HPLC

Principle & Instrumentations
V. Namasivayam

Spinco Biotech Pvt. Ltd. Chennai

Course Outline

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Concept and scope of HPLC Separation Mechanisms Instrumentation

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Concept of Chromatography
• • Chromatography is an analytical method that the compounds are physically separated prior to measurement The main purpose of chromatography is to separate and quantify the target sample in the matrix
Liquid Chromatography

Chromatography

Gas Chromatography

Supercritical-fluid Chromatography
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Why use HPLC? • Simultaneous Analysis • High Resolution • High Sensitivity (ppm-ppb) • Good repeatability • Small sample size • Moderate analysis condition .no need to vaporize the sample like GC • Easy to fractionate the sample and purify • No destructive for many detectors 4 .

poisons. enzymes. amino acids.Scope of HPLC Field Pharmaceuticals Biochemical Food products Industrial chemicals Forensic chemistry Environmental field Clinical medicine Typical mixtures Antibiotics. crude drugs. antibacterials Condensed aromatics. carbohydrates. phenols. analgesics. Bile acids. agricultural chemicals. cosmetics Amino acids. fatty acid. steroids. organic acids. drug metabolites. proteins. pesticides. blood alcohol. additives. hormone Mycotoxins. polymers. herbicides. propellants. surfactants. dyes. urine extracts. narcotics Inorganic ions. coloring agents. vitamins. saccharides. plasticizers Drugs. sedatives. estrogens 5 . peptides. medicines. lipids.

Flow Diagram of HPLC Column Injector Pump Mobile Phase Oven Detector 6 .

Chromatogram tR Signal Peak tR : Retention time A : Area h : Height h A Time 7 .

Qualitation: An analysis process which is designed to identify the components of a substance or mixture. • • • 8 . Retention time: The time taken by the analyte peak to reach the detector after sample injection. Peak: The visual representation on the chromatogram based on the detector's electrical response due to the presence of a sample component inside the flow cell. Quantitation: An analysis process which is designed to determine the amounts or proportion of the components of a substance. Stationary phase: The packing material of the column. Mobile phase: The liquid that moves the solute through the column.Some Important Terms • • • • Chromatogram: A plot of detector signal output versus time or elution volume. which is the immobile phase involved in the chromatographic process.

Tswett : first developer of chromatography Petroleum ether Chlorophylls CaCO3 9 .History of chromatography M.

1 2 column 10 .Separation Mechanism Compounds are separated because the molecules move at different rates in the column.

therefore separation can be done.Separation Mechanism Due to different interaction between stationary phase and different sample. Mobile Phase 1 Stronger interaction Weaker interaction 2 Stationary Phase 11 . the molecules move at different rate.

Separation Modes • • • • • Normal phase chromatography Reversed phase chromatography Ion chromatography Size exclusion chromatography Affinity chromatography 12 .

Reversed Phase Mode Stationary phase: Non-polar property Mobile phase : Polar property This combination is defined as Reversed Phase Mode 13 .

Stationary Phase in Reversed Phase Column • C18 (ODS) type • C8 (octyl) type • C4 (butyl) type • Phenyl type • TMS type • Cyano type Reversed phase HPLC • • 14 CH3 Si O Si CH3 Non-polar C18 H37 Stationary phase: Mobile phase: Non-polar property Polar property .

Mobile Phase for Reversed Phase HPLC • Water / buffer + Organic solvent – – – – – – – – Organic solvents: Methanol Acetonitrile THF Buffer: Phosphate buffer Acetate buffer etc • Ratio of aqueous and organic solvents is important 15 .

they are eluted last. 16 .What Is the Interaction? Hydrophobic Interaction A Less polar analyte B More polar analyte B A A A A A B B B B B A Support particle Nonpolar bonded phase Interstitial area (mobile phase) Less polar (more hydrophobic) analytes are more attracted and spend more time associated with the hydrophobic bonded phase. therefore.

Hydrophobicity • If the sample has more – CH3CH2CH2--– : Carbon chain : Aromatic group – Hydrophobicity is stronger • If the sample has more – -COOH : Carboxyl group : Amino group – -NH2 – -OH : Hydroxyl group Hydrophobicity is weaker 17 .

Retention Time and Hydrophobicity OH 1 C18 (ODS) Strong OH Weak 1 2 2 18 .

Increase of Solvent Polarity H2O/MeOH=20/80 H2O/MeOH=30/70 H2O/MeOH=40/60 1 2 3 4 19 : : : : p-Hydoxymethylbenzoate p-Hydoxyethylbenzoate p-Hydoxypropylbenzoate p-Hydoxybutylbenzoate .

