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

The Detector is the “Eye” of the HPLC System 300

6.Mannose 5.Glucose 4.Galactose 3.Glucosamine 2.Galactosamine 1.Fucose 1

Detector

2 6 3

mV

4

5

0.00

Control & Data Processing

0. 50

Minutes

20.00

Waste

Fraction Collector

a b c d Pump

Auto Sampler

flows 50-5000µL/min)

HPLC Column in Oven

Optical Bench of UV-VIS Detector

Detectors

Optical Light Path Taper-Cell Flow Cell

UV/VIS Beam Splitter Mirrors

Refractive index Fluorescence Electrochemical Conductivity Mass-spectrometric (LC/MS)

Dual Photodiode

Reference side Sample side

Beam-Defining Apparatus

Evaporative light scattering

Illumination Lens Deuterium Arc Lamp

Dr. Shulamit Levin,

Aperture Slit Rotating Diffraction Grating 190 to 600nm

Detection in HPLC UV Chromophores

Beer's Law Absorbance = Extinction Coefficient x Pathlength x Concentration

Reduce Pathlength

Reduce Concentration

UV Chromophores

UV-Vis chromophores

max Adenine Guanine Cytosine Thymine Uracil NADH NAD

Dr. Shulamit Levin,

260.5 275 267 264.5 259.5 340 260

Em x 10-3 @max E = 13.4 E = 8.1 E = 6.1 E = 7.9 E = 8.2 E = 6.23 E = 18

Detection in HPLC UV spectrum of 10 nM mobile phase

U.V. Cut-offs for some Common Solvents

1.00

1.00 0.90

0.90 0.80

0.80

TFA

0.70

AU

0.50 0.40

Acetic Acid

0.20 0.10

Water Methanol N-Propanol Acetonitrile THF Acetone Methyl acetate Ethyl Acetate Nitromethane

0.00

0.00

210.00220.00230.00240.00 250.00260.00270.00280.00 290.00 nm

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00 nm

pH 0.94

pH 2.78

1.00 0.90

1.00 0.90

0.80

0.80 0.70

Sodium Phosphate

0.60 AU

0.50

0.70

Formic Acid

0.60 205.2

0.40 0.30

0.50 0.40

0.30 0.20

0.20

UV Cutoff 180 205 205 190 225 330 260 260 380

Solvent

0.10 0.00 210.00 220.00

230.00

240.00

250.00 260.00 nm

270.00

280.00

0.00

290.00

265.1 210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

295.8

All wavelengths reported in nm.

nm

pH 2.26

pH 2.0

Diode Array Detector UV Detection of AccQ-Tag Amino Acid Derivatives

0.002

Asparagine

0.000 15.00

20.00

25.00

30.00

35.00 Minutes

40.00

45.00

50.00

Tryptophan

0.004

Lysine

0.006

Serine

0.008

Glutamic Acid

0.010

Hydroxyproline

0.012

Phenylalanine

0.016

Isoleucine

0.018

AU 0.014

Ornithine Leucine

0.020

Tyrosine Cysteic Acid Vaine Mtehionine

0.022

Glycine AMQ Glutamine Histidine NH3 Arginine Threonine Alanine

0.024

55.00

Dr. Shulamit Levin,

UV Cutoff

N-Heptane Cyclohexane Carbon tetrachloride Chloroform Benzene Toluene Methylene chloride Tetrachloroethylene 1,2-Dichloroethane

0.10

Aspartic Acid

AU

Solvent

0.40 0.30 0.20 0.10

0.30

AU

Remember Solvents chosen can affect detection!!

0.70 0.60 0.50

Proline Alpha-aminobutyric acid

AU

0.60

197 200 265 245 280 285 232 280 225

Detection in HPLC Extraction of 3D Data PDA Spectrum Index Plot DNPH Derivatives 0.25 ng Each Peak

Chromatogram Absorbance

2

Absorbance

Spectrum

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

1

nm

440.00 420.00 400.00 380.00

440.00 420.00 400.00 380.00

360.00 340.00 320.00 300.00 280.00 260.00

360.00 340.00 320.00 300.00 280.00 260.00

Time 0.0006

0.0006

0.0004 0.0004 AU

AU 0.0002

0.0002 0.0000 0.0000

Wavelength

Coelutionof 2 Peaks

A

B

Peak Purity Measurement

Coelution detection at a single wavelength

300.00

nm 250.00

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

200.00

Spectrum at maximum impurity is different

0.40

Maximum Impurity

AU

AU

Spectra at apex and inflection points are displayed

0.20

0.00 2.20

Dr. Shulamit Levin,

2.40

2.60

2.80

3.00

Detection in HPLC Maximum Impurity Detection

Determination of Peak Purity

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm 440.00 420.00 400.00 380.00

