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Detection in HPLC
Detection Qualification and Types of Detectors in HPLC Dr. Shulamit Levin Medtechnica http://www.forumsci.co.il/HPLC
Dr. Shulamit Levin, Medtechnica
Detection in HPLC
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Beer's Law Absorbance = Extinction Coefficient x Pathlength x Concentration Only for monochromatic light
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Reduce Pathlength
Single Wavelength UV Detector
Beer's Law
Optical Light Path
Absorbance = Extinction Coefficient x Pathlength x Concentration
A
A
Reduce Concentration
Wavelength Aperture Plate
B Mercury, Zinc or Cium Source Lamps
B
Wavelength Filter
Reference side
Absorbance AU
Transmittance %
Extinction Coefficient T=solvent/sample
* * * * Concentration
Dr. Shulamit Levin, Medtechnica
*
*
A= log(solvent/sample)
* * * * Concentration
AU 0 1.0 2.0 3.0
%T 100 10 1 0.1
T 1.000 0.100 0.010 0.001
Sample side Flow Cell
Dual Photodiode
Detection in HPLC
UV Detection of AccQ-Tag Amino Acid Derivatives
UV-VIS Detector Optical Bench
SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard
Optical Light Path Taper-Cell Flow Cell
0.024
Tyrosine Cysteic Acid Vaine Mtehionine
Lysine
Proline Alpha-aminobutyric acid
Glycine Glutamine
0.002
Tryptophan
Rotating Diffraction Grating 190 to 600nm
Phenylalanine
0.004
Deuterium Arc Lamp
Isoleucine Ornithine Leucine
0.006
Serine
0.008
Aperture Slit
Glutamic Acid
Illumination Lens
0.010
Hydroxyproline
0.012 Beam-Defining Apparatus
Asparagine
0.016 AU 0.014 Aspartic Acid
Sample side
0.018
NH3
AMQ
0.020 Dual Photodiode
Reference side
Histidine Arginine Threonine Alanine
0.022
Beam Splitter Mirrors
0.000 15.00
20.00
25.00
30.00
35.00 Minutes
40.00
45.00
50.00
55.00
Extraction of 3D Data
Absorbance
Absorbance
Chromatogram
Liquid from column
Dr. Shulamit Levin, Medtechnica
λ1 λ2
Spectrum Time
Wavelength
Detection in HPLC
PDA Spectrum Index Plot
Maximum Impurity Detection
DNPH Derivatives 0.25 ng Each Peak Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm
nm
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm
440.00
440.00
420.00
420.00
440.00
440.00
400.00 380.00
400.00
360.00
360.00
340.00
340.00
320.00
320.00
300.00 280.00
300.00 280.00
260.00
260.00
420.00 400.00 380.00 360.00 340.00 320.00 300.00 280.00
420.00 400.00 380.00 360.00 340.00 320.00 300.00 280.00
260.00
260.00
380.00
0.0006 0.0004 AU
nm
nm
0.0006
0.00030
0.0004 AU
0.00020
0.00030 0.00020
AU
AU 0.00010
0.0002
0.0002
Coelution of DNPH Hexaldehyde and 2,5-Dimethylbenzaldehyde Hexaldehyde
0.00010
2,5-Dimethylbenzaldehyde
0.00000
0.00000
0.0000
0.0000
-0.00010
0.00010 18.40
4.00
6.00
8.00
10.00
12.00 Minutes
14.00
16.00
18.60
18.80 Minutes
18.00
PDA and fluorescent Detector Comparisons for Aflatoxin Analysis
19.00
19.20
Photo-diode array Chromatographic and Spectral Sensitivity
SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Type: Standard
55.00
45.00 40.00 35.00 mV 30.00
Aflatoxin B1
25.00 20.00 15.00
1.6
10.00
2.4
Minutes
5.00 0.00 4.00
5.00
Dr. Shulamit Levin, Medtechnica
6.00
7.00 8.00 Minutes
0.07mAU 0.530 AU
0.07 mAU 0.15 ng Ethylparaben
UV at 360 nm 31 point smoothed UV at 360 nm Fluorescence 365 ex 455 em Aflatoxin B2 Aflatoxin G1 Aflatoxin G2
Absorbance
50.00
9.00
10.00
11.00
12.00
nm
3.2
210.0
250.0
nm
290.0
Detection in HPLC
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Differential Refractive Index Detector No sample = n
S R LAMP
With sample = n+∆ν
LED or Incandescent
Less common:
S
Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
To Amplifier
R ∆X = Const x ∆n
Refractive Index Detection with Differential RI - Sugars
Refractive Index Detection with Differential RI - Sugars
SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard 120.00
SampleName: Sugar Stds -500 ng each
Fructose
100.00
Dextrose