Effect of Stationary Phase C8 Medium sample Strong sample Weak sample C4 C18 (ODS) 20 .

THF. MeOH Compound type Neutral or non-ionised compounds which can be dissolved in water/organic mixtures Ion-Pair RP Same as above with addition of Ionic or ionizable compounds ion-pair reagent Mixture of isomers and compounds not soluble in organic/water mixtures Inorganic ions. proteins. CHCl3. organic acids. ACN. High molecular weight compounds Normal Phase Organic solvents Ion exchange H2O/Buffer SEC H2O.Choice of LC mode Mode Reversed Phase Solvent type used H2O/Buffer. DMF 21 . nucleic acids.

Scope of HPLC
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Gel permeation

Size exclusion

Gel filtration

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Normal phase

Reversed phase

Ion exchange

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Nonionic polar Nonpolar Water-insoluble Ionic Water-soluble

Increasing polarity
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Instrumentations
Modular HPLC • Possible configurations • Solvent delivery pumps • Sample injectors • Column ovens • Detectors Integrated HPLC • LC-2010
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Possible Configuration
Isocratic system Low-pressure gradient system High-pressure gradient system
Isocratic
B% B%

Gradient

Time

Time

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Elution Modes MeOH / H2O = 6 / 4 Long Time Analysis Isocratic Bad Separation MeOH / H2O = 8 / 2 Isocratic Volts Gradient MeOH% Time 25 ( Column : ODS ) .

solvents should be premixed.Isocratic System Column Injector Pump Mobile Phase Oven Detector Data processor Simple system with one pump and one solvent reservoir. If more than one solvent is used. 26 .

A B C D •On-line degasser is necessary. 27 . •Mixing is done before pump.Low-pressure Gradient System Injector low pressure gradient valve Column Oven Detector Pump Data processor •One pump used to control 4 reservoirs.

On-line degassing may not be critical. 2-3 pumps required . 28 .High-pressure Gradient System A pump Mixer pump Injector B Column Oven Detector Data processor C pump • • • Excellent gradient accuracy.one pump per solvent used.

LC-10Avp Series Layout System Controller SCL-10Avp Auto-injector SIL-10ADvp Detector SPD-10A(V )vp SPD-M10Avp RF-10Axl RID-10A etc. LC Work Station Solvent delivery unit LC-10ATvp LC-10ADvp CLASS-VP LCsolution Low pressure GE Unit Column Oven CTO-10ASvp CTO-10ACvp CTO-10Avp 29 .

Shimadzu HPLC Integrated LC      Modular LC LC-2010 LC-20A 30 .

Outline of LC-2010 Reservior Tray System Controller Auto sampler Column Oven Degassing Unit Pump Unit Low pressure gradient device 31 UV detector .

384well) 32 .5mL vial) –100 x Samples (4 mL vial) – 4 x Microtiter Plates (96.LC-2010 Concept: HAVE We HA VE it a ll! H : High Throughput •High Speed Injection – 15 seconds (when injecting 10µ L) •Multiple Sample Processing –350 x Samples (1mL vial) –210 x Samples (1.

LC-2010 Concept: HAVE A : Automation Automated Analytical Operation – Auto Start Up – Auto Purge – Auto Baseline Check – Auto Shutdown We HA VE it a ll! 33 .

LC-2010 Concept: HAVE We HA VE it a ll! V : Validation Auto Validation (1) Approximately 1.5 hours with the Isocratic mode (2) Approximately 3 hours with Gradient mode Guarantee of system performance 34 .

Validation Support ・ Performance Check  - Validation support through the Wizard ・ System Check  - Validation report ・ IQ/OQ documents are attached as standard We HA VE it a ll! 35 .

LC-2010 Concept: HAVE

We HA VE

it a ll!

E : Ease of Use
•Graphical Operation System –GUI Capability –Wizard Function •Front access for maintenance

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Prominence overview
Low volume degasser DGU-20A Solvent delivery units -Low pulsation LC-20AD -General purpose LC-20AT -Binary LC-20AB

World's highest sensitivity! World's highest sensitivity! High Sensitive detector SPD-20A/20A V / M20A

Rack Changer Oven CTO-20A/C

Fast autosampler SIL-20A/C World' fastest! World' fastest!
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Controller CBM-20A / CBM card World first! World first!