360.00

360.00

340.00 320.00 300.00 280.00 260.00

340.00 320.00 300.00 280.00 260.00

0.00030 0.00020

Coelution of DNPH Hexaldehyde and 2,5-Dimethylbenzaldehyde

Peak Purity

Standard

0.00030 0.00020

AU

AU 0.00010

Hexaldehyde

Spectral Matching

nm

0.00010

2,5-Dimethylbenzaldehyde

0.00000

Time

0.00000

-0.00010

Time

0.00010 18.40

18.60

19.00

18.80 Minutes

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

19.20

Different Spectra – 53 deg

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

10 deg of Spectral Contrast

Ethylparaben EthylPaba

Theophylline Dyphylline

Absorbance

Absorbance

200.00

240.00

280.00

Unknown

Absorbance

nm

440.00 420.00 400.00 380.00

320.00

nm

Dr. Shulamit Levin,

Similar spectra for structurally related compounds

230.00

250.00

270.00

nm

290.00

310.00

Detection in HPLC Spectral Contrast 0.5 Degrees

Analyte and 2 Impurities Very similar spectra, CH2 difference Spectral Contrast can differentiate these spectra

200.00

240.00

280.00

nm

320.00

Absorbance

Absorbance

Methylparaben Ethylparaben

Spectra of non-UV Active Compounds

210.00

230.00

250.00

nm

270.00

Refractive Index Detector Detectors

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

Dr. Shulamit Levin,

290.00

Detection in HPLC Differential Refractive Index Detector

Refractive Index Detector No sample = n

S R LAMP With sample = n+

LED

To Amplifier

S R

X = Const x n

Polymer Analysis

Sugar Analysis

60.00 40.00

Maltose

Lactose

20.00 0.00 mV

10300

600.00 550.00 500.00 450.00 MV 400.00 350.00 300.00 250.00 200.00

-20.00 -40.00 -60.00 -80.00 -100.00 -120.00

150.00 100.00

-140.00 -160.00

Bagel Extract 5.00

6.00

7.00

8.00 Minutes

50.00 0.00 9.00

10.00

11.00

Dr. Shulamit Levin,

96400

750.00 700.00 650.00

Sucrose

190000

Dextrose

1260000

100.00 80.00

2890000

120.00

800.00

5570

SampleName: GPC STDS SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard Fructose

192300 Dow 1683 18.00

20.00

22.00

24.00 Minutes

26.00

28.00

30.00

Detection in HPLC Lipids

Detectors 250 ng on column

1

1=Tristearin 2=Myristic acid

2

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

Del RIU

dRI sensitivity = 32X, 32°C 5.0

6.0

7.0

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

8.0

Minutes

Excitation-Emission Spectra

Fluorescence Process

Lifetime= 10-9 – 10-15 sec

Excitation

Energy Levels

 Maximum of Excitation Spectrum

 Maximum of Emission Spectrum

Stoke’s  shift

Excited States

Emission

Ground State

Dr. Shulamit Levin,

Detection in HPLC Fluorescence Detectors

Fluorescence Detector Optical Bench

Excitation filter

Photomultiplier tube

Cell Emission Grating

LAMP

Mirror

Emission Slit

Flow Cell

Torroidal Mirror

Emission filter

Excitation Grating

Beam Splittter Excitation Slit

Photo diode

Photomultiplier Torroidal Mirror Mirror

UV vs Fluorescence Sensitivity

Detectors AccQ-Tag amino acid analysis

AMQ

Fluorescence Excitation=250 nm Emission=395 nm

Response

UV 254 nm

20.00

40.00

60.00

Minutes

Dr. Shulamit Levin,

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

Detection in HPLC Electrochemical Detector

Electrochemical Detection of Catecholamines & Related Compounds

1

Reference Electrode

3

Working Electrode Analyte is oxidized or reduced

-

+

2

1. Norepinepherine 2. Epinepherine

150 ppb 200 ppb

3. Normetanepherine 4. Dopamine

50 ppb 200 ppb

5. Metanepherine 6. 3-Methoxytyramine 7. 4-Methoxytyramine

200 ppb 75 ppb 500 ppb

4

nAmps

5 7 6

Electrolyte (mobile phase) Auxiliary Electrode 0.00

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

2.00

4.00

6.00

8.00

Minutes

Pulsed Amperometric Detection of Monosaccharides

1. 2. 3. 4. 5.