Sucrose
80.00
0.0000004
Sucrose
60.00 Maltose
40.00
Lactose
20.00 0.00 mV
Dextrose Fructose
del RIU
-20.00
0.0000002
-40.00 -60.00 -80.00 -100.00 -120.00
00000000
-140.00 -160.00
Bagel Extract 5.00
6.00
Dr. Shulamit Levin, Medtechnica
7.00
8.00 Minutes
5.00 9.00
10.00
11.00
5.50
6.00
6.50
7.00 Minutes
7.50
8.00
8.50
9.00
Detection in HPLC
Refractive Index Detection with Differential RI - Polymers
Sensitivity Refractive Index Detector
10300
800.00 750.00 700.00 96400
190000
Styragel HR 0.5, 4.6 x 300 mm, 35°C, 0.35 mL/min dRI sensitivity = 32X, 32°C
Del RIU
300.00 250.00 200.00 150.00
192300 Dow 1683
5.0 18.00
20.00
22.00
24.00 Minutes
26.00
28.00
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
7.0
8.0
Fluorescence Detectors
The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
6.0
Minutes
30.00
Detectors
Dr. Shulamit Levin, Medtechnica
1=Tristearin 2=Myristic acid
2
1260000
2890000
450.00 400.00 350.00
100.00 50.00 0.00
250 ng on column 1
650.00 600.00 550.00 500.00 MV
5570
SampleName: GPC STDS
Excitation filter Cell
LAMP
Emission filter
Photomultiplier Short - transmits all wavelengths below a specified cutoff Long - transmits all wavelengths above a specified cutoff Band - blocks all wavelengths outside a specified band
Detection in HPLC
Sensitivity Fluorescence Detector Optical Bench
Emission Grating
Photomultiplier tube Mirror
Emission Slit
Flow Cell
Torroidal Mirror
Excitation Grating
Fluorescence Detector
Beam Splittter Excitation Slit
Photo diode
0.1 pg Anthracene Excitation = 251 nm Emission = 406 nm
mV
5 mV Torroidal Mirror LAMP
Mirror
0.0
1.0
2.0
3.0
4.0
5.0
Minutes
Fluorescence vs. UV Detection
The most common HPLC detectors:
AMQ
AccQ-Tag amino acid analysis
Response
Fluorescence Excitation=250 nm Emission=395 nm UV 254 nm
20.00
40.00
Minutes
Dr. Shulamit Levin, Medtechnica
Detectors
60.00
UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Detection in HPLC
Electrochemical Detection of Catecholamines & Related Compounds
Electrochemical Detector Reference Electrode
2
1
Working Electrode
3
Analyte is oxidized or reduced
-
+
1. 2. 3. 4. 5. 6. 7.
Norepinepherine Epinepherine Normetanepherine Dopamine Metanepherine 3-Methoxytyramine 4-Methoxytyramine
4
nAmps
150 ppb 200 ppb 50 ppb 200 ppb 200 ppb 75 ppb 500 ppb
5 7 6
Electrolyte (mobile phase) Auxiliary Electrode As compounds are oxidized or reduced, a current proportional to concentration is produced.
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Minutes
Pulsed Amperometric Detection of Monosaccharides 1. 2. 3. 4. 5. 6.
300
1
4 6
Less common:
5
0.00 5.00
Dr. Shulamit Levin, Medtechnica
Minutes
The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Fucose Galactosamine Glucosamine Galactose Glucose Mannose
2
3
mV
Detectors
20.00
Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Detection in HPLC
Conductivity Detection of Seven Anion Standard
Conductivity Detector
1. 2. 3. 4. 5. 6. 7.
1.40 3 2
Mobile phase
Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm 4 ppm
Column: Eluent: Flow rate: Injection vol.: Detection:
1
1.05
4
µS
5 7
6
Mobile phase plus sample
0.70 0.00
5.00
Conductivity and UV Detectors in Series 1.60
1
2
Detection:
3
Direct Conductivity after Suppression
1.20 4
5
0.80 µS
7 6
0.40 0.00 3
0.05
Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
0.03 AU 0.02
Flow rate: Injection vol.:
0.01
4 12.00 16.00 Minutes
Dr. Shulamit Levin, Medtechnica
Minutes
20.00
24.00
15.00
Waters IC-Pak Anion HR 1.2 mM Sodium Carbonate/ 1.2 mM Sodium Bicarbonate 1.0 mL/min 50 µL
20.00
25.00
Applications 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:
Reversed Phase and Ion-Pair RP No normal phase separations
0.00 8.00
10.00
Ion Exchange Column: Eluent:
5
4.00
1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm 4 ppm
Detection: UV (PDA) at 214 nm
0.04
0.00
1. 2. 3. 4. 5. 6. 7.