….the future of HPLC

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World’s first web-based instrument control World’s only system with Data Buffering capability World’s quietest HPLC pump World’s lowest degassing volume World’s fastest Auto-sampler World’s cleanest Auto-sampler World’s highest sensitive PDA detector World’s most sensitive UV-VIS detector World’s best Front End HPLC for LC-MS/MS World’s best LC Virtual advisor multimedia tool

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Isocratic system 39 Fully automated gradient system .Flexibility  Modular type provides excellent flexibility.

LC-20AD/20AT/20AB Features ◆ Excellent solvent delivery performance ◆ Improved stability ◆ Improved durability ◆ Space saving design Low pressure GE valve LC-20AD 40 LC-20AT LC-20AB .

50µL plunger plunger seal check valve in 41 Mobile phase .Plunger Reciprocating Pump out pump head check valve motor and cam 5 .

Plunger Reciprocating Pump • Consists of a small chamber in which the solvent is pumped by the back and forth motion of a motor-driven piston • Advantage – Low pressure fluctuation – Very easy to replace other solvent • Disadvantage – Change the plunger seal 42 .

Dual Plunger with Tandem Flow Line LC-20AT Low pressure fluctuation UV / PDA detector Fluorescence detector Sub plunger Main plunger check valve The number of maintenance parts is less. 43 . So this design is suitable for routine analysis.

check valve plunger plunger check valve 44 .Dual Plunger with Parallel Flow Line LC-20AD/AB Very low pressure fluctuation Refractive index detector Conductivity detector Electrochemical detector MS detector The number of maintenance parts is more.

Do not use more than pH 10 solution. Turn the knob to the INJECT position. – Must change rotor seal. Remove the syringe. 45 . Load the sample. Wash the injection port.HPLC Manual Injector How to inject sample • • • • • • Insert a syringe at INJECT position. Rheodyne Manual injector • • Cautions Do not use pointed or beveled needle tip. Turn the knob to the LOAD position. – Must use square end type.

6 Port Valve System Typical sample loop volume is 5-200 µl. 46 .

HPLC Auto Injectors SIL-10ADvp Inside of SIL-10Avp 47 .

HPLC Auto Injectors SIL-20AC Inside of SIL-20AC 48 .

Principle of Auto Injectors (1) Sample Aspiration 49 .

Principle of Auto Injectors (2) Start of analysis 50 .

Using optional rinsing kit. 51 . ◆ In MTP/DWP setting. multi-liquid rinse is possible.5mL vials is available.SIL-20A/20AC Overview ◆ World's fastest sample injection 10µL injection -. continuous analysis is possible. so fast! ! ◆ Near zero sample carry over World's best low sample carry over performance is achieved.10 seconds Wow. vials can be used Control rack to accommodate 10 x 1. ◆ Rack Changer for large number of sample processing Switching max 12x MTP/DWP plates.

sample carryover is not detected. 0.08 % SIL-HT Remained sample SIL-20A reduced to 1/4 Minimized touch area SIL-20A provides good result even with adsorptive compound. With rinsing pump.0007 % Not detected 52 Sample carryover test using Chlor-hexidine . with HPLC from vendor A: 0.Low sample carryover ◆ Data reliability improvement for ultra trace sample analysis ・ Surface treatment of needle and shape of the needle and injection port are optimized to reduce carry over.

5 1 1.5 2 2. 0 0.5 3 min 53 .SIL-20A/20AC High Throughput World's fastest sample injection makes analysis cycle less than 1 minute possible.

Column temperature control devices are functioning to keep the column temperature constant.Column Ovens The temperature fluctuation of column will influence retention time reproducibility. CTO-10ASvp 54 CTO-10A/10ACvp CTO-20A/20AC .

CTO-20A/20AC Overview ◆ Wide temperature control range Max 85ºC. CDD detector cell are accommodated for easy system expansion. applicable to sugar analysis       CTO-20A : (ambient +10ºC) .85ºC ◆ Column management with CMD Optional column management device automatically records column usage history.85ºC CTO-20AC : (ambient . flow switching valves. 55 . ◆ Large inner space Manual injector.10ºC) . mixer.

Ultra Fast Ultra Flexible Ultra Fidelity High Throughput not High Pressure Ultra Quick Method Transfer A Ultra Durable XR-ODS Column Technology Ultra Performance “Prominence” Proven Platform tr it s sh ly u ul o be d 56 .

Shimadzu LCMS-2010EV system LC-20A MS detector LCMSsolution Probe holder with a Source Window Connector panel pilot lump API probe 57 Ion inlet port Shutter .

Multipurpose 58 .Integrated and Modular HPLC Integrated LC-2010 Simplicity and automation Ease of operation Ease of support Routine analysis Modular Prominence Flexibility and expandability Dependent on budget Dependent on application R&D.