300

1

mV

Fucose Galactosamine Glucosamine Galactose Glucose

UV/VIS Refractive index

6. Mannose

Fluorescence

2

3

Detectors

Electrochemical Conductivity Mass-spectrometric (LC/MS)

4 6 5

Evaporative light scattering 0.00 5.00

Minutes

20.00

Dr. Shulamit Levin,

10.00

12.00

Detection in HPLC Conductivity Detector

Conductivity Equations Ohm’s Law

Mobile phase

+

Mobile phase plus sample

Conductivity Detector

Anion Analysis by IC

Mobile phase

1. 2. 3. 4.

1.40 3 2

Mobile phase plus sample

1.05

Fluoride Chloride Nitrite Bromide

1 ppm 2 ppm 4 ppm 4 ppm

Column: Eluent: Flow rate: Injection vol.:

Waters IC-Pak Anion HC Borate/Gluconate 2.0 mL/min 100µL

5. Nitrate

4 ppm

Detection:

Direct Conductivity

6. Phosphate 7. Sulfate

6 ppm 4 ppm

1 4

S

5 7

6

0.70 0.00

Dr. Shulamit Levin,

5.00

10.00

Minutes

15.00

20.00

25.00

Detection in HPLC Anion analysis by IC

1.60

1

2 3

Detection:

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

Direct Conductivity after Suppression

1.20 4

5 7

0.80 S 0.40

6

0.00

1. 2. 3. 4. 5. 6.

Fluoride Chloride Nitrite Bromide Nitrate Phosphate

1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm

7. Sulfate

3

0.05

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

4 ppm

Detection: UV (PDA) at 214 nm

0.04

5

Column:

Ion Exchange

Waters IC-Pak Anion HR

Eluent:

1.2 mM Sodium Carbonate/ 1.2 mM Sodium Bicarbonate Flow rate: 1.0 mL/min Injection vol.: 50 µL

0.03 AU 0.02 0.01

Reversed Phase and Ion-Pair RP

4 0.00 0.00

4.00

8.00

12.00 16.00 Minutes

20.00

No normal phase separations

24.00

Typical LC/MS System Progression

Detectors ANALYZER

ION DETECTOR +

UV/VIS Refractive index

+ +

SOURCE +

+ +

Fluorescence

++ +

-

+ +

+

DETECTION OF IONS

+

+

+ +

+

+

+

SORTING OF IONS

+

SAMPLE DESOLVATION AND IONIZATION

Electrochemical Conductivity

LC/MS INTERFACE

Mass-spectrometric (LC/MS) Evaporative light scattering

HPLC MASS SPECTRUM

DATA SYSTEM

CHROMATOGRAM

Dr. Shulamit Levin,

Detection in HPLC Transition from LC to MS

Typical LC/MS System Progression ANALYZER

ION DETECTOR + + +

SOURCE +

+

+ +

+ +

-+ -

+

• State of Matter: Liquid to Gas

+

+

+

+

+ +

+

+

SORTING OF IONS

DETECTION OF IONS

+

• Charge State: “Neutral” to Ion SAMPLE DESOLVATION AND IONIZATION

LC/MS INTERFACE

HPLC

DATA SYSTEM

MASS SPECTRUM

• Pressure: 760 torr to 10-5 to 10-8 torr

CHROMATOGRAM

APCI Mechanism Electrospray Ionization

Ionization produces solvent ions x xH+

x M x

x

M x

XH+

xH+ M x

x x

Heated Nebulizer

X = Solvent Molecules e.g.H2O, MeCN M = Sample Molecule

xH+

MH+ x

x Corona Needle

MH+

x

The solvent ions react with analyte molecules forming clusters

Dr. Shulamit Levin,

Detection in HPLC Recognizing Multiply Charged Ions Positive or Negative?

Basic Compounds (-NH2)

(M+H)+

Acidic Compounds (-CO2H, -OH)

(M-H)-

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 n charge

Mass = 1/2 (M+2H) Mass = 1/n (M+nH)

Isotopes of doubly charged ions are separated by 0.5 Da

Hemoglobin Spectrum Presence of More Than One Charged Envelope

Mass Range Multiply Charged Molecules

1081.60

Horse Heart Myoglobin

100

n = 23, m/z = 738 n = 22 n = 21 n = 20

1164.52 1009.36 1261.64

n = 19 n = 18 n = 17 n = 16, m/z = 1060

%

1133.92

1376.08

1221.18 1323.14

1058.83

0

Acquired Mass range

Calculated Mass

Dr. Shulamit Levin,

1000

1050

1100

1150

1200

1250

1300

1350

m/z 1400

Detection in HPLC Multiply Charged Ions – How Many Charges?