Waters IC-Pak Anion HC Borate/Gluconate 2.0 mL/min 100 µL Direct Conductivity
Detection in HPLC
EVAPORATIVE LIGHT SCATTERING
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
The scattered light is 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.
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
How LC-MS Works
Radioactive Detector Primarily used for the measurement of 3H, 14C, and 32P, beta-emitters and many soft gamma and positron emitters encountered in bio-medical research and pharmaceutical quality control.
Ionization
Source
x
Sorting of Ions
Detection
Ion Detector
Analyzer
Data System
LC/MS Interface Desolvation
HPLC
Separation
Dr. Shulamit Levin, Medtechnica
Date Processing
Mass Spectrum
Detection in HPLC
TTiim M A mee O Off FFlliigghhtt M Maaassssss A Annnaaalllyyyzzzeeerrrsss
FT--ICR FT ICR--Spectrometer
SSOOUURRCCEE
LC-MS
R RE EF F LL E EC C TT R RO ON NO OFFFF
D R II FFTT TTUUBBEE
Magnetic Fielt B
DDEETT E ORR EC C TT O LLI INNEE A R MMO OD DE
D DE ET TE EC CT TO OR R R RE EF FL LE EC CT TR RO ON N M MO OD DE E
Y Z
X
Source
Sender
R RE EF F LL EECCTTRROONN OONN
SSOOUURRCCEE
Trapping Plates Electrodes
Elektroden
D RR DE E TTEECCTTOO L L II N NEEAARR M MO ODDEE
DRIFT D R I F T TUBE TUBE
DC
D DE ET TE EC CT TO OR R RE EF FL LE EC CTTRRO ONN M R MO OD DE E
Receiver Plates
Filament DC
Total-Ion-Current Chromatogram with poor resolution
Transmitter Plates
1: ScanES+ 4.34e5
262.87
100
Ion Traps
DC
Transferoptic
Mix (10.696)
199
Types of Mass Spectrometer’s Analyzers
59.99
213.90 %
222.87 235.87
Mixture
263.87
1: Mass Chromatogram 195.98
4.65 5.05
Int.
240.88
264.85
120.80
68.92
98.85
267.91
128.82
76.87
8.62
287.01
309.02
170.92
333.84
0 60
80
100
120
140
160
180
200
220
240
260
280
300
320
m/z
End Cap Electrode
Axial Modulation
+ + + ++ + ++
Ring Electrode, R f
Inlet
ElectronMultiplier
340
190
The Quadrupole Quadrupole Analysator
5.65
S e c t o r Mass Sector Mass Spectrometers Spectrometers
8.02 Nier -J o h n s o n -G e o m e t r y ( E B )
resonant Ion
Slit
non resonant Ion
10.62
Magnetic sector
Detector
Electrostatic Sector
3.82
(ESA)
0.74 Detector
Slit dcand Rf Voltages
Ion Source IonSource
77
0
Time 2.00
4.00
6.00
8.00
10.00
Fast LC-MS Analysis 2.1 x 50 mm ( 5 µm) 2.56
100
MW=295 8.11e4
0 MW=280 2.21e5
0 MW=264 1.26e5
(4)
N
1.29
MW=260 1.53e5
0
0
Ding
280 1.13e5
N
100
(2)
281
O
100
264
(3)
1.57 2.16 1.29 2.56
1.00
2.00
TIC TIC 3.50e5
3.00
Time (min)
Dr. Shulamit Levin, Medtechnica
4.00
5.00
7.67e4
265
NH
0 100
O OH
100
296
233
0 100
100
0
2.16
100
295 4.59e4
0
1.57
100
HARDWARE HARDWARE -- ES/APCI ES/APCI Ion Ion Source Source N
N H
(1)
260
9.25e4
261
0 100 125 150 175 200 225 250 275 300 Mass/Charge (m/z) 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 / MeCN 0.2 mL/min
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
220
Detection in HPLC
ESI-MS 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
Acquired Mass range
Liquid Chromatography- Mass Spectrometery (LC-MS)
APCI APCI Probe Probe Equipped With a Heated Heated Nebulizer Nebulizer
Makeup Gas
Heater Block From LC Column
SMSSM S SSSMM S
Nebulizer Gas
+ + SH M SH
SH+ + + SH M SH+
+ MH + S
760 torr