HPLC Detectors V. Namasivayam Spinco Biotech Pvt. Ltd. Chennai .

Detectors for HPLC • UV-VIS • PDA • CDD • ECD • ELSD Ultraviolet / Visible detector Photodiode Array detector • RF Fluorescence detector Conductivity detector • RID Refractive Index detector Electrochemical detector Evaporative light scattering detector • MS Mass spectrometer detector 60 .

Selection of Detectors Detectors Type of compounds can be detected UV-Vis & PDA Compounds with chromophores. RF CDD ECD Fluorescent compounds. eg. sccharides. RID & ELSD For compounds that do not show characteristics usable by the other detectors. For easily oxidized compounds like quinones or amines. such as aromatic rings or multiple alternating double bonds. usually with fused rings or highly conjugated planar system. such as inorganic ions and organic acid. 61 . polymers. Charged compounds.

Ultraviolet / Visible Detector (1) 62 .

log (Eout / Ein ) Ideal 2.5 Actual ra ng e A : absorbance ε : molar absorptivity C : analyte concentration L : path length of the flow cell E : energy ec nab os b A r Backgroud absorbance Concentration 63 lin ea r .Ultraviolet / Visible Detector (2) Lambert-Beer’s Law A = ε C L = .

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Dual Wavelength mode Wavelength Time Program mode Wavelength Scan mode Additional Functions • • • 65 .Ultraviolet / Visible Detector (4) Advantage: • • • Sensitivity is high Relative robust to temperature and flow rate change Compatible with gradient elution Disadvantage: • Only compounds with UV or visible absorption could be detected.

512 Elements Photodiode Array 66 .Photodiode Array Detector (1) Sample Cell Grating D2 / W lamp One element detects one absorbance at one wavelength.

Photodiode Array Detector 3-D Data Spectrum Chromatogram ec na b os b A r Time 67 hg t ne le va W .

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UV Visible spectra of many compounds could be stored in the spectrum libraries. Relatively robust to temperature and flow rate fluctuations Compatible with gradient elution. UV Visible spectra are useful for compound identification. checking peak purity. as well as finding the optimum absorbance for the compounds. which are useful for compound identification.PDA Detector Advantages: • • PDA Detector could analyze a sample simultaneously at many different wavelengths. . • • • Disadvantages: • 78 Slightly less sensitive than UV-Visible detector.

Fluorescence of Compounds Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds Non-radiation transition S1 T1 Light absorption Non-radiation transition Fluorescence Phosphorescence S0 79 .

Relationship Between Fluorescence Intensity & Concentration F = 2.3 Φf I0εb c F Φf I0 ε b c :Relative fluorescence intensity :Quantum efficiency :Intensity of incident radiation :Molar absorptivity :Pathlength of flow cell :Concentration 80 .

Fluorescence Detector 81 .

Fluorescence Detector Advantage • • • Sensitivity is higher than UV-Vis detector Selectivity is high because relatively few compounds fluorescence Compatible with gradient elution Disadvanage • • Difficult to predict fluorescence Greatly affected by environment – Solvent – pH – Temperature – Viscosity – Ionic strength – Dissolved gas 82 .

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Refractive Index Detector (1) Photodiode Reference Refraction W Lamp Sample 84 .

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Refractive Index Detector (3) Advantage Responds to nearly all solutes Unaffected by flow rate Disadvantage Not as sensitive as most other types of detectors Could not be used with gradient elution 86 .

Xylose 3. Lactose • 87 . Glucose 5. Manose 7. Fructose 4. Sucrose 6.Refractive Index Detector (4) Application Example • Analytical Conditions – Column : Shim-pack CLC-NH2 – Mobile phase : Acetonitrile / water = 70/30 – Flow rate : 1. Glycerol 2.0 mL/min – Temperature : Ambient Peaks 1.

Conductivity Detector Principle V I K (conductivity) = I [A] / E [V] =A [cm2] / L [cm] * k (k : specific conductivity) k= (I/E)*(L/A) A A Electrodes L 88 .

Must keep the cell in the temperature control devise.Temperature Control of Conductivity Detector • • Conductivity is very affected by temperature. 89 .

compounds and suitable for ion High sensitivity for low concentration range Disadvantages: • • Sensitive to the fluctuations in the solvent flow and mobile phase composition Not compatible with gradient elution. 90 .Conductivity Detector Advantages: • • Respond to ionic chromatography.