Deconvolution by MaxEnt Hemoglobin

1 Da

15125.0

100

(M+H)+

% 15857.0 15866.0

0.5 Da

(M+2H)2+

15149.0 0

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

mass

Time Of Flight Mass Analyzers

Mass Spectrometer’s Analyzers

FT-ICR-Spectrometer

Typical LC/MS System Progression

Magnetic Fielt B

REFLECTRON OFF

SOURCE

DRIFT TUBE

DETECTOR LINEAR MODE

DETECTOR REFLECTRON MODE

Y SOURCE

Z X

Source

ANALYZER

+

+ + +

-+ -

Filament

+ +

DC

+

+

TransferopticDC

+

+

Transmitter Plates

Ion Traps

199

+

+ +

DRIFT TUBE

DETECTOR REFLECTRON MODE

Receiver Plates

+ +

REFLECTRON ON

TrappingPlates Electrodes DC

ION DETECTOR + +

SOURCE

Elektroden Sender

SORTING OF IONS

+

End Cap Electrode

Axial Modulation

DETECTION OF IONS

+ Ring Electrode, Rf

++ + + + +++ Inlet

SAMPLE DESOLVATION AND IONIZATION

Starting with the quadrupole

LC/MS INTERFACE

Electron Multiplier 190

Resonant Ion Nonresonant Ion

HPLC MASS SPECTRUM

DATA SYSTEM

Detector

Nier-Johnson-Geometry(EB) Slit

Electrostatic Sector

CHROMATOGRAM

Magnetic sector

V(t) =- Vdc- Vrf cost

(ESA) V(t) =Vdc + Vrf cost Detector dc andRfvoltages Source

Dr. Shulamit Levin,

Slit Ion Source

DETECTOR LINEAR MODE

Detection in HPLC Typical LC/MS System Progression ANALYZER

MS Detectors Electron Multiplier

ION DETECTOR + + +

SOURCE

+ +

-

+ +

+

+

+

Mass Analyser

+ +

-

+

+

+

+

+

+

+

SORTING OF IONS

+ + + +

+ + -

ConversionDynode (Voltage 1- 20 kV)

DETECTION OF IONS

+

Electron Multiplier (voltage setting lower than Dynode)

SAMPLE DESOLVATION AND IONIZATION

Photomultiplier

Currentismeasured

LC/MS INTERFACE

HPLC

dynode

DATA SYSTEM

MASS SPECTRUM

CHROMATOGRAM

phosphor photomultiplier

Mass Spectrometer 3D Run Int.

Mixture Abs

Mix

59.99

294.00

213.90

%

222.87

nm 0 200210220230240250260270280290300310

8.62

235.87 263.87

5.65 195.98

8.02

68.92 76.87

0 60

Mixture

4.655.05

Int.

10.62 3.82

80

120.80 98.85 100

120

128.82 140

170.92 160

240.88

200

220

4.00

6.00

8.00

Mix

1: Scan4.34e5 ES+

(10.696) 100

Total-Ion-Current MS Chromatogram with poor resolution

262.87

0

2.00

4.00

6.00

10.00

8.00

Time

Mix

213.90

%

222.87

100 195.98

0

8.62

60

263.87 264.85 240.88

98.85 120.80 128.82

76.87

100 80

120

140

160

267.91

287.01

280

300

309.02

170.92

200 180

220

240

1: Scan ES+ 262.87 4.59e5

10.60

235.87

1: Mass Chromatogram 68.92

4.65 5.05

m/z

320 333.84 340

59.99

Mixture Int.

