detection by hplc

8/9/2019 Detection by Hplc

1/20

Detection in HPLC

Dr. Shulamit Levin, Medtechnica

Selecting the Right Detector:Selecting the Right Detector:

Types of Detectors in HPLCTypes of Detectors in HPLC

Shulamit LevinShulamit Levin

[email protected]

[email protected]

/HPLCwww.forumsci.co.ilhttp://

UV/VISRefractive 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 theThe Detector is the EyeEye of the HPLC Systemof the HPLC System

HPLC Column

in OvenAuto

Sampler

Pumpflows 50-5000L/min)

Fraction

Collector

Waste

DetectorControl &

DataProcessing

1.Fuc os e2.Galactosami ne

3.Glucosami ne4.Galactose

5.Glucose6.Mannos e

20.00Minutes5.00

mV

0.00

300

12

34

5

6

a bc d

DetectorsDetectors

UV/VISUV/VIS

Refractive indexRefractive index

FluorescenceFluorescence

ElectrochemicalElectrochemical

ConductivityConductivity

MassMass--spectrometric (LC/MS)spectrometric (LC/MS)Evaporative light scatteringEvaporative light scattering

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

DeuteriumArc Lamp

Rotating

DiffractionGrating

190 to 600nm

Taper-Cell

FlowCell

Beam Splitter

Mirrors

Dual

Photodiode

ApertureSlit

Illumination

Lens

Beam-Defining

Apparatus

Optical Light Path

Sample side

Reference side


2/20

Detection in HPLC


Beer's LawBeer's Law

Reduce Pathlength Reduce Concentration

Absorbance = Extinction Coefficient x Pathlength x Concentration

UVUV ChromophoresChromophores

UVUV ChromophoresChromophores UVUV--VisVis chromophoreschromophores

max Emx 10-3 @max

Adenine 260.5 E = 13.4

Guanine 275 E = 8.1Cytosine 267 E = 6.1

Thymine 264.5 E = 7.9

Uracil 259.5 E = 8.2NADH 340 E = 6.23NAD 260 E = 18


3/20

Detection in HPLC


UV spectrum of 10UV 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

nm

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

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

nm210.00220.00 230.00240.00 250.00 260.00270.00280.00 290.00

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

nm

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

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

nm210.00220.00230.00240.00250.00260.00270.00280.00290.00

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 Cutoff

WaterWater 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 ofUV Detection ofAccQAccQ--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

AsparticAcid

GlutamicAcid

Hydroxypro

line

Serine

Asparagine

AMQ

Glycine

Glutamine H

istidine

NH3

Threonine

Arginine

Alanine

Proline

Alpha-aminobutyricacid

Tyrosine

CysteicAcid

Vaine

Mtehionine

Ornithin

e

Leucine

Lysin

e

Phenylalanine

Tryptophan

Isoleuc

ine

Diode Array DetectorDiode Array Detector


4/20

Detection in HPLC


Extraction of 3D DataExtraction of 3D Data

Time

Absorbance

Chromatogram

1

2

Wavelength

Absorbance Spectrum

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

260.00 260.00

280.00 280.00

300.00 300.00

320.00 320.00

340.00 340.00

360.00 360.00

380.00 380.00

400.00 400.00

420.00 420.00

440.00 440.00

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

Coelutiondetection at asingle wavelength

Coelution is the sum ofabsorbance of 2 peaksA 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

Maximum

Impurity

Spectra at apex andinflection points aredisplayed

Spectrum atmaximum impurityis different

AU

nm


5/20

Detection in HPLC


Maximum Impurity DetectionMaximum Impurity Detection

260.00 260.00

280.00 280.00

300.00 300.00

320.00 320.00

340.00 340.00

360.00 360.00380.00 380.00

400.00 400.00

420.00 420.00

440.00 440.00

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 ng360nm 996PDA 360.0 nm

Hexaldehyde 2,5-Dimethylbenzaldehyde

Coelutionof DNPHHexaldehyde and

2,5-Dimethylbenzaldehyde

Determination of Peak PurityDetermination of Peak Purity

Absorbance

Time

S t a n d a r d

T i m 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 users library.