NO3(431 ppm) – 6.0 mM p-hydroxybenzoic acid 3. F(1.2 mM Bis-Tris * – Flow rate : 1. Br(43 ppm) (44 ppm) – 5.Application Example (Anions) • Analytical Conditions – Column : Shim-pack IC-A3 – Mobile phase : 8.5 mL/min – Temperature : 40ºC – Injection Volume : 100 µL Peaks – 1. SO42- • Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane 91 . Cl(10200 ppm) (10 ppm) – 3. NO2– 4.4 ppm) – 2.

25 ppm) (0. Na+ 2. Ca2+ (8.66 ppm) (2.85 ppm)  Peaks      92 . NH4+ 3.01 ppm) (1.Application Example (Cations)  Analytical Conditions      Column : Shim-pack IC-C3 Mobile phase : 2.0 mM Oxalic Acid Flow rate : 1. Mg2+ 5. K+ 4.0 mL/min Temperature : 40ºC Injection volume : 100µL 1.22 ppm) (11.

Electrochemical Detector Working electrode Reference electrode AUX electrode 93 .

such as phenols. Ag. aldehydes. ketones.Principle of ECD Detection Electrochemical detector responds to compounds that can be oxidized or reduced. Au [ Applications ] GC : phenol compounds general use Pt : H2O2 Ag : halogen ion Au : sugar analysis 94 A . R eO + H+ Electrode Glassy Carbon (GC) Pt. aromatic amines.

red: -) multiple steps: – mass transport by diffusion – electron transfer reaction – follow-up reactions (electro) chemical reaction detector potential (E) is driving force • 95 .LC-EC Detection Reaction Principle • red ⇔ ox + n e– n determines signal – potential sign determines direction (ox: +.

96 .Electrochemical Detector Advantages: • • Selective as relatively few compounds are electro-active. Excellent sensitivity for low concentration range. Aqueous or other polar solvents containing dissolved electrolytes are required and they must be rigorously free from oxygen. Disadvantages: • • • Sensitive to temperature and flow rate fluctuations Not compatible with gradient elution.

Noradrenalin ( 5 ppb)  2.8 V  Temperature : 40 C  Injection volume : 10 uL Peaks  1.Application Example (catecolamines)   Analytical Conditions  Column : Shim-pack CLC-ODS  Mobile phase : 80 mM phosphate buffer (pH=2. Dopamine (5 ppb) 97 .7) 100 mM NaNO3. 200 mg/l SOS 5 mg/l EDTA.0 mL/min  Applied Potential : + 0. Adrenalin (5 ppb)  3. 4 % acetonitrile  Flow rate : 1.

Evaporative Light Scattering Detector Detection Pinciple Three steps • Nebulization • Evaporation • Detection ELSD responds to compound that is less volatile than that of the mobile phase Shimadzu ELSD-LT 98 .

Evaporative Light Scattering Detector Mobile phase PC Light Gas Detection Nebulization Evaporation 99 .

Applications of ELSD Food ( Saccharides. surfactants ) Pharmaceutical ( Impurities ) 100 . fatty acids )   Chemical Industry ( Polymers.

Mobile Phase & Nebulizing Gas Mobile Phase • Water • Methanol • Acetonitrile • THF • etc Nebulizing Gas • Nitrogen • Compressed air • etc 101 .

Evaporative Light Scattering Detector Advantages: • • Most compounds can be detected (universal detector) Compatible with gradient elution Disadvantages: • • Mobile phase must be volatile. 102 . Nebulizing gas is required.

  Single Quadrupole LC/MS   System HPLC Interface Ionization probe Q-array Octopole MS Quadrupole Detector Atmospheric Pressure 80 ~ 150 Pa 10-3 ~ 10-4 Pa TMP 1 TMP 2 Rotary Pump 103 .

 API interfaces: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) 104 . API (atmospheric pressure ionization) sources were commercialized in 1987.Interface of LC-MS Key Technology HPLC  Aqueous/organic solvent with buffers  Non-volatile compounds Interface  To Remove solvent  To Ionize analyte molecules MS  High vacuum  Analyze ions. m/z  Research on interfacing HPLC to MS began in the 1970s.

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001-1 ml/min Ions ESI probe +++ + + + + ++   + ++ + + + ++ + + + + + + + + + + [M+H] + 107 Ionization in liquid phase Ionization at room temperature .Principles of ESI Electro Spray Ionization Nebulizing gas 3-5 kV Drying gas Vacuum HPLC 0.

ion-molecule reaction  108 .05 .Principles of APCI Atmospheric Pressure Chemical Ionization Heater (400oC) