Detection in HPLCDetection Qualification and Types of Detectors in HPLC Dr. Shulamit Levin Medtechnica http://www.forumsci.co.il/HPLC Dr. Shulamit Levin, Medtechnica 0 2.001 Sample side Flow Cell Dual Photodiode .0 %T 100 10 1 0.0 3.010 0.100 0.1 T 1. Medtechnica * * A= log(solvent/sample) * * * * Concentration AU 0 1. Shulamit Levin. Zinc or Cadmium Source Lamps B Wavelength Filter Reference side Absorbance AU Transmittance % Extinction Coefficient T=solvent/sample * * * * Concentration Dr.000 0.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. 016 AU 0.024 Tyrosine Cysteic Acid Vaine Mtehionine Lysine Proline Alpha-aminobutyric acid Glycine Glutamine 0.000 15. Medtechnica λ1 λ2 Spectrum Time Wavelength .002 Tryptophan Rotating Diffraction Grating 190 to 600nm Phenylalanine 0.00 35.00 50.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 0.022 Beam Splitter Mirrors 0.00 30.00 20.00 25.010 Hydroxyproline 0. Shulamit Levin.008 Aperture Slit Glutamic Acid Illumination Lens 0.00 Extraction of 3D Data Absorbance Absorbance Chromatogram Liquid from column Dr.006 Serine 0.020 Dual Photodiode Reference side Histidine Arginine Threonine Alanine 0.00 55.00 45.012 Beam-Defining Apparatus Asparagine 0.014 Aspartic Acid Sample side 0.004 Deuterium Arc Lamp Isoleucine Ornithine Leucine 0.018 NH3 AMQ 0.00 Minutes 40. 00 19.00 12.00 11.25 ng 360nm 996PDA 360.00 15.4 Minutes 5.00000 0.00030 0.00 9.00 360.00 400.0002 Coelution of DNPH Hexaldehyde and 2.00 440.00 20.00 360. Shulamit Levin.00030 0.00 340.00 420.00 280.00 360.00 300.0 nm 440.00000 0.00 300.00 16.6 10.00 420.00 Minutes 0.00 360.00 4.530 AU 0.00 260.0 nm 290.00 Aflatoxin B1 25.00 mV 30.00 5.00 400.00 Minutes 14.00 35.00 10.00 380.0000 -0.20 Photo-diode array Chromatographic and Spectral Sensitivity SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Type: Standard 55.00 1.00 0.00 10.00 260.00 380.00 260.SampleWeight 0.0000 0.00 PDA and fluorescent Detector Comparisons for Aflatoxin Analysis 19.00 320.00010 18.5-Dimethylbenzaldehyde Hexaldehyde 0.00 340.25 ng Each Peak Millennium PDA Spectrum Index Plot .00 nm 3.07mAU 0.60 18.00 300.00 8.00020 0.40 4.00 18.00 440.00 420.00 440.00 400.SampleWeight 0.00 320.0004 AU 0.00010 2.00 7.15 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 45.2 210.00 40.00 260.00 280.0 nm nm Millennium PDA Spectrum Index Plot .5-Dimethylbenzaldehyde 0.0002 0.00 300.0004 AU nm nm 0.0 .0 250.00 420.00010 0.00 340.07 mAU 0.00 12.00 0.00 Dr.0006 0.0006 0.PDA 360.25 ng .00 280.Detection in HPLC PDA Spectrum Index Plot Maximum Impurity Detection DNPH Derivatives 0.00 380. Medtechnica 6.80 Minutes 18.00 280.00 8.00 320.00 2.00 340.00010 0.00020 AU AU 0.00 6.00 380.00 400.00 320. 00 9.00 5.0000004 Sucrose 60.00 -60.00 Lactose 20.50 6.00 0.00 Dr. Medtechnica 7.00 SampleName: Sugar Stds -500 ng each Fructose 100.0000002 -40.50 8. Shulamit Levin.00 00000000 -140.00 11.00 -100.00 -120.00 Minutes 7.Sugars Refractive Index Detection with Differential RI .00 8.00 0.00 .00 10.00 Bagel Extract 5.50 7.00 Dextrose Sucrose 80.00 6.00 8.00 -80.00 6.00 Maltose 40.00 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 SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard 120.50 9.00 mV Dextrose Fructose del RIU -20.00 Minutes 5.00 -160. 0.00 250.00 500.00 100. 4.00 200.0 18.00 150. Medtechnica 1=Tristearin 2=Myristic acid 2 1260000 2890000 450.00 Minutes 26.00 700.Detection in HPLC Refractive Index Detection with Differential RI .Polymers Sensitivity Refractive Index Detector 10300 800.00 550.00 28.00 20.00 600.00 400.transmits all wavelengths below a specified cutoff Long pass .0 Minutes 30.00 50.00 MV 5570 SampleName: GPC STDS Excitation filter Cell LAMP Emission filter Photomultiplier Short pass . Shulamit Levin.00 750.blocks all wavelengths outside a specified band .6 x 300 mm.0 8.00 22.0 Fluorescence Detectors The most common HPLC detectors: UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical 6.00 Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering 7.00 96400 190000 Styragel HR 0.00 250 ng on column 1 650.00 192300 Dow 1683 5.00 Detectors Dr.35 mL/min dRI sensitivity = 32X. 32°C Del RIU 300.00 350.00 24.00 0.transmits all wavelengths above a specified cutoff Band pass . 35°C.5. 00 40. UV Detection The most common HPLC detectors: AMQ AccQ-Tag amino acid analysis Response Fluorescence Excitation=250 nm Emission=395 nm UV 254 nm 20.0 3.00 Minutes Dr.0 1.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 0.0 2.00 UV/Vis Fixed wavelength Variable wavelength Diode array Refractive index Fluorescence Electrochemical Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering . Shulamit Levin.0 4.1 pg Anthracene Excitation = 251 nm Emission = 406 nm mV 5 mV Torroidal Mirror LAMP Mirror 0.0 Minutes Fluorescence vs. Medtechnica Detectors 60.0 5. 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 8. 6. 5. 6. 7. 0. 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. 4.00 Dr.00 6.00 12.00 2. 2.00 Minutes Pulsed Amperometric Detection of Monosaccharides 1. 300 1 4 6 Less common: 5 0. 5. a current proportional to concentration is produced. 2.00 5. 3.00 Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering . 3.Detection in HPLC Electrochemical Detection of Catecholamines & Related Compounds Electrochemical Detector Reference Electrode 2 1 Working Electrode 3 Analyte is oxidized or reduced - + 1. 4.00 10.00 4. Shulamit Levin. 2 mM Sodium Bicarbonate 1. Medtechnica Minutes 20. 3. catecholamines.2 mM Sodium Carbonate/ 1.00 Applications Sensitivities for compounds such as phenol.00 Conductivity and UV Detectors in Series 1.00 24. 5. 7. 4.70 0.05 Fluoride Chloride Nitrite Bromide Nitrate Phosphate Sulfate 0.Detection in HPLC Conductivity Detection of Seven Anion Standard Conductivity Detector 1. usually by the addition of a suitable salt: Reversed Phase and Ion-Pair RP No normal phase separations 0.00 Minutes Dr. The mobile phase must be made electrically conductive.01 4 12.00 15.00 Ion Exchange Column: Eluent: 5 4.0 mL/min 100 µL Direct Conductivity . and organic acids are in the picomole (nanogram) range. 2.40 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.20 4 5 0.03 AU 0.0 mL/min 50 µL 20. 6. 5. 2.00 16.02 Flow rate: Injection vol. nitrosamines.05 4 µS 5 7 6 Mobile phase plus sample 0. Waters IC-Pak Anion HC Borate/Gluconate 2. 7. Shulamit Levin.: 0.00 1.00 3 0.00 8.40 0.00 Waters IC-Pak Anion HR 1. 6.60 1 2 Detection: 3 Direct Conductivity after Suppression 1.00 5.00 10.: Detection: 1 1.00 25.00 1 ppm 2 ppm 4 ppm 4 ppm 4 ppm 6 ppm 4 ppm Detection: UV (PDA) at 214 nm 0.04 0.80 µS 7 6 0. 3. 1. 4. Shulamit Levin. Ionization Source x Sorting of Ions Detection Ion Detector Analyzer Data System LC/MS Interface Desolvation HPLC Separation Dr.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. beta-emitters and many soft gamma and positron emitters encountered in bio-medical research and pharmaceutical quality control. A light trap is located 180º from the laser to collect any light not scattered by particles in the aerosol stream. and 32P. The photodiode produces a signal which is sent to the analog outputs for collection. 14C. Less common: Conductivity Mass-spectrometric (LC/MS) Evaporative light scattering How LC-MS Works Radioactive Detector Primarily used for the measurement of 3H. Medtechnica Date Processing Mass Spectrum . 16 100 295 4.02 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. Medtechnica 4.1=Propranolol.85 267.74 Detector Slit dcand Rf Voltages Ion Source IonSource 77 0 Time 2.87 235.00 4.82 (ESA) 0.34e5 262.87 Mixture 263.85 120.1 x 50 mm ( 5 µm) 2.88 264.29 2.92 333.00 2.00 Time (min) Dr.00 7.87 100 Ion Traps DC Transferoptic Mix (10. 240.13e5 N 100 (2) 281 O 100 264 (3) 1.11e4 0 MW=280 2.80 68.ES/APCI ES/APCI Ion Ion Source Source N N H (1) 260 9.87 8.50e5 3.87 1: Mass Chromatogram 195.00 6.1 % Formic Acid / MeCN 0.56 100 MW=295 8.62 Magnetic sector Detector Electrostatic Sector 3.56 1. 4=Trimipramine.53e5 0 0 Ding 280 1.98 4.26e5 (4) N 1.05 Int. R f Inlet ElectronMultiplier 340 190 The Quadrupole Quadrupole Analysator 5.92 98.29 MW=260 1.57 2.00 8.99 213.2 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 .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.90 % 222.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+ 4.67e4 265 NH 0 100 O OH 100 296 233 0 100 100 0 2. Shulamit Levin.65 S e c t o r Mass Sector Mass Spectrometers Spectrometers 8. 65/35 0.25e4 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).16 1.00 TIC TIC 3.57 100 HARDWARE HARDWARE -.65 5.82 76.00 Fast LC-MS Analysis 2. 3=Nortriptyline. 2=Doxepin.59e4 0 1.00 10.91 128.01 309.21e5 0 MW=264 1.00 5.62 287.696) 199 Types of Mass Spectrometer’s Analyzers 59.02 170. 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. Shulamit Levin.Detection in HPLC ESI-MS ESI-MS Ion Formation Mass Range Multiply Charged Molecules Horse Heart Myoglobin n = 23.2 to 2mL/minute Option: Crossflow interface Dr. Medtechnica Calculated Mass . 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. 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). so that detection can be optimized for different compounds. Fast response for high performance systems. Shulamit Levin. Insensitivity to changes in type of solvent. Low dead volume (minimal peak broadening & remixing of the separated bands). flow rate. PROPERTIES OF DETECTORS Detector Criteria Selectivity Sensitivity and detection limit Stability Linear range Dynamic Range Reproducibility Effect on peak shape Maintenance Dr. Preferably non-destructive. High sensitivity. and temperature. Medtechnica SELECTIVITY SPECIFIC UNIVERSAL A selective detector allows one to see only components of interest despite of their co-elution with any others. Operational simplicity and reliability. Wide linear dynamic range (quantitation). . Tuneable. 2 Minutes Dr. Medtechnica 3.2 AU Limit of quantitation No apparent noise Lowest concentration that can be determined with acceptable precision Signal-to-noise ratio of 10:1 2.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.0 3.001 AU 0.00 3.8 3.4 Noise 2. Shulamit Levin. 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 0.00 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 0.0 4.004 0. Medtechnica 6.0 Minutes Dr.000 LONG RANGE -0.0 4. Shulamit Levin.0 2.006 NOISE 0.002 0.0 0.0 Minutes 6.0 DRIFT .010 Maxplot 220 nm 0.002 0.008 SHORT RANGE AU 0.0 2. and smallest detectable peak. Medtechnica PROPERTIES OF DETECTORS FLOW-CELL VOLUME . drift.Detection in HPLC Noise and drift PROPERTIES OF DETECTORS LINEAR RANGE Noise. Shulamit Levin. 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. ELECTRONICS SAFETY Dr. Shulamit Levin. Medtechnica .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.
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