=Myristic acid

Styragel HR 0.5, 4.6 x 300 mm, 35°C, 0.35 mL/min

dRI sensitivity = 32X, 32°C

DetectorsDetectors


Fluorescence ProcessFluorescence Process

Excitation

Emission

Energy Levels

Ground State

Excited States

ExcitationExcitation--Emission SpectraEmission Spectra

λ Maximum of Excitation Spectrum

λ Maximum of Emission Spectrum

Stoke’s λ shift

Lifetime= 10-9 – 10-15 sec

Detection in HPLC

Dr. Shulamit Levin9

Fluorescence DetectorsFluorescence DetectorsExcitation filter

LAMPCell

Emission filter

Photomultiplier

Fluorescence Detector Optical BenchFluorescence Detector Optical Bench

Photom ultiplier tube

Em issionG rating

ExcitationGrating

M irror

Torroidal M irror

Beam Splittter

FlowCell

M irror

Torroidal M irror

Photodiode

ExcitationSlit

Em ission Slit

UV UV vsvs Fluorescence SensitivityFluorescence Sensitivity

Minutes

AMQ

20.00 40.00 60.00

Res

pons

e

AccQ-Tag amino acid analysis

FluorescenceExcitation=250 nmEmission=395 nm

UV 254 nm

DetectorsDetectors


Detection in HPLC

Dr. Shulamit Levin10

Electrochemical DetectorElectrochemical Detector

Reference Electrode

Working Electrode

Electrolyte (mobile phase)

Auxiliary Electrode

Analyte is oxidized or reduced

+ -

As compounds are oxidized or reduced, a current proportional to concentration is produced.

Electrochemical Detection of Electrochemical Detection of CatecholaminesCatecholamines & Related Compounds & Related Compounds

1. Norepinepherine 150 ppb2. Epinepherine 200 ppb3. Normetanepherine 50 ppb4. Dopamine 200 ppb5. Metanepherine 200 ppb6. 3-Methoxytyramine 75 ppb7. 4-Methoxytyramine 500 ppb

2.00 4.00 6.00 8.00 10.00 12.00

Minutes

0.00

nAmps

1 2

3

45

6

7

Pulsed Pulsed AmperometricAmperometric Detection of Detection of MonosaccharidesMonosaccharides

1. Fucose2. Galactosamine3. Glucosamine4. Galactose5. Glucose6. Mannose

20.00Minutes5.00

mV

0.00

300

12

34

5

6

DetectorsDetectors


Detection in HPLC


Conductivity DetectorConductivity Detector

M o bile ph ase

M o bile p hase p lu s sam ple

++ --

Conductivity DetectorConductivity Detector

Mobile phase

Mobile phase plus sample

Anion Analysis by ICAnion Analysis by IC

0.70

1.05

1.40

µS

0.00 5.00 10.00 15.00 20.00 25.00Minutes

1

2

3

45

6 7

1. Fluoride2. Chloride3. Nitrite4. Bromide5. Nitrate6. Phosphate7. Sulfate

1 ppm2 ppm4 ppm4 ppm4 ppm6 ppm4 ppm

Column:Eluent:Flow rate:Injection vol.:Detection:

Waters IC-Pak Anion HCBorate/Gluconate2.0 mL/min100 µLDirect Conductivity

Anion analysis by ICAnion analysis by IC

µS

0.00

0.40

0.80

1.20

1.60 1 23

4 5

67

0.00 4.00 8.00 12.00 16.00 20.00 24.00Minutes

0.00

0.01

0.02

0.03

0.04

0.05

AU

3

4

5 Column:Eluent:

Flow rate:Injection vol.:

Waters IC-Pak Anion HR1.2 mM Sodium Carbonate/1.2 mM Sodium Bicarbonate1.0 mL/min50 µL

Detection: Direct Conductivity after Suppression

Detection: UV (PDA) at 214 nm

1. Fluoride2. Chloride3. Nitrite4. Bromide5. Nitrate6. Phosphate7. Sulfate

1 ppm2 ppm4 ppm4 ppm4 ppm6 ppm4 ppm

Detection in HPLC


ApplicationsApplications

Sensitivities for compounds such as phenol, catecholamines, nitrosamines, and organic acids are in the picomole (nanogram) range.

The mobile phase must be made electrically conductive, usually by the addition of a suitable salt:

Ion Exchange

Reversed Phase and Ion-Pair RP

No normal phase separations

DetectorsDetectors


DATA SYSTEM

DETECTION OF IONS

+

+

SAMPLE DESOLVATIONAND IONIZATION

LC/MSINTERFACE

SOURCE

ANALYZERION DETECTOR

HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

+ +

++

-

+

+

+

CHROMATOGRAM

Typical LC/MS System ProgressionTypical LC/MS System Progression

DATA SYSTEM

DETECTION OF IONS

+

+


LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

+ +

++

-

+

+

+

CHROMATOGRAM


Detection in HPLC


Transition from LC to MSTransition from LC to MS

• State of Matter: LiquidLiquid to GasGas

• Charge State: “NeutralNeutral” to IonIon

• Pressure: 760 760 torrtorr to 1010--55 to 10to 10--88 torrtorr

APCI MechanismAPCI Mechanism

Ionization produces solvent ions

The solvent ions reactwith analyte moleculesforming clusters

Corona Needle

X = Solvent Molecules e.g.H2O, MeCNM = Sample Molecule

Heated Nebulizer

xx

xH+ M

xx

xH+

Mx

xM

MH+

xx

xx

XH+x

xH+

MH+

ElectrosprayElectrospray IonizationIonization Positive or Negative?Positive or Negative?

Basic Compounds (-NH2) (M+H)+

Acidic Compounds (-CO2H, -OH) (M-H)-

Detection in HPLC


Recognizing Multiply Charged Ions

Mass spectrometers operate on the basis of mass-to-charge ratio (m/z). Mass assignments are normally made assuming a single charge per ion (i.e. m/z = m)

Single charge Mass = (M+H)

Double charge Mass = 1/2 (M+2H)

n charge Mass = 1/n (M+nH)

Isotopes of doubly charged ions are separated by 0.5 Da

n = 20

n = 22

n = 18

n = 16, m/z = 1060

n = 23, m/z = 738

n = 21

n = 19

n = 17

Horse Heart Myoglobin

Mass RangeMass RangeMultiply Charged MoleculesMultiply Charged Molecules

Calculated MassAcquired Mass range

Hemoglobin SpectrumHemoglobin SpectrumPresence of More Than One Charged EnvelopePresence of More Than One Charged Envelope

1000 1050 1100 1150 1200 1250 1300 1350 1400m/z0

100

%

1081.60

1009.36

1058.83

1164.52

1133.92

1261.64

1221.181376.08

1323.14

DeconvolutionDeconvolution by by MaxEntMaxEntHemoglobinHemoglobin

15000 15100 15200 15300 15400 15500 15600 15700 15800 15900 16000 16100mass0

100

%

15125.0

15857.0

15149.0

15866.0

Detection in HPLC


Multiply Charged Ions – How Many Charges?

(M+H)+

(M+2H)2+

1 Da

0.5 Da

DATA SYSTEM

DETECTION OF IONS

+

+


LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

+ +

++

-

+

+

+

CHROMATOGRAM


Mass Mass SpectrometerSpectrometer’’ss

AnalyzersAnalyzers

T im e O f F lig h t M a s s A n a ly ze rsT im e O f F lig h t M a s s A n a ly ze rs

S O U R C E

D E T E C T O RR E F L E C T R O N M O D E

R E F L E C T R O N O ND E T E C T O RL IN E A R M O D E

D R IF T T U B E

S O U R C E


R E F L E C T R O N O F F D E T E C T O RL IN E A R M O D ED R IF T T U B E

T im e O f F lig h t M a s s A n a ly ze rsT im e O f F lig h t M a s s A n a ly ze rs

S O U R C E


R E F L E C T R O N O ND E T E C T O RL IN E A R M O D E

D R IF T T U B E

S O U R C E


R E F L E C T R O N O F F D E T E C T O RL IN E A R M O D ED R IF T T U B E

199

FTFT--ICRICR--SpectrometerSpectrometer

DCDC

DCSource

Filament

Transferoptic

Trapping Plates

Transmitter Plates

Receiver Plates

Sender

Elektroden Electrodes

Y

ZX

Magnetic Fielt B

Starting with the Starting with the quadrupolequadrupole

Source

DetectorNonresonant Ion

Resonant Ion

dc and Rf voltages

V(t) = - Vdc - Vrf cosΩt

V(t) = Vdc + Vrf cosΩt

190

Electron Multiplier

Inlet

Ring Electrode, Rf

End Cap Electrode

Axial Modulation

IonIon TrapsTraps

+ + ++++ ++

IonSource

Slit

Magnetic sectorElectrostatic Sector

(ESA)

Detector

SlitNier-Johnson-Geometry (EB) DATA SYSTEM

DETECTION OF IONS

+

+


LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

+ +

++

-

+

+

+

CHROMATOGRAM


Detection in HPLC


MS DetectorsMS Detectors

++ ++ -

-

-Electron Multiplier(voltage setting lower than Dynode)

Conversion Dynode (Voltage 1- 20 kV)

Current is measured

+ +Mass Analyser

dynodedynode

phosphorphosphorphotomultiplierphotomultiplier

Photomultiplier

Electron Multiplier

Mass Spectrometer 3D RunMass Spectrometer 3D Run

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Int.1: Mass Chromatogram

5.054.65

3.820.74

8.62

5.65

8.02

10.62

Total-Ion-Current MS Chromatogramwith poor resolution

Mix

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z0

100

%

(10.696) 1: Scan ES+ 4.34e5262.87

59.99

213.90

195.98120.8068.92

98.8576.87 128.82

170.92

222.87

235.87

240.88

263.87

264.85

267.91 287.01 309.02333.84

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Abs1: Diode Array

5.054.65

3.820.74

8.62

5.65

8.02

10.62

200210220230240250260270280290300310nm0

Int. 210.00

246.00

294.00

UV-Diode Array Chromatogramwith poor resolution

Mix

2.00 4.00 6.00 8.00 10.00 12.00 14.00Time0

100

%

1: Scan ES+ 262.874.59e5

10.60

Mixture

2.00 4.00 6.00 8.00 10.00 Time0

Int. 5.054.65

3.820.74

8.625.65

8.0210.62

Selectivity of Mass Spectrometer DetectorSelectivity of Mass Spectrometer Detector

Extracted Ion Chromatogramof a single Component from

a mixture of Components

Mix

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z0

100

%

(10.696) 1: Scan ES+ 4.34e5262.87

59.99

213.90

195.98120.8068.92 98.85

76.87 128.82 170.92

222.87

235.87

240.88

263.87

264.85

267.91 287.01 309.02333.84

LC-MS Analysis

XTerra™ MS C18, 2.1 x 50 mm ( 5 µm)

10 µL injection of 200 ng/mL sample (in 40% MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline, 4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN0.2 mL/min

Time (min)

1.00 2.00 3.00 4.00 5.000

100

0

100

0

100

0

100

0

100 2958.11e4

2.56

2802.21e5

1.57

2641.26e5

2.16

2601.53e5

1.29

Total Ion Chromatogram3.50e5

1.571.29 2.16

2.56

100 125 150 175 200 225 250 275 300Mass/Charge (m/z)

0

100

0

100

100

0

1004.59e4295

296

1.13e5280

281

7.67e4264

233 265

9.25e4260

261

0O

N

N

N

NH

O NHOH

(1)

(2)

(3)

(4)

Ding

Detection in HPLC


Triple Triple QuadrupolesQuadrupoles: MS: MS--MS ModesMS Modes

Multiple Reaction Monitoring

Typically used in Quantitative Work of

Triple Quadrupoles



Triple Quadrupoles

MS1 Collision

Cell

MS2

StaticStatic



Triple Quadrupoles



Triple Quadrupoles

MS1 Collision

Cell

MS2

StaticStatic

Daughter (Product) Ion SpectraDaughter (Product) Ion Spectra

MS1 CollisionCell

MS2

ScanningStatic

Daughter (Product) Ion SpectraDaughter (Product) Ion Spectra

MS1 CollisionCell

MS2

ScanningStatic

Parent (Precursor) Ion SpectraParent (Precursor) Ion Spectra

MS1 CollisionCell

MS2

StaticScanning

Parent (Precursor) Ion SpectraParent (Precursor) Ion Spectra

MS1 CollisionCell

MS2

StaticScanning

Constant Neutral Loss SpectraConstant Neutral Loss Spectra

MS1 CollisionCell

MS2

ScanningScanning

Constant Neutral Loss SpectraConstant Neutral Loss Spectra

MS1 CollisionCell

MS2

ScanningScanning

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Time

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

Std_017 MRM of 12 Channels ES+ 208 > 162.9

1.73e75.10

Std_017 MRM of 12 Channels ES+ 149.95 > 91

1.49e73.80


4.29e62.95


2.05e72.25


4.13e63.20

Std_017 MRM of 12 Channels ES+ 193.65 > 162.7

6.48e63.95

MDEA

Ephedrine

MDMA

MDA

Amphetamine

Methamphetamine

Typical Quantitative Analysis Using Triple Quadrupoles:Simultaneous MRM analysis of 6 amphetamines

Highly specific and sensitive chromatograms

DetectorsDetectors


Evaporative Light Scattering Evaporative Light Scattering -- ELSELS

Nebulization Desolvation

DetectionTo detector cell

Nebulizer

Lamp

Detection in HPLC


Rayleigh Scattering Rayleigh Scattering –– Why the Sky is blueWhy the Sky is blue

• Scattering is independent of the particle’s chemical properties, where:– N = # of particles– α = Polarizability i.e. the sum of the dipoles of all the molecules in the particle.

For a homogeneous particle this is proportional to the particle volume. – R = Distance of observer from scatterer– Dependence on wavelength of incident light, shorter wavelengths produce

greater scattering

λ4R2

I = I0 8 π4 Nα2 (1 +cos2θ)

Scattering ModelsScattering Models

If D/λ< 0.1then I = f (D6)

0.1<D/λ< 10then I = f (D4)

D/λ>10then I = f (D2)

I = Intensity of the scattered lightλ = wavelength of the light

Scattering is dependent on particle size “D” Increasing particle size

D α C1/3

(Often see solute density)I α (Cb)

With 2>b>2/32 is the limiting value for Rayleigh

symmetrical scattering

Depends on which type of scattering is predominant

Non-linear mass detectoruse chromatography data software

quadratic curve or log/log curve to fit calibration curve

ELSD ELSD vsvs UVUV ELSD ELSD vsvs RIRI

Detection in HPLC


ELSD Used with Other DetectorsELSD Used with Other Detectors

ELSD to monitor all compounds and determine purity levels

Diode Array TIC

Min

ES+TIC

ELSD

24.5 7

Diode array TIC

PDA to monitor UV/Vis friendly compounds

Mass Spec to verify that compound has been synthesized

Not a Universal DetectorNot a Universal DetectorTypically Used with Other DetectorsTypically Used with Other Detectors

UV TIC

MS ES+ TIC

ELSD

Erythromycins Separation

Min1 3 5

ELSD

Diode Array TIC

See NonSee Non--UV Absorbing CompoundsUV Absorbing Compounds See Your Peaks FasterSee Your Peaks FasterUse of Gradients Versus IsocraticUse of Gradients Versus Isocratic

RI Detection

ELSD Detection

Detection in HPLC

20

Estrogen analogues and saltm

V

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

110.00

Minutes0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

NaC

l-0.

207

Estr

adio

l-0.

849

Estr

iol-

1.21

0

Evaporative LightEvaporative Light--scattering Detectorscattering Detector

detection in hplc -...

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