To Mass Analyzer
vacuum
Corona Discharge Needle
Generates molecular weight and structural information APCI flow rate: 0.2 to 2mL/minute Option: Crossflow interface
Dr. Shulamit Levin, Medtechnica
Calculated Mass
Detection in HPLC
D D aa uu gg hhhttteeerrr (( P P rr oo dd uu cc ttt))) II oo nn S S pp ee cc tttrrraaa M S 11
C o l l ii ss iioonn CCee ll ll
S t aatti icc
M M SS22
Collision Collision CCell ell
MS2
Multiple Reaction Reaction Monitoring Reaction Monitoring Monitoring
Typically Typically used used in in in Quantitative Quantitative Work Work Work of of of Triple Quadrupoles Quadrupoles Quadrupoles Quadrupoles MS1 MS1
S Sc c aa n nnniinngg
Collision Collision Cell Cell
MS2 MS2
Static Static
C S pp ee cc tt rr aa Coo nn ss tt aa nn tt N e u t r a l L o s s S M S 11 MS
BASIC DETECTOR REQUIREMENTS An ideal LC detector should have the following properties:
SSccaa nn nn ii n g
P a r e n t ( P r e ccuurrssoorr)) IIoonn SSppeeccttrraa M S 11
Triple Quadrupoles Quadrupoles MSMSMS – Modes of Operation
CCoolllliissiioonn CCee ll ll
S Sc c aa n n nn iinngg
Static Static
Static Static
M MS S2 2
S c aa n n nn ii nngg Sc
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 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.
PROPERTIES OF DETECTORS Detector Criteria Selectivity Sensitivity and detection limit Stability Linear range Dynamic Range Reproducibility Effect on peak shape Maintenance
Dr. Shulamit Levin, Medtechnica
SELECTIVITY
SPECIFIC
UNIVERSAL
A selective detector allows one to see only components of interest despite of their co-elution with any others.
Detection in HPLC
PROPERTIES OF DETECTORS SENSITIVITY R E S P O N S
PROPERTIES OF DETECTORS DETECTION LIMIT
h signal = 2 x h noise
h signal
E CONCENTRATION
Sensitivity of a detector is not the minimum amount that can be detected.
Detector Sensitivity
h noise
Chromatographic Sensitivity Signal-to-Noise Ratio
Limit of detection Lowest concentration that can be detected Signal-to-noise ratio of 2:1 or 3:1 0.001 AU
0.2 AU
Limit of quantitation No apparent noise
Lowest concentration that can be determined with acceptable precision Signal-to-noise ratio of 10:1 2.8
3.0
3.2
Minutes
Dr. Shulamit Levin, Medtechnica
3.4
Noise
2.00
3.00 Minutes
4.00
Detection in HPLC
Factors Increasing UV Signal Increase Signal-to-Noise Ratio 6:1
3:1
Signal-to-noise (S/N) is peak height to noise
8:1
Increase S/N by increasing peak height Increase S/N by decreasing noise
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 Lamp energy Wavelengths Mobile phase composition Pump pulsation Electronics
Chromatographic Sensitivity
PROPERTIES OF DETECTORS
Single Wavelength vs Maxplo t
BASELINE STABILITY
0.010
Maxplot
220 nm
0.008
SHORT RANGE
AU
0.006
NOISE
0.004 0.002 0.000
LONG RANGE
-0.002 0.0
2.0
4.0
Minutes
Dr. Shulamit Levin, Medtechnica
6.0
0.0
2.0
4.0
Minutes
6.0
DRIFT
Detection in HPLC
Noise and drift
PROPERTIES OF DETECTORS LINEAR RANGE
Noise, drift, and smallest detectable peak. R E S P O N S E
Max linear response
2 x detector noise
CONCENTRATION
The linear dynamic range of a detector is the maximum linear response divided by the detector noise.
PROPERTIES OF DETECTORS DYNAMIC RANGE
R E S P O N S E
CONTRIBUTION TO BAND BROADENING
RESPONSE TIME
TIME (MIN)
Dr. Shulamit Levin, Medtechnica
PROPERTIES OF DETECTORS
FLOW-CELL VOLUME
Detection in HPLC
PROPERTIES OF DETECTORS REPEATABILITY OF RESPONSE
PROPERTIES OF DETECTORS MAINTENANCE AND COST
EASY HANDLING OF FLOW-CELL EASY A/D CONVERSION TEMPERATURE, FLOW RATE, ELECTRONICS
SAFETY
Dr. Shulamit Levin, Medtechnica
Detection Qualification and Types of Detectors in HPLC Dr. Shulamit Levin Medtechnica http://www.forumsci.co.il/HPLC
Dr. Shulamit Levin, Medtechnica
Detection in HPLC
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Beer's Law Absorbance = Extinction Coefficient x Pathlength x Concentration Only for monochromatic light
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Reduce Pathlength
Single Wavelength UV Detector
Beer's Law
Optical Light Path
Absorbance = Extinction Coefficient x Pathlength x Concentration
A
A
Reduce Concentration
Wavelength Aperture Plate
B Mercury, Zinc or Cium Source Lamps
B
Wavelength Filter
Reference side
Absorbance AU
Transmittance %
Extinction Coefficient T=solvent/sample
* * * * Concentration
Dr. Shulamit Levin, Medtechnica
*
*
A= log(solvent/sample)
* * * * Concentration
AU 0 1.0 2.0 3.0
%T 100 10 1 0.1
T 1.000 0.100 0.010 0.001
Sample side Flow Cell
Dual Photodiode
Detection in HPLC
UV Detection of AccQ-Tag Amino Acid Derivatives
UV-VIS Detector Optical Bench
SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard
Optical Light Path Taper-Cell Flow Cell
0.024
Tyrosine Cysteic Acid Vaine Mtehionine
Lysine
Proline Alpha-aminobutyric acid
Glycine Glutamine
0.002
Tryptophan
Rotating Diffraction Grating 190 to 600nm
Phenylalanine
0.004
Deuterium Arc Lamp
Isoleucine Ornithine Leucine
0.006
Serine
0.008
Aperture Slit
Glutamic Acid
Illumination Lens
0.010
Hydroxyproline
0.012 Beam-Defining Apparatus
Asparagine
0.016 AU 0.014 Aspartic Acid
Sample side
0.018
NH3
AMQ
0.020 Dual Photodiode
Reference side
Histidine Arginine Threonine Alanine
0.022
Beam Splitter Mirrors
0.000 15.00
20.00
25.00
30.00
35.00 Minutes
40.00
45.00
50.00
55.00
Extraction of 3D Data
Absorbance
Absorbance
Chromatogram
Liquid from column
Dr. Shulamit Levin, Medtechnica
λ1 λ2
Spectrum Time
Wavelength
Detection in HPLC
PDA Spectrum Index Plot
Maximum Impurity Detection
DNPH Derivatives 0.25 ng Each Peak Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm
nm
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm
440.00
440.00
420.00
420.00
440.00
440.00
400.00 380.00
400.00
360.00
360.00
340.00
340.00
320.00
320.00
300.00 280.00
300.00 280.00
260.00
260.00
420.00 400.00 380.00 360.00 340.00 320.00 300.00 280.00
420.00 400.00 380.00 360.00 340.00 320.00 300.00 280.00
260.00
260.00
380.00
0.0006 0.0004 AU
nm
nm
0.0006
0.00030
0.0004 AU
0.00020
0.00030 0.00020
AU
AU 0.00010
0.0002
0.0002
Coelution of DNPH Hexaldehyde and 2,5-Dimethylbenzaldehyde Hexaldehyde
0.00010
2,5-Dimethylbenzaldehyde
0.00000
0.00000
0.0000
0.0000
-0.00010
0.00010 18.40
4.00
6.00
8.00
10.00
12.00 Minutes
14.00
16.00
18.60
18.80 Minutes
18.00
PDA and fluorescent Detector Comparisons for Aflatoxin Analysis
19.00
19.20
Photo-diode array Chromatographic and Spectral Sensitivity
SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Type: Standard
55.00
45.00 40.00 35.00 mV 30.00
Aflatoxin B1
25.00 20.00 15.00
1.6
10.00
2.4
Minutes
5.00 0.00 4.00
5.00
Dr. Shulamit Levin, Medtechnica
6.00
7.00 8.00 Minutes
0.07mAU 0.530 AU
0.07 mAU 0.15 ng Ethylparaben
UV at 360 nm 31 point smoothed UV at 360 nm Fluorescence 365 ex 455 em Aflatoxin B2 Aflatoxin G1 Aflatoxin G2
Absorbance
50.00
9.00
10.00
11.00
12.00
nm
3.2
210.0
250.0
nm
290.0
Detection in HPLC
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Differential Refractive Index Detector No sample = n
S R LAMP
With sample = n+∆ν
LED or Incandescent
Less common:
S
Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
To Amplifier
R ∆X = Const x ∆n
Refractive Index Detection with Differential RI - Sugars
Refractive Index Detection with Differential RI - Sugars
SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard 120.00
SampleName: Sugar Stds -500 ng each
Fructose
100.00
Dextrose
Sucrose
80.00
0.0000004
Sucrose
60.00 Maltose
40.00
Lactose
20.00 0.00 mV
Dextrose Fructose
del RIU
-20.00
0.0000002
-40.00 -60.00 -80.00 -100.00 -120.00
00000000
-140.00 -160.00
Bagel Extract 5.00
6.00
Dr. Shulamit Levin, Medtechnica
7.00
8.00 Minutes
5.00 9.00
10.00
11.00
5.50
6.00
6.50
7.00 Minutes
7.50
8.00
8.50
9.00
Detection in HPLC
Refractive Index Detection with Differential RI - Polymers
Sensitivity Refractive Index Detector
10300
800.00 750.00 700.00 96400
190000
Styragel HR 0.5, 4.6 x 300 mm, 35°C, 0.35 mL/min dRI sensitivity = 32X, 32°C
Del RIU
300.00 250.00 200.00 150.00
192300 Dow 1683
5.0 18.00
20.00
22.00
24.00 Minutes
26.00
28.00
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
7.0
8.0
Fluorescence Detectors
The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
6.0
Minutes
30.00
Detectors
Dr. Shulamit Levin, Medtechnica
1=Tristearin 2=Myristic acid
2
1260000
2890000
450.00 400.00 350.00
100.00 50.00 0.00
250 ng on column 1
650.00 600.00 550.00 500.00 MV
5570
SampleName: GPC STDS
Excitation filter Cell
LAMP
Emission filter
Photomultiplier Short - transmits all wavelengths below a specified cutoff Long - transmits all wavelengths above a specified cutoff Band - blocks all wavelengths outside a specified band
Detection in HPLC
Sensitivity Fluorescence Detector Optical Bench
Emission Grating
Photomultiplier tube Mirror
Emission Slit
Flow Cell
Torroidal Mirror
Excitation Grating
Fluorescence Detector
Beam Splittter Excitation Slit
Photo diode
0.1 pg Anthracene Excitation = 251 nm Emission = 406 nm
mV
5 mV Torroidal Mirror LAMP
Mirror
0.0
1.0
2.0
3.0
4.0
5.0
Minutes
Fluorescence vs. UV Detection
The most common HPLC detectors:
AMQ
AccQ-Tag amino acid analysis
Response
Fluorescence Excitation=250 nm Emission=395 nm UV 254 nm
20.00
40.00
Minutes
Dr. Shulamit Levin, Medtechnica
Detectors
60.00
UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Detection in HPLC
Electrochemical Detection of Catecholamines & Related Compounds
Electrochemical Detector Reference Electrode
2
1
Working Electrode
3
Analyte is oxidized or reduced
-
+
1. 2. 3. 4. 5. 6. 7.
Norepinepherine Epinepherine Normetanepherine Dopamine Metanepherine 3-Methoxytyramine 4-Methoxytyramine
4
nAmps
150 ppb 200 ppb 50 ppb 200 ppb 200 ppb 75 ppb 500 ppb
5 7 6
Electrolyte (mobile phase) Auxiliary Electrode As compounds are oxidized or reduced, a current proportional to concentration is produced.
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Minutes
Pulsed Amperometric Detection of Monosaccharides 1. 2. 3. 4. 5. 6.
300
1
4 6
Less common:
5
0.00 5.00
Dr. Shulamit Levin, Medtechnica
Minutes
The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
Fucose Galactosamine Glucosamine Galactose Glucose Mannose
2
3
mV
Detectors
20.00
Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
Detection in HPLC
Conductivity Detection of Seven Anion Standard
Conductivity Detector
1. 2. 3. 4. 5. 6. 7.
1.40 3 2
Mobile phase
Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm 4 ppm
Column: Eluent: Flow rate: Injection vol.: Detection:
1
1.05
4
µS
5 7
6
Mobile phase plus sample
0.70 0.00
5.00
Conductivity and UV Detectors in Series 1.60
1
2
Detection:
3
Direct Conductivity after Suppression
1.20 4
5
0.80 µS
7 6
0.40 0.00 3
0.05
Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate
0.03 AU 0.02
Flow rate: Injection vol.:
0.01
4 12.00 16.00 Minutes
Dr. Shulamit Levin, Medtechnica
Minutes
20.00
24.00
15.00
Waters IC-Pak Anion HR 1.2 mM Sodium Carbonate/ 1.2 mM Sodium Bicarbonate 1.0 mL/min 50 µL
20.00
25.00
Applications 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:
Reversed Phase and Ion-Pair RP No normal phase separations
0.00 8.00
10.00
Ion Exchange Column: Eluent:
5
4.00
1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm 4 ppm
Detection: UV (PDA) at 214 nm
0.04
0.00
1. 2. 3. 4. 5. 6. 7.
Waters IC-Pak Anion HC Borate/Gluconate 2.0 mL/min 100 µL Direct Conductivity
Detection in HPLC
EVAPORATIVE LIGHT SCATTERING
Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical
The scattered light is 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.
Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering
How LC-MS Works
Radioactive Detector Primarily used for the measurement of 3H, 14C, and 32P, beta-emitters and many soft gamma and positron emitters encountered in bio-medical research and pharmaceutical quality control.
Ionization
Source
x
Sorting of Ions
Detection
Ion Detector
Analyzer
Data System
LC/MS Interface Desolvation
HPLC
Separation
Dr. Shulamit Levin, Medtechnica
Date Processing
Mass Spectrum
Detection in HPLC
TTiim M A mee O Off FFlliigghhtt M Maaassssss A Annnaaalllyyyzzzeeerrrsss
FT--ICR FT ICR--Spectrometer
SSOOUURRCCEE
LC-MS
R RE EF F LL E EC C TT R RO ON NO OFFFF
D R II FFTT TTUUBBEE
Magnetic Fielt B
DDEETT E ORR EC C TT O LLI INNEE A R MMO OD DE
D DE ET TE EC CT TO OR R R RE EF FL LE EC CT TR RO ON N M MO OD DE E
Y Z
X
Source
Sender
R RE EF F LL EECCTTRROONN OONN
SSOOUURRCCEE
Trapping Plates Electrodes
Elektroden
D RR DE E TTEECCTTOO L L II N NEEAARR M MO ODDEE
DRIFT D R I F T TUBE TUBE
DC
D DE ET TE EC CT TO OR R RE EF FL LE EC CTTRRO ONN M R MO OD DE E
Receiver Plates
Filament DC
Total-Ion-Current Chromatogram with poor resolution
Transmitter Plates
1: ScanES+ 4.34e5
262.87
100
Ion Traps
DC
Transferoptic
Mix (10.696)
199
Types of Mass Spectrometer’s Analyzers
59.99
213.90 %
222.87 235.87
Mixture
263.87
1: Mass Chromatogram 195.98
4.65 5.05
Int.
240.88
264.85
120.80
68.92
98.85
267.91
128.82
76.87
8.62
287.01
309.02
170.92
333.84
0 60
80
100
120
140
160
180
200
220
240
260
280
300
320
m/z
End Cap Electrode
Axial Modulation
+ + + ++ + ++
Ring Electrode, R f
Inlet
ElectronMultiplier
340
190
The Quadrupole Quadrupole Analysator
5.65
S e c t o r Mass Sector Mass Spectrometers Spectrometers
8.02 Nier -J o h n s o n -G e o m e t r y ( E B )
resonant Ion
Slit
non resonant Ion
10.62
Magnetic sector
Detector
Electrostatic Sector
3.82
(ESA)
0.74 Detector
Slit dcand Rf Voltages
Ion Source IonSource
77
0
Time 2.00
4.00
6.00
8.00
10.00
Fast LC-MS Analysis 2.1 x 50 mm ( 5 µm) 2.56
100
MW=295 8.11e4
0 MW=280 2.21e5
0 MW=264 1.26e5
(4)
N
1.29
MW=260 1.53e5
0
0
Ding
280 1.13e5
N
100
(2)
281
O
100
264
(3)
1.57 2.16 1.29 2.56
1.00
2.00
TIC TIC 3.50e5
3.00
Time (min)
Dr. Shulamit Levin, Medtechnica
4.00
5.00
7.67e4
265
NH
0 100
O OH
100
296
233
0 100
100
0
2.16
100
295 4.59e4
0
1.57
100
HARDWARE HARDWARE -- ES/APCI ES/APCI Ion Ion Source Source N
N H
(1)
260
9.25e4
261
0 100 125 150 175 200 225 250 275 300 Mass/Charge (m/z) 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 / MeCN 0.2 mL/min
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
220
Detection in HPLC
ESI-MS 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
Acquired Mass range
Liquid Chromatography- Mass Spectrometery (LC-MS)
APCI APCI Probe Probe Equipped With a Heated Heated Nebulizer Nebulizer
Makeup Gas
Heater Block From LC Column
SMSSM S SSSMM S
Nebulizer Gas
+ + SH M SH
SH+ + + SH M SH+
+ MH + S
760 torr
To Mass Analyzer
vacuum
Corona Discharge Needle
Generates molecular weight and structural information APCI flow rate: 0.2 to 2mL/minute Option: Crossflow interface
Dr. Shulamit Levin, Medtechnica
Calculated Mass
Detection in HPLC