Different SpectraDifferent Spectra 53 deg53 deg

200.00 240.00 280.00 320.00

nm

EthylPabaEthylparaben

Absorbance

10 deg of Spectral Contrast10 deg of Spectral Contrast

Similar spectra for

structurally related

compounds

230.00 250.00 270.00 290.00 310.00

nm

Theophylline

Dyphylline

Absorbance


6/20

Detection in HPLC


Spectral Contrast 0.5 DegreesSpectral Contrast 0.5 Degrees

200.00 240.00 280.00 320.00nm

MethylparabenEthylparaben

Absorbance

Very similar spectra,CH2 difference

Spectral Contrast candifferentiate thesespectra

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

210.00 230.00 250.00 270.00 290.00nm

Absorbance

Analyte and 2 Impurities

DetectorsDetectors

UV/VISUV/VIS






Refractive Index DetectorRefractive Index Detector


7/20

Detection in HPLC


Refractive Index DetectorRefractive Index DetectorDifferential Refractive Index DetectorDifferential Refractive Index Detector

LAMP

SLED

R

RS

To Amplifier

No sample = n

With sample = n+

X = Const x n

Sugar AnalysisSugar 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

0.00

50.00

100.00

150.00

200.00

250.00300.00

350.00

400.00

450.00

500.00

550.00

600.00

650.00

700.00

750.00

800.00

18.00 20.00 22.00 24.00 26.00 28.00 30.00

MV

Minutes

1260000

96400

5570

SampleName: GPC STDS

Dow 1683

28

90000

190000

10300

192300


8/20

Detection in HPLC


LipidsLipids

DelRIU

5.0 6.0 7.0 8.0

Minutes

1 2

250 ng on column1=Tristearin

2=Myristic acid

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

dRI sensitivity =32X, 32C

DetectorsDetectors

UV/VISUV/VIS





MassMass--spectrometric (LC/MS)spectrometric (LC/MS)

Evaporative light scatteringEvaporative light scattering

Fluorescence ProcessFluorescence Process

Excitation

Emission

Energy Levels

Ground State

Excited States

ExcitationExcitation--Emission SpectraEmission Spectra

Maximum ofExcitation Spectrum

Maximum of

Emission Spectrum

Stokes shift

Lifetime= 10-9 10-15 sec


9/20

Detection in HPLC


Fluorescence DetectorsFluorescence Detectors

Excitation filter

LAMP

Cell

Emission filter

Photomultiplier

Fluorescence Detector Optical BenchFluorescence Detector Optical Bench

Photomultipliertube

Emission

Grating

ExcitationGrating

Mirror

Torroidal Mirror

Beam Splittter

FlowCell

Mirror

Torroidal Mirror

Photodiode

Excitation

Slit

Emission

Slit

UVUV vsvs Fluorescence SensitivityFluorescence Sensitivity

Minutes

AMQ

20.00 40.00 60.00

Response

AccQ-Tag amino acidanalysis

Fluorescence

Excitation=250 nm

Emission=395 nm

UV 254 nm

DetectorsDetectors

UV/VISUV/VIS







10/20

Detection in HPLC


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 ofElectrochemical Detection ofCatecholaminesCatecholamines & Related Compounds& Related Compounds

1 . N or ep in ep he ri ne 1 50 pp b

2. Epinepherine 200 ppb

3. Normetanepheri ne 50 ppb

4. Dopamine 200 ppb5. Me tan ep he ri ne 200 p pb

6 . 3 -M e th o xy ty r am i ne 7 5 p pb

7 . 4 - Me t ho x yt y ra m in e 5 00 pp b

2.00 4.00 6.00 8.00 10.00 12.00

Minutes

0.00

nAmps

12

3

45

6

7

PulsedPulsed AmperometricAmperometric Detection ofDetection ofMonosaccharidesMonosaccharides

1. Fucose

2. Galactosamine

3. Glucosamine

4. Galactose

5. Glucose

6. Mannose

20.00Minutes5.00

mV

0.00

300

12

34

5

6

DetectorsDetectors

UV/VISUV/VIS







11/20

Detection in HPLC


Conductivity DetectorConductivity Detector

Mobile phase

Mobile phase plus sample

++ --

Conductivity EquationsConductivity Equations

Ohms Law

Conductivity DetectorConductivity Detector

Mobile phase

Mobile phase plus sample

Anion Analysis by ICAnion Analysis by IC

0.70

1.05

1.40

S

0.00 5.00 10.00 15.00 20.00 25.00Minutes

1

2

3

45

6 7

1. Fluoride2. Chloride

3. Nitrite

4. Bromide5. Nitrate

6. Phosphate

7. Sulfate

1 ppm2 ppm

4 ppm

4 ppm4 ppm

6 ppm

4 ppm

Column:

Eluent:

Flow rate:Injection vol.:

Detection:

Waters IC-Pak Anion HC

Borate/Gluconate2.0 mL/min

100L

Direct Conductivity


12/20

Detection in HPLC


Anion analysis by ICAnion analysis by IC

S

0.00

0.40

0.80

1.20

1.60 12

3

45

6

7

0.00 4.00 8.00 12.00 16.00 20.00 24.00

Minutes

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 HR

1.2 mM Sodium Carbonate/

1.2 mM Sodium Bicarbonate

1.0 mL/min

50L

Detection: Direct Conductivity after

Suppression

Detection: UV (PDA) at 214 nm

1. Fluoride

2. Chloride

3. Nitrite

4. Bromide

5. Nitrate

6. Phosphate

7. Sulfate

1 ppm

2 ppm

4 ppm

4 ppm

4 ppm

6 ppm

4 ppm

ApplicationsApplications

Sensitivities for compounds such as phenol, catecholamines,nitrosamines, and organicacidsare in thepicomole (nanogram)

range.

Themobilephasemustbe made electrically conductive, usuallyby the addition of a suitable salt:

Ion Exchange

Reversed Phase and Ion-Pair RP

No normal phase separations

DetectorsDetectors

UV/VISUV/VIS





MassMass--spectrometric (LC/MS)spectrometric (LC/MS)Evaporative light scatteringEvaporative light scattering DATA SYSTEM

DETECTION OF IONS

+

+

SAMPLE DESOLVATION

AND IONIZATION

LC/MSINTERFACE

SOURCE

ANALYZERION DETECTOR

HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ +

+

-+

+

++

++

-

+

+

+

CHROMATOGRAM

Typical LC/MS System ProgressionTypical LC/MS System Progression


13/20

Detection in HPLC


DATA SYSTEM

DETECTION OF IONS

+

+

SAMPLE DESOLVATIONAND IONIZATION

LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

++

++

-

+

+

+

CHROMATOGRAM

Typical LC/MS System ProgressionTypical LC/MS System Progression Transition from LC to MSTransition from LC to MS

State of Matter: LiquidLiquid to GasGas

Charge State: NeutralNeutral to IonIon

Pressure: 760760 torrtorr to 1010--55 to 10to 10--88 torrtorr

APCI MechanismAPCI Mechanism

Ionization producessolvent ions

The solvent ions reactwith analyte moleculesforming clusters

CoronaNeedle

X = Solvent Molecules e.g.H 2O, MeCN

M = Sample Molecule

Heated Nebulizer

xx

xH+ M

xx

xH+

M

x

x

MMH+

xx

xx

XH+x

xH+

MH+

ElectrosprayElectrospray IonizationIonization


14/20

Detection in HPLC


Positive or Negative?Positive or Negative?

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

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

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 Spectrum

Presence 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


15/20

Detection in HPLC


DeconvolutionDeconvolution byby MaxEntMaxEnt

HemoglobinHemoglobin

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

100

%

15125.0

15857.0

15149.0

15866.0

Multiply Charged Ions How Many Charges?

(M+H)+

(M+2H)2+

1 Da

0.5 Da

DATA SYSTEM

DETECTION OF IONS

+

+

SAMPLE DESOLVATION

AND IONIZATION

LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ +

+

-+

+

++

++

-

+

+

+

CHROMATOGRAM

Typical LC/MS System ProgressionTypical LC/MS System ProgressionMassMass

SpectrometerSpectrometerss

AnalyzersAnalyzers

T ime O f F l igh t Ma s s Ana ly ze rsT ime O f F l igh t Ma s s Ana ly ze rs

SOURCE

DETECTORREFLECTRON MODE

R E F LE C T R ON ONDETECTOR

LI NEAR MODEDRI FT TUBE

SOURCE

DETECTORREFLECTRON MODE

R E F LE C T R ON OF FDETECTORLI NEAR MODE

DRI FT TUBE

199

FTFT--ICRICR --SpectrometerSpectrometer

DCDC

DC

Source

Filament

Transferoptic

TrappingPlates

Transmitter Plates

ReceiverPlates

Sender

Elektroden Electrodes

Y

ZX

Magnetic FieltB

Starting with theStarting with the quadrupolequadrupole

Source

DetectorNonresonantIon

Resonant Ion

dc and Rfvol tages

V(t) = -Vdc

-Vrf

cost

V(t) = Vdc

+Vrf

cost

190

ElectronMultiplier

Inlet

RingElectrode,Rf

End Cap Electrode

AxialModulation

IonIon TrapsTraps

+ + ++++

++

IonSource

Slit

Magnetic sectorElectrostatic Sector

(ESA)

Detector

SlitNier-Johnson-Geometry(EB)


16/20

Detection in HPLC


DATA SYSTEM

DETECTION OF IONS

+

+

SAMPLE DESOLVATIONAND IONIZATION

LC/MSINTERFACE

SOURCE


HPLCMASS SPECTRUM

SORTING OF IONS

+

++

++ ++ ++

-+

+

++

++

-

+

+

+

CHROMATOGRAM

Typical LC/MS System ProgressionTypical LC/MS System Progression MS DetectorsMS Detectors

++ ++

--

-

Electron Multiplier(voltage setting lower than

Dynode)

Conversion Dynode(Voltage 1- 20 kV)

Current is measured

++

Mass Analyser

dynodedynode

phosphorphosphor

photomultiplierphotomultiplier

Photomultiplier

Electron Multiplier

Mass Spectrometer 3D RunMass Spectrometer 3D Run

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Int.

1:Mass Chromatogram5.054.65

3.82

0.74

8.62

5.65

8.02

10.62

Total-Ion-Current MS Chromatogram

with poor resolution

Mix

6 0 8 0 1 0 0 1 2 0 1 4 0 1 60 1 80 2 00 2 20 2 40 2 60 2 80 3 00 3 20 3 40m/z0

100

%

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

59.99

213.90

195.98

120.8068.9298.85

76.87128.82

170.92

222.87

235.87

240.88

263.87

264.85

267.91287.01 309.02

333.84

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Abs1:DiodeArray

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 Chromatogram

with 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

8.02

10.62

Selectivity of Mass Spectrometer DetectorSelectivity of Mass Spectrometer Detector

Extracted Ion Chromatogramof a single Component from

a mixture of Components

Mix

60 8 0 1 00 1 20 14 0 1 60 1 80 2 00 2 20 24 0 2 60 28 0 3 00 3 20 3 40m/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

2 6 7 .9 1 2 8 7 .0 1309.02

333.84


17/20


18/20

Detection in HPLC


Evaporative Light ScatteringEvaporative Light Scattering -- ELSELS

NebulizationDesolvation

DetectionTo detector cell

Nebulizer

Lamp

RayleighRayleigh ScatteringScattering Why the Sky is blueWhy the Sky is blue

Scattering is independent of the particles chemical properties, where:

N = # of particles

= 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 producegreater scattering

4

R2

I = I0

8 p4 Na2 (1 +cos2?)

Scattering ModelsScattering Models

If D/< 0.1then I = f (D6)

0.110then I = f (D2)

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

Scattering is dependent on particle size D Increasing

particle size

D C1/3

(Often see solute density )I a (Cb)

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

symmetrical scattering

Depends on which type of scattering ispredominant

Non-linear mass detector

vuse chromatography data softwarequadratic curve or log/log curve to fitcalibration curve

ELSDELSD vsvs UVUV


19/20

Detection in HPLC


ELSDELSD vsvs RIRI ELSD Used with Other DetectorsELSD Used with Other Detectors

ELSD to monitor allcompounds and determinepurity levels

Diode Array TIC

Min

ES+TIC

ELSD

24.5 7

Diode array TIC

PDA to monitor UV/Visfriendly compounds

Mass Spec to verify thatcompound has beensynthesized

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 ArrayTIC

See NonSee Non--UV Absorbing CompoundsUV Absorbing Compounds


20/20

Detection in HPLC


See Your Peaks FasterSee Your Peaks FasterUse of Gradients Versus IsocraticUse of Gradients Versus Isocratic

RI Detection

ELSD Detection

Estrogen analogues and salt

mV

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

110.00

Minutes

0.100. 200. 300. 40 0.500. 60 0.700. 800. 901. 00 1.10 1.201. 301. 40 1.501. 601. 70 1.801. 902. 00

NaCl-0

.207

Estradiol-0

.849

Estriol-1

.210

Evaporative LightEvaporative Light--scattering Detectorscattering Detector

detection by hplc

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