Detection Qualification and Types of Detectors in HPLC.pdf

7/26/2019 Detection Qualification and Types of Detectors in HPLC.pdf

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Detection Qualification and Typesof Detectors in HPLC

Dr. Shulamit LevinMedtechnica

http://www.forumsci.co.il/HPLC

Detection in HPLC

Dr. Shulamit Levin, Medtechnica


2/17

The most common HPLC detectors:UV/Vis

Fixed wavelengthVariable wavelengthDiode array

Refractive indexFluorescenceElectrochemical

Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering

DetectorsBeer's LawAbsorbance = Extinction Coefficient x

Pathlength x Concentration

Reduce Pathlength Reduce Concentration

Only for monochromatic light

Beer's Law

Absorbance = Extinction Coefficient x Pathlength x Concentration

ABExtinction Coefficient

Concentration Concentration

A b s o r b a n c e

A U

T r a n s m

i t t a n c e

%AU %T T0 100 1.0001.0 10 0.1002.0 1 0.0103.0 0.1 0.001

T=solvent/sample A=log(solvent/sample)

A B

*

*

*

*

**

*

*

*

*

Single Wavelength UV Detector

Optical Light Path

Mercury, Zincor Cadmium

Source Lamps

Dual

Photodiode

Sample side

Reference side

Wavelength Aperture Plate Wavelength

Filter

Flow Cell

Detection in HPLC



3/17

UV-VIS Detector Optical Bench

Deuterium Arc Lamp

RotatingDiffraction

Grating190 to 600nm

Taper-CellFlowCell

Beam Splitter Mirrors

DualPhotodiode

ApertureSlit

IlluminationLens

Beam-Defining Apparatus

Optical Light Path

Sample side

Reference side

UV Detection of AccQ-Tag AminoAcid Derivatives

SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard

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

A s p a r t

i c A c i

d

G l u t a m

i c A c i

d

H y d r o x y p r o l

i n e

S e r

i n e

A s p a r a g

i n e

A M Q

G l y c i n e

G l u t a m

i n e

H i s t i d i n e

N H 3

T h r e o n i n e

A r g

i n i n e

A l a n i n e

P r o

l i n e

A l p h a - a m

i n o b u t y r

i c a c

i d

T y r o s i n e

C y s

t e i c A c i

d

V a

i n e

M t e h i o n

i n e

O r n

i t h i n e

L e u c

i n e L y

s i n e

P h e n y

l a l a n i n e

T r y p t o p

h a n I s

o l e u c i n e

Liquid fromcolumn

Extraction of 3D Data

Wavelength

A b s o r b a n c e Spectrum

Time

A

b s o r b a n c e

Chromatogram

1

2

Detection in HPLC



4/17

Minutes

nm

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

260.00 260.00280.00 280.00300.00 300.00

320.00 320.00

340.00 340.00360.00 360.00

380.00 380.00400.00 400.00420.00 420.00

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

PDA Spectrum Index Plot DNPH Derivatives 0.25 ng Each Peak

Maximum Impurity Detection

260.00 260.00

280.00 280.00300.00 300.00320.00 320.00340.00 340.00360.00 360.00380.00 380.00

400.00 400.00420.00 420.00

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 ng 360nm 996PDA 360.0 nm

Hexaldehyde 2,5-Dimethylbenzaldehyde

Coelution of DNPHHexaldehyde and2,5-Dimethylbenzaldehyde

PDA and fluorescent DetectorComparisons for Aflatoxin Analysis

SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Typ e: Standard

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

mV

Minutes

AflatoxinG2

AflatoxinG1

AflatoxinB2

AflatoxinB1

UV at 360 nm 31 point smoothedUV at 360 nmFluorescence 365 ex 455 em

Photo-diode arrayChromatographic and Spectral Sensitivity

1.6 2.4 3.2 A b s o r b a n c e

Minutes

0.07 mAU0.15 ng Ethylparaben

210.0 250.0 290.0nm

0.07mAU0.530 AU

Detection in HPLC



5/17





Detectors

LAMP

SLED or Incandescent

R

R

S

To Amplifier

No sample = n

With sample = n+

X = Const x n

Differential Refractive Index Detector

Refractive Index Detection withDifferential RI - Sugars

-160.00

-140.00

-120.00

-100.00

-80.00

-60.00