309.02

280

Extracted Ion Chromatogram ofasingleComponentfrom amixtureofComponents

Time

10.00

260

8.02 10.62

2.00

240

300

3.82

0.74

264.85 267.91 287.01

180

8.62 5.65

0.74

0

1: Scan 4.34e5 ES+

262.87

(10.696) 100

246.00

1:DiodeArray

4.655.05

Selectivity of Mass Spectrometer Detector

210.00

UV-DiodeArrayChromatogram with poor resolution

260

333.84 340

m/z

320

5.65 8.02

% 10.62 3.82 0.74

0 0

2.00

4.00

6.00

8.00

10.00

Time

Dr. Shulamit Levin,

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Time

Detection in HPLC LC-MS Analysis

Triple Quadrupoles: MS-MS Modes Daughter (Product) Ion Spectra

XTerra™ MS C18, 2.1 x 50 mm ( 5 µm)

MS1 MS1

N

2.56

100

295 8.11e4

295

100

Collision Collision Cell

MS2 MS2

4.59e4

N

(4)

Multiple Multiple MultipleReaction Reaction ReactionMonitoring Monitoring Monitoring

296 Static Static

Typically used in Quantitative Work of

Scanning Scanning

0

0 1.57

100

280 2.21e5

Triple Quadrupoles N

100

280 1.13e5

(2)

Parent (Precursor) Ion Spectra

O

281

0

0 2.16

100

264 1.26e5

100

260 1.53e5

100

264

(3)

Scanning O OH

260

N H

(1) 0 100

1.29

1.57 2.16

0 100 125

TIC 3.50e5

2.56

2.00

3.00

4.00

Static

261

175 200 225 Mass/Charge (m/z)

250

275

Constant Neutral Loss Spectra

300

MS1

Collision Collision Cell Cell

MS2

10 L injection of 200 ng/mLsample (in 40% 5.00

Scanning Scanning

Scanning Scanning

Time (min)

Ding

Typical Quantitative Analysis Using Triple Quadrupoles: SimultaneousMRManalysisof6amphetamines Std_017 100

MDEA

0 Std_017 100 %

Methamphetamine

0 Std_017 100 %

Amphetamine

3.80

0 Std_017 100 % 0 Std_017 100 %

2.95

1.00

1.50

2.00

2.50

MRMof12ChannelsES+ 135.8>91 4.29e6

Refractive index Fluorescence

MRMof12ChannelsES+ 193.65>162.7 6.48e6

3.95

MDMA 0.50

UV/VIS

MRMof12ChannelsES+ 179.8>163 4.13e6

3.20

MDA

MRMof12ChannelsES+ 149.95>91 1.49e7

MRMof12ChannelsES+ 165.85>148 2.05e7

2.25

Ephedrine

0 Std_017 100 % 0

Detectors

MRMof12ChannelsES+ 208>162.9 1.73e7

5.10

%

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

MS2

Static

9.25e4

MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline, 4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN 0.2 mL/min

0 1.00

150

Collision Cell

MS2

7.67e4

0 1.29

Collision Cell

265

233

NH

0 100

MS1

MS1

8.00

Time

Highly specific and sensitive chromatograms

Dr. Shulamit Levin,

Electrochemical Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering

Static

Detection in HPLC Rayleigh Scattering – Why the Sky is blue

Evaporative Light Scattering - ELS

I = I0 8 p4 Na2 (1 +cos2?) 4R2

Lamp

• Scattering is independent of the particle’s chemical properties, where: To detector cell

Detection

– 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 produce greater scattering

Nebulizer

Nebulization

Desolvation

ELSD vs UV

Scattering Models Scattering is dependent on particle size “D” Increasing particle size If D/ 0.1 then I = f (D6)

0.110 then I = f (D2)

D  C1/3 (Often see solute density) I a (Cb) With 2>b>2/3 2 is the limiting value for Rayleigh symmetricalscattering

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

Depends on which type of scattering is predominant Non-linear mass detector vuse chromatography data software quadratic curve or log/log curve to fit calibration curve

Dr. Shulamit Levin,

Detection in HPLC ELSD vs RI

ELSD Used with Other Detectors PDAtomonitorUV/Vis friendlycompounds DiodeDiodeArrayarrayTICTIC

MassSpectoverifythat compoundhasbeen ES+TIC

synthesized

ELSD to monitor all compounds and determine purity levels

ELSD

2

7

4.5 Min

Not a Universal Detector

See Non-UV Absorbing Compounds

Typically Used with Other Detectors

ELSD

UV TIC

Diode Array TIC

MS ES+ TIC

ELSD 1

3

Min

Erythromycins Separation

Dr. Shulamit Levin,

5

Detection in HPLC See Your Peaks Faster Evaporative Light-scattering Detector

Use of Gradients Versus Isocratic

90.00 80.00

Estriol - 1.210

100.00

Estradiol - 0.849

110.00

RI Detection

NaCl - 0.207

Estrogen analogues and salt

mV

70.00 60.00 50.00 40.00 30.00 20.00

ELSD Detection

10.00 0.00 0.10 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 Minutes

Dr. Shulamit Levin,