detection qualification and types of detectors in...

Detection Qualification and Types of Detectors in HPLC

Dr. Shulamit Levin

Detection in HPLC

Dr. Shulamit Levin,

Tra

nsm

itta

nce

%

Ab

sorb

ance

AU

Conductivity Mass-spectrometric (LC/MS)

Evaporative light scattering

Detectors The most common HPLC detectors: UV/Vis

Fixed wavelength

Variable wavelength

Diode array

Refractiveindex

Fluorescence

Electrochemical

Less common:

Reduce Pathlength Reduce Concentration

Beer's Law Absorbance = Extinction Coefficient x

Pathlength xConcentration

Only for monochromatic light

Beer's Law Absorbance = Extinction Coefficientx Pathlength x Concentration

A B Extinction Coefficient

AU

0

%T

100

T

1.000

1.0 10 0.100

2.0 3.0

1 0.1

0.010 0.001

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

A B

*

*

*

*

Concentration *

*

*

*

*

Concentration

*

Mercury, Zinc

orCadmium SourceLamps

Dual

Photodiode

Sample side

Reference side

Single Wavelength UV Detector Optical Light Path

Wavelength

Aperture Plate Wavelength

Filter

FlowCell

Detection in HPLC

Dr. Shulamit Levin,

AM

Q

Gly

cin

e G

luta

min

e

Asp

arti

c A

cid

Glu

tam

ic A

cid

His

tidin

e A

rgin

ine Th

reon

ine

Ala

nin

e

Pro

lin

e

Alp

ha-

amin

obuty

ric

acid

Hyd

roxyp

roli

ne

Ser

ine

Isole

uci

ne

Orn

ithin

e L

euci

ne Lys

ine

Ab

sorb

an

ce

Ab

sorb

an

ce

Try

pto

ph

an

Ph

enyl

alan

ine

Tyr

osi

ne

Cys

teic

Aci

d

Vai

ne

Mte

hio

nin

e

Asp

arag

ine

NH

3

Deuterium

Arc Lamp Rotating

Diffraction Grating

190 to 600nm

Flow

Cell

Beam Splitter Mirrors

Dual

Photodiode

Aperture Slit Illumination Lens

UV-VIS Detector Optical Bench Optical Light Path Taper-Cell

Reference side

Sample side

Beam-Defining Apparatus

UV Detection of AccQ-Tag Amino Acid Derivatives SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard

0.004

0.002

0.000

0.006

0.024

0.022

0.020

15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

0.018 0.016

AU 0.014 0.012 0.010 0.008

Liquid from column

Wavelength

Spectrum

Time

Minutes Extraction of 3D Data Chromatogram

1

2

Detection in HPLC

Dr. Shulamit Levin,

CoelutionofDNPH

Hexaldehydeand

2,5-Dimethylbenzaldehyde Hexaldehyde

2,5-Dimethylbenzaldehyde

Ab

sorb

an

ce

nm

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

360.00

340.00

320.00

360.00

340.00

320.00

440.00

420.00

400.00

380.00

nm

0.0000 0.0000

0.0004

0.0002

300.00

280.00

260.00 0.0006

300.00

280.00

260.00 0.0006

AU

0.0004 AU

0.0002

380.00

360.00

340.00

380.00

360.00

340.00

440.00

420.00

400.00

nm nm

-0.00010 0.00010

0.00020

0.00010

0.00000

0.00020

0.00010

0.00000

320.00 300.00 280.00 260.00 0.00030

320.00

300.00

280.00

260.00 0.00030

18.40 18.60 18.80 Minutes

19.00 19.20

AU AU

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

440.00

420.00

400.00

Minutes PDA and fluorescent Detector Comparisons for Aflatoxin Analysis SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Type: Standard