-40.00

-20.000.00

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

Refractive Index Detection withDifferential RI - Sugars

SampleName: Sugar Stds -500 ng each

0.0000002

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

Minutes

Sucrose

DextroseFructose

00000000

0.0000004

del

RIU

Detection in HPLC



6/17

Refractive Index Detection withDifferential RI - Polymers

0.0050.00

100.00

150.00200.00250.00300.00

350.00400.00450.00

500.00550.00600.00650.00

700.00750.00800.00

18.00 20.00 22.00 24.00 26.00 28.00 30.00

MV

Minutes

1 2 6 0 0 0 0

9 6 4 0 0

5 5 7 0

SampleName: GPC STDS

Dow 1683

2 8 9 0 0 0 0

1 9 0 0 0 0

1 0 3 0 0

192300

SensitivityRefractive Index Detector

D e

l R I U

5.0 6.0 7.0 8.0Minutes

1 2

250 ng on column1=Tristearin2=Myristic acid

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

dRI sensitivity =32X, 32C



Refractive index

FluorescenceElectrochemical


DetectorsFluorescence Detectors

Excitation filter

LAMP

Cell

Emission filter

Photomultiplier

Short pass - transmits all wavelengths below a specified cutoff Long pass - transmits all wavelengths above a specified cutoff Band pass - blocks all wavelengths outside a specified band

Detection in HPLC



7/17

Fluorescence Detector Optical Bench

LAMP

Photomultipliertube

EmissionGrating

ExcitationGrating

Mirror

Torroidal Mirror

Beam Splittter

FlowCell

Mirror

Torroidal Mirror

Photodiode

ExcitationSlit

Emission

Slit

SensitivityFluorescence Detector

0.1 pg Anthracene

Excitation = 251 nmEmission = 406 nm

Minutes

0.0 1.0 2.0 3.0 4.0 5.0

5 mV

mV

Fluorescence vs. UV Detection

Minutes

AMQ

20.00 40.00 60.00

R e s p

o n s e

AccQ-Tag amino acidanalysis

FluorescenceExcitation=250 nm

Emission=395 nm

UV 254 nm



Refractive index



Detectors

Detection in HPLC



8/17

Electrochemical 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 ofCatecholamines & Related Compounds

1 . N or ep in eph er in e 1 50 pp b2. Epinepherine 200 ppb3. Normetanepherine 50 ppb4. Dopamine 200 ppb5. Metanepherine 2 00 ppb6. 3-Methoxytyramine 75 ppb

7. 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 Amperometric Detectionof Monosaccharides

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

20.00Minutes5.00

mV

0.00

300

12

34

5

6



Refractive index



Detectors

Detection in HPLC



9/17

Conductivity Detector

Mobile phase

Mobile phase plus sample

Conductivity Detection of SevenAnion Standard

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. 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 LDirect Conductivity

Conductivity and UV Detectors inSeries

S

0.00

0.40

0.80

1.20

1.60 1 2 3

45

6

7

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/min50L

Detection: Direct Conductivity afterSuppression

Detection: UV (PDA) at 214 nm

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

1 ppm2 ppm4 ppm4 ppm4 ppm6 ppm4 ppm

Applications

Sensitivities for compounds such as phenol, catecholamines,nitrosamines, andorganic acids arein thepicomole (nanogram)range.

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

Ion Exchange

Reversed Phase and Ion-Pair RP

No normal phase separations

Detection in HPLC



10/17





Detectors

The scattered light is detected by asilicone photodiode located at a

90 angle from the laser. Thephotodiode produces a signalwhich is sent to the analogoutputs for collection. A light trapis located 180 from the laser tocollect any light not scattered byparticles in the aerosol stream.

EVAPORATIVE LIGHT SCATTERING

Radioactive Detector Primarily used for the measurement of 3H, 14C, and 32P,beta-emitters and many soft gamma and positron emittersencountered in bio-medical researchand pharmaceuticalqualitycontrol.

How LC-MS Works

MassSpectrum

HPLC

Source Analyzer IonDetector

DataSystem

LC/MSInterface

Separation

Desolvation

Ionization Sorting of Ions Detection DateProcessing

x

Detection in HPLC



11/17

Mixture

2.00 4.00 6.00 8.00 10.00Time0

Int.1: MassChromatogram

5.054.65

3.82

0.74

8.62

5.65

8.02

10.62

Mixture

2.00 4.00 6.00 8.00 10.00Time0


Mixture

2.00 4.00 6.00 8.00 10.00Time0


5.054.65

3.82

0.74

8.62

5.65

8.02

10.62

T otal- I on-Current Chromatogram

with poor resolution

Mix

60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0

100

%

(10.696) 1: ScanES+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

267.91287.01 309.02333.84

Mix

60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0

100

%

(10.696) 1: ScanES+4.34e5

Mix

60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0

100

%

(10.696) 1: ScanES+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

267.91287.01 309.02333.84

LC-MS

190

Electron Multiplier

I n l e t

RingElectrode,Rf

EndCapElectrode

AxialModulation

IonIon TrapsTraps

+ + +++ + ++

Types of Mass

SpectrometersAnalyzers

Types of Mass

SpectrometersAnalyzers

Ti m e O f F l i g h t M a s s A n a l y z e r sTi m e O f F l i g h t M a s s A n a l y z e r s

SOURCE

DETECTORREFLECTRON MODE

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

DRIFT TUBE

SOURCE


R E F L E C T R O N O F F DETECTORLINEAR MODEDRIFT TUBE

Ti m e O f F l i g h t M a s s A n a l y z e r sTi m e O f F l i g h t M a s s A n a l y z e r s

SOURCE


R E F L E C T R O N O NDETECTORLINEAR MODEDRIFT TUBE

SOURCE


R E F L E C T R O N O F F D E T E C T O RL I N E A R M O D EDRIFT TUBE

2 2 0

IonSource

Slit

Magnetic sector

Electrostatic Sector

( E S A )

Detector

SlitNier-Johnson-Geometry (EB)

SectorSector Mass SpectrometersMass S pectrometers

199

FTFT -- ICR ICR --SpectrometerSpectrometer

DCDC

DC

Source

Filament

Transferoptic

Trapping Plates

TransmitterPlates

Receiver Plates

Sender

Elektroden Electrodes

Y

ZX

Magnetic Fielt B

77

IonSource

Detector

nonresonant Ion

resonantIon

dcandRfVoltages

TheThe Quadrupole AnalysatorQuadrupole Analysator

Time (min)

1.00 2.00 3.00 4.00 5.000

100

0

100

0

100

0

100

0

100 MW=2958.11e4

2.56

MW=2802.21e5

1.57

MW=2641.26e5

2.16

MW=2601.53e51.29

TIC3.50e5

1.571.29 2.16

2.56

Ding Time (min)

1.00 2.00 3.00 4.00 5.000

100

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

1008.11e4

2.56

2.21e51.57

1.26e52.16

1.53e51.29

TIC3.50e5

1.571.29 2.16

2.56

Ding

10 L 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

1 00 1 25 1 50 1 75 2 00 2 25 2 50 2 75 3 00Mass/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 NH

OH (1)

(2)

(3)

(4)

10 L 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

1 00 1 25 1 50 1 75 2 00 2 25 2 50 2 75 3 00Mass/Charge (m/z)

0

100

0

100

100

0

1004.59e4295

296

1.13e5280

281

7.67e4264

233 265

9.25e4260

261

0O

N

O

N

N

N

NHNH

O NH

OH

O NH

OH (1)

(2)

(3)

(4)

Fast LC-MS Analysis2.1 x 50 mm ( 5 m) HARDWARE - ES/APCI Ion SourceHARDWARE - ES/APCI Ion Source

ESI and APCI are easily interchangeable in seconds withoutventing the system

ESI and APCI use a unique counter electrode to optimizesampling from the liquid spray and to aid sample desolvation

Automatic Probe recognition

Detection in HPLC



12/17

ESI-MS Ion FormationESI-MS Ion Formation

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

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 rangeAcquired Mass range

APCI Probe Equipped With aAPCI Probe Equipped With aHeated Nebulizer Heated Nebulizer

From LCColumn

NebulizerGas

Heater Block

Makeup Gas

Corona Discharge Needle

S M S S MSS S S M MS

MSH+SH+

SH+

SH+

SH +M

+ MH +S

+ ToMass Analyzer

760 torr vacuum

Generates molecular weight and structural information APCI flow rate: 0.2 to 2mL/minute Option: Crossflow interface

Liquid Chromatography- Mass Spectrometery(LC-MS)