D D aa uu gg hhhttteeerrr (( P P rr oo dd uu cc ttt))) II oo nn S S pp ee cc tttrrraaa M S 11
C o l l ii ss iioonn CCee ll ll
S t aatti icc
M M SS22
Collision Collision CCell ell
MS2
Multiple Reaction Reaction Monitoring Reaction Monitoring Monitoring
Typically Typically used used in in in Quantitative Quantitative Work Work Work of of of Triple Quadrupoles Quadrupoles Quadrupoles Quadrupoles MS1 MS1
S Sc c aa n nnniinngg
Collision Collision Cell Cell
MS2 MS2
Static Static
C S pp ee cc tt rr aa Coo nn ss tt aa nn tt N e u t r a l L o s s S M S 11 MS
BASIC DETECTOR REQUIREMENTS An ideal LC detector should have the following properties:
SSccaa nn nn ii n g
P a r e n t ( P r e ccuurrssoorr)) IIoonn SSppeeccttrraa M S 11
Triple Quadrupoles Quadrupoles MSMSMS – Modes of Operation
CCoolllliissiioonn CCee ll ll
S Sc c aa n n nn iinngg
Static Static
Static Static
M MS S2 2
S c aa n n nn ii nngg Sc
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 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.
PROPERTIES OF DETECTORS Detector Criteria Selectivity Sensitivity and detection limit Stability Linear range Dynamic Range Reproducibility Effect on peak shape Maintenance
Dr. Shulamit Levin, Medtechnica
SELECTIVITY
SPECIFIC
UNIVERSAL
A selective detector allows one to see only components of interest despite of their co-elution with any others.
Detection in HPLC
PROPERTIES OF DETECTORS SENSITIVITY R E S P O N S
PROPERTIES OF DETECTORS DETECTION LIMIT
h signal = 2 x h noise
h signal
E CONCENTRATION
Sensitivity of a detector is not the minimum amount that can be detected.
Detector Sensitivity
h noise
Chromatographic Sensitivity Signal-to-Noise Ratio
Limit of detection Lowest concentration that can be detected Signal-to-noise ratio of 2:1 or 3:1 0.001 AU
0.2 AU
Limit of quantitation No apparent noise
Lowest concentration that can be determined with acceptable precision Signal-to-noise ratio of 10:1 2.8
3.0
3.2
Minutes
Dr. Shulamit Levin, Medtechnica
3.4
Noise
2.00
3.00 Minutes
4.00
Detection in HPLC
Factors Increasing UV Signal Increase Signal-to-Noise Ratio 6:1
3:1
Signal-to-noise (S/N) is peak height to noise
8:1
Increase S/N by increasing peak height Increase S/N by decreasing noise
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 Lamp energy Wavelengths Mobile phase composition Pump pulsation Electronics
Chromatographic Sensitivity
PROPERTIES OF DETECTORS
Single Wavelength vs Maxplo t
BASELINE STABILITY
0.010
Maxplot
220 nm
0.008
SHORT RANGE
AU
0.006
NOISE
0.004 0.002 0.000
LONG RANGE
-0.002 0.0
2.0
4.0
Minutes
Dr. Shulamit Levin, Medtechnica
6.0
0.0
2.0
4.0
Minutes
6.0
DRIFT
Detection in HPLC
Noise and drift
PROPERTIES OF DETECTORS LINEAR RANGE
Noise, drift, and smallest detectable peak. R E S P O N S E
Max linear response
2 x detector noise
CONCENTRATION
The linear dynamic range of a detector is the maximum linear response divided by the detector noise.
PROPERTIES OF DETECTORS DYNAMIC RANGE
R E S P O N S E
CONTRIBUTION TO BAND BROADENING
RESPONSE TIME
TIME (MIN)
Dr. Shulamit Levin, Medtechnica
PROPERTIES OF DETECTORS
FLOW-CELL VOLUME
Detection in HPLC
PROPERTIES OF DETECTORS REPEATABILITY OF RESPONSE
PROPERTIES OF DETECTORS MAINTENANCE AND COST
EASY HANDLING OF FLOW-CELL EASY A/D CONVERSION TEMPERATURE, FLOW RATE, ELECTRONICS
SAFETY
Dr. Shulamit Levin, Medtechnica
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