5.00

0.00

10.00

20.00

15.00

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

55.00

50.00

45.00

40.00

35.00

mV30.00

25.00

Minutes

Aflatoxin B1

UV at 360 nm 31 point smoothed UV at 360 nm

Fluorescence 365 ex 455 em Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

Photo-diode array Chromatographic and Spectral Sensitivity

1.6 2.4 3.2

Minutes

0.07 mAU

0.15 ng Ethylparaben

210.0 250.0 290.0

nm

0.07mAU

0.530 AU

Detection in HPLC PDA Spectrum Index Plot DNPH Derivatives 0.25 ng Each Peak Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm 440.00 420.00 400.00 380.00

Dr. Shulamit Levin,

Fluorescence Electrochemical

Less common:

Conductivity

Mass-spectrometric (LC/MS)

Detectors

The most common HPLC detectors:

UV/Vis

Fixed wavelength

Variable wavelength

Diode array

Refractive index

LAMP

LED or

Incandescent ToAmplifier

With sample = n+�

S

R

Differential Refractive Index Detector No sample = n

S

R

-160.00

-140.00

-20.00 -40.00 -60.00 -80.00

-100.00

-120.00

20.00 0.00

40.00

60.00

100.00 80.00

120.00

5.00 6.00 7.00 8.00 9.00 10.00 11.00

mV

Minutes

Fructose

Dextrose Sucrose

Maltose Lactose

Evaporative light scattering Refractive Index Detection with Differential RI - Sugars SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard

Bagel Extract

�X = Const x �n

Refractive Index Detection with Differential RI - Sugars

SampleName: Sugar Stds -500 ng each

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

Minutes

Sucrose

Dextrose

Fructose

0.0000002 00000000

0.0000004

del RIU

Detection in HPLC

Dr. Shulamit Levin,

1260000

190000

96400

10300

5570

2890000

Del

RIU

50.00 0.00

150.00

100.00

550.00

500.00

450.00

400.00

350.00

300.00

250.00

200.00

700.00

650.00

600.00

20.00 22.00 24.00 26.00 28.00 30.00

MV

Dow 1683 18.00

192300

5.0 7.0 8.0 6.0 Minutes

1 2

Sensitivity Refractive Index Detector 250 ng on column

1=Tristearin

2=Myristic acid

Styragel HR 0.5, 4.6 x 300 mm,

35°C, 0.35 mL/min dRI sensitivity =

32X, 32°C

Fixed wavelength

Minutes Detectors


UV/Vis

Fluorescence Detectors

Excitation filter

Cell

LAMP 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 Refractive Index Detection with

Differential RI - Polymers SampleName: GPC STDS

800.00 750.00

Variable wavelength Diode array

Refractiveindex

Fluorescence

Electrochemical

Less common: Conductivity


Evaporative light scattering Dr. Shulamit Levin,

Res

po

nse

LAMP

Fluorescence Detector Optical Bench Photomultiplier

Emission

Grating

Excitation Grating

Mirror

Torroidal Mirror

Beam Splittter

Flow Cell

tube

Mirror

Photo

diode Excitation Slit

Emission Slit

Torroidal Mirror

Sensitivity Fluorescence Detector

0.1 pg Anthracene

Excitation = 251 nm

Emission = 406 nm

0.0 1.0 2.0 3.0 4.0 5.0

5 mV

mV

20.00 40.00

Minutes

60.00

Fluorescence vs. UV Detection AMQ

AccQ-Tag aminoacid

analysis

Fluorescence

Excitation=250 nm

Emission=395 nm

UV 254 nm

Evaporative light scattering

Minutes Detectors


UV/Vis

Fixed wavelength

Variable wavelength

Diode array

Refractiveindex

Fluorescence

Electrochemical



Detection in HPLC

Dr. Shulamit Levin,

Electrochemical Detector

Reference Electrode

Working Electrode

Analyte is oxidized or reduced

+ -

Electrolyte (mobile phase) Auxiliary Electrode

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

Electrochemical Detection of

Catecholamines & Related Compounds

1.

2.

Norepinepherine

Epinepherine

150ppb

200ppb

3.

4.

Normetanepherine

Dopamine

50 ppb

200ppb

5.

6.

7.

Metanepherine

3-Methoxytyramine

4-Methoxytyramine

200ppb 75 ppb 500 ppb

2.00 4.00 6.00 8.00 10.00 12.00

Minutes

0.00

nAmps

1 2

3

4 5

6

7

Pulsed Amperometric Detection of Monosaccharides

1.

2.

3.

4.

5.

Fucose

Galactosamine

Glucosamine

Galactose

Glucose

6. Mannose

20.00 Minutes

mV

0.00 5.00

300

1 2

3 4

5

6

UV/Vis

Fixed wavelength

Variable wavelength

Diode array Refractiveindex

Fluorescence

Electrochemical


Mass-spectrometric (LC/MS) Evaporative light scattering

Detectors The most common HPLC detectors:

Detection in HPLC

Dr. Shulamit Levin,

Conductivity Detector

Mobile phase

Mobile phase plus sample

Conductivity Detection of Seven Anion Standard

0.70

1.05

1.40

S

0.00 5.00 10.00 15.00 20.00 25.00 Minutes

1

2

3

4 5

6 7

1. 2.

3.

4.

Fluoride Chloride

Nitrite

Bromide

5. Nitrate

6. 7.

Phosphate Sulfate

1 ppm 2 ppm

4 ppm

4 ppm

4 ppm

6 ppm 4 ppm

Column:

Eluent:

Flow rate:

Injectionvol.:

Detection:

Waters IC-Pak Anion HC Borate/Gluconate

2.0 mL/min

100 µL

Direct Conductivity

Conductivity and UV Detectors in Series

1.20 0.80 S 0.40

1.60 1 2

3

4 5

6

7

0.00 4.00 8.00 12.00 16.00 20.00 24.00

Minutes

0.01

0.00

0.00

0.05

0.04 0.03 AU 0.02

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

50µL

Detection: Direct Conductivity after

Suppression

Detection: UV (PDA) at 214 nm

1.

2.

3. 4.

5.

Fluoride

Chloride

Nitrite Bromide

Nitrate

6.

7.

Phosphate

Sulfate

1 ppm

2 ppm

4 ppm

4 ppm

4 ppm

6 ppm

4 ppm

Applications

Sensitivities for compounds such as phenol, catecholamines,

nitrosamines,and organic acids arein thepicomole(nanogram) range. The mobile phasemust be made electrically conductive, usually

by the addition of a suitable salt: Ion Exchange

Reversed Phase and Ion-Pair RP

No normal phase separations

Detection in HPLC

Dr. Shulamit Levin,

beta-emitters and many soft gamma and positron emitters

encounteredin bio-medical researchand pharmaceuticalquality

control.

EVAPORATIVE LIGHT SCATTERING The scattered lightis detected by a

silicone photodiode located at a 90º angle from the laser. The

photodiode produces a signal

which is sent to the analog outputs for collection. A light trap is located 180º from the laser to

collect any light not scattered by

particles in the aerosol stream. How LC-MS Works

Mass

Spectrum

Source Analyzer Ion

Detector

Data

System

LC/MS

Interface

HPLC Separation

Desolvation

Ionization Sorting of Ions Detection Date

Processing

x

Detection in HPLC Detectors


UV/Vis

Fixed wavelength

Variable wavelength

Diode array

Refractiveindex

Fluorescence

Electrochemical



Evaporative light scattering Radioactive Detector Primarily used for the measurement of 3H, 14C, and 32P,

Dr. Shulamit Levin,

D DE ET TE ECT TO OR R

DETECTOR

REFLECTRON MODE

DE ET TE EC C TT OO RR

DETECTOR

REF FL LE EC CT TR RO ON N M O D E MODE

++ +

128.82

2.00 4.00 6.00 8.00 10.00 Time 0

Mixture

Int. 4.65 5.05

3.82

0.74

8.62

5.65

8.02

10.62

Total-Ion-Current Chromatogram with poor resolution

60 m/z 0

1: ScanES+ 4.34e5 262.87

Mix (10.696)