Detection in HPLC



13/17

Triple Quadrupoles MS-MS

Modes of Operation

Triple Quadrupoles MS-MS

Modes of Operation

Multiple Reaction Monitoring

Typically used in Quantitative Work ofTriple Quadrupoles



MS 1 CollisionCell

MS2

StaticStatic





MS1 CollisionCell

MS2

StaticStatic

D a u g h t e r ( P r o d u c t ) I o n S p e c t r aD a u g h t e r ( P r o d u c t ) I o n S p e c t r a

M S 1 C o l l i s i o nC e l l

M S 2

S c a n n i n gS t a t i c

D a u g h t e r ( P r o d u c t ) I o n S p e c t r aD a u g h t e r ( P r o d u c t ) I o n S p e c t r a

M S 1 C o l l i s i o nC e l l

MS 2

S c a n n i n gS t a t i c

Parent (Precursor) Ion Spect raParent (Precursor) Ion Spect ra

M S1 Col li sionCell

MS 2

Sta t i cScann ing

Parent (Precursor) Ion Spect raParent (Precursor) Ion Spect ra

M S1 Col li sionC e l l

MS 2

StaticScanning

C o n s t a n t N e u t r a l L o s s S p e c t r aC o n s t a n t N e u t r a l L o s s S p e c t r a

M S 1 Col li s i onCell

MS 2

S c a n n i n gS c a n n i n g

C o n s t a n t N e u t r a l L o s s S p e c t r aC o n s t a n t N e u t r a l L o s s S p e c t r a

M S1 Co l li s i onC e l l

MS 2

S c a n n i n gScann ing

BASIC DETECTOR REQUIREMENTS

Low drift and noise level (trace analysis).High sensitivity.

Fast response for high performance systems.Wide linear dynamic range (quantitation).Low dead volume (minimal peak broadening & remixing of theseparated bands).Insensitivity to changes in type of solvent, flow rate, andtemperature.Operational simplicity and reliability.Tuneable, so that detection can be optimized for differentcompounds.Preferably non-destructive.

An ideal LC detector should have the following properties:

Detector Criteria

SelectivitySensitivity and detection limitStabilityLinear range

Dynamic RangeReproducibilityEffect on peak shapeMaintenance

PROPERTIES OF DETECTORS

SELECTIVITY

UNIVERSALSPECIFIC

A selective detector allows one to seeonly components of interest despite oftheir co-elution with any others.

Detection in HPLC



14/17


CONCENTRATION

RE

SPONSE

Sensitivity of a detector is not the minimumamount that can be detected.

SENSITIVITY PROPERTIES OF DETECTORS

h noise

h signal

h signal

= 2 x

h noise

DETECTION LIMIT

Limit of detectionLowest concentration that can be detectedSignal-to-noise ratio of 2:1 or 3:1

Limit of quantitationLowest concentration that can be determinedwith acceptable precisionSignal-to-noise ratio of 10:1

Detector Sensitivity Chromatographic SensitivitySignal-to-Noise Ratio

Noapparentnoise

Minutes

2.8 3.0 3.2 3.4

0.2 AU0.001AU

Minutes

2.00 3.00 4.00

Noise

Detection in HPLC



15/17

Increase Signal-to-Noise Ratio

Signal-to-noise (S/N)is peak height to

noise

Increase S/N byincreasing peakheight

Increase S/N bydecreasing noise

3:1

6:1

8:1

Factors Increasing UV SignalIncrease sample concentrationIncrease injection volumeChoice of wavelength (s)Low volume flow cell

Flow cell pathlength

Optics bench designLamp energyWavelengthsMobile phase compositionPump pulsationElectronics

Factors Affecting Noise in UV Detectors

Chromatographic SensitivitySingle Wavelength vs Maxplo t

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

A U

0.0 2.0 4.0 6.0

Minutes

220 nm

0.0 2.0 4.0 6.0Minutes

Maxplot


BASELINE STABILITY

SHORT RANGE

LONG RANGE

DRIFT

NOISE

Detection in HPLC



16/17


17/17


TEMPERATURE, FLOW RATE, EL ECTRONICS

REPEATABILITY OF RESPONSE


MAINTENANCE AND COST

EASY HANDLING OF FLOW-CELL

EASY A/D CONVERSION

SAFETY

Detection in HPLC


Documents

Detection Qualification and Types of Detectors in HPLC.pdf