100 59.99

%

195.98

68.92 76.87 80

1: MassChromatogram 120.80 98.85 170.92 100 120 140 160 180 200 220

213.90 222.87

235.87 240.88 240 260

263.87

264.85

267.91 280

287.01 300

309.02 320 333.84 340

LC-MS

ElectronMultiplier

Inlet

End Cap Electrode Ring Electrode, R f

Axial Modulation

Ion Traps + + + ++

Types of

Mass

Spectrometer’s Analyzers

ll r Tiime Off Flliightt Mas ss s Ana aly yzze ers s

S S O O U U R R C C E E

DETECTOR RE

REFLECTRON NO O N N D

LINEAR RMODE E

DRIFT TTUBE E

S S O O U U R R C C E E

DETECTOR REFLECTRON MODE

REFLECTRON OFF C LLIN E A R M O D E

I DRIFT TUB B E E

220

Ion Source

Slit

Magnetic sector

Electrostatic Sector

(ESA)

Detector

Slit Nier -Johnson-Geometry (EB)

190

Sector Mass Spectrometers

199

FT-ICR-Spectrometer

DC

Source

Filament

DC DC Transferoptic Transmitter Plates

Receiver Plates

Elektroden Sender

Trapping Plates Electrodes

Y

Z X

Magnetic Fielt B

77

IonSource

Detector

non resonant Ion

resonant Ion

dcand Rf Voltages

The Quadrupole Analysator

1.00 2.00 3.00

Time (min)

4.00 5.00 0

0

100

0

100

0

100

0

100

100 MW=295 8.11e4

2.56

MW=280 2.21e5

1.57

MW=264 1.26e5

2.16

MW=260 1.53e5

1.29

TIC 3.50e5

1.57 1.29 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 / MeCN

0.2mL/min

0 100 125 150 175 200 225 250 275 300 Mass/Charge (m/z)

0

100

0

100

100 295 4.59e4 296

280 1.13e5

281 7.67e4 264

233

9.25e4

265

260

261

0

100

O

N

N

N

NH

O N

OH H

(1)

(2)

(3)

(4)

Fast LC-MS Analysis

2.1 x 50 mm ( 5 µm) HARDWARE --ES/APCI Ion Source

ESI and APCI are easily interchangeable in seconds without venting the system

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

Automatic Probe recognition

Detection in HPLC

Dr. Shulamit Levin,

+ SH M SH + +

SH M

MH +

ESI-MS Ion Formation Mass Range Multiply Charged Molecules

Horse Heart Myoglobin

n = 23, m/z = 738 n = 22 n = 21 n = 20 n = 19 n = 18 n = 17 n = 16, m/z = 1060

Calculated Mass Acquired Mass range

From LC Column

Nebulizer Gas

Heater Block

APCI Probe Equipped With a Heated Nebulizer MakeupGas

SMSSM S

SSSMM

S

SH

+

+

SH+

S

+ To Mass Analyzer

760 torr vacuum

Liquid Chromatography- Mass Spectrometery

(LC-MS)

Detection in HPLC

Corona Discharge Needle

Generates molecular weight and structural information

APCI flow rate: 0.2 to 2mL/minute

Option: Crossflow interface

Dr. Shulamit Levin,

C Celll

MS1 Collision MS2

Cell

Sc ca an nn niin ng g Static

M S S1 1 M S 2 2

Sc ca an nn niin ng g Sc ca an nn niin ng g

Static

Triple QuadrupolesMS- MS –

Modes of Operation

Multiple Reaction Monitoring

Cell

Typically used in Quantitative Work of Triple Quadrupoles MS1 Collision MS2

Static

i

MS1 MS2 Collision el

S Static Constant Neutral Loss Spectra

M Colllliisiion C e ll

MS

S S

Fast response for high performance systems.

Wide linear dynamic range (quantitation).

Low deadvolume (minimal peak broadening & remixing of the

separated bands).

Insensitivity to changes in type of solvent, flow rate, and

temperature.

Operational simplicity and reliability.

Tuneable, so that detection can be optimized for different

compounds.

Preferably non-destructive.

BASIC DETECTOR REQUIREMENTS An ideal LC detector should have the following properties: Low drift and noise level (trace analysis).

High sensitivity.

Detector Criteria Selectivity

Sensitivity and detection limit

Stability

Linear range Dynamic Range

Reproducibility

Effect on peak shape Maintenance

PROPERTIES OF DETECTORS

SELECTIVITY

UNIVERSAL SPECIFIC

A selective detector allows one to see only components of interest despite of their co-elution with any others.

Detection in HPLC D a u g h tte rr ( P r o d u c tt)) IIo n S p e c ttrra a M S 1 C o l l i s iio n M S S22 C e ll Statticc S c a n n i n g Parent (Precursor)) Ion Spectra

Dr. Shulamit Levin,


SENSITIVITY R

E

S

P

O

N

S

E

CONCENTRATION

Sensitivity of a detector is not the minimum

amount that can be detected.

PROPERTIES OF DETECTORS DETECTION LIMIT

hsignal = 2 x h noise

h signal

h noise

Limit of quantitation

Lowest concentration that can be determined with acceptable precision

Signal-to-noise ratio of 10:1

Detector Sensitivity Limit of detection Lowest concentration that can be detected

Signal-to-noise ratio of 2:1 or 3:1

No apparent

noise

Minutes

2.8 3.0 3.2 3.4

0.2 AU

Chromatographic Sensitivity Signal-to-Noise Ratio 0.001

AU

Minutes

2.00 3.00 4.00

Noise

Detection in HPLC

Dr. Shulamit Levin,

AU

Increase Signal-to-Noise Ratio

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

Increase S/N by increasing peak height

3:1

6:1

8:1

Factors Increasing UV Signal

Increase sample concentration

Increase injection volume

Choice of wavelength (s) Low volume flow cell

Flow cell pathlength

Factors Affecting Noise in UV Detectors

Optics bench design

Increase S/N by decreasing noise Chromatographic Sensitivity

Single Wavelength vs Maxplot

-0.002

0.000

0.006 0.004 0.002

0.008

0.010

0.0 2.0 4.0 6.0

220 nm

0.0 2.0 4.0 6.0

Minutes

Maxplot

Lamp energy Wavelengths

Mobile phase composition

Pump pulsation

Electronics PROPERTIES OF DETECTORS

BASELINE STABILITY

SHORT RANGE

LONG RANGE

DRIFT

NOISE

Detection in HPLC

Minutes

Dr. Shulamit Levin,

E S

P

O

N

S

E

Max linear response 2 x detector noise


LINEAR RANGE R

TIME (MIN)

R E S P O N S E

PROPERTIES OF DETECTORS DYNAMIC RANGE

RESPONSE TIME FLOW-CELL VOLUME

CONCENTRATION

The linear dynamic range of a detector is the maximum linear response divided by the detector noise. PROPERTIES OF DETECTORS CONTRIBUTION TO BAND BROADENING

Detection in HPLC Noise and drift Noise, drift, and smallest detectable peak.

Dr. Shulamit Levin,

PROPERTIES OF DETECTORS REPEATABILITY OF RESPONSE TEMPERATURE, FLOW RATE, ELECTRONICS

PROPERTIES OF DETECTORS MAINTENANCE AND COST EASY HANDLING OF FLOW-CELL

EASY A/D CONVERSION SAFETY

Detection in HPLC

Dr. Shulamit Levin,

detection qualification and types of detectors in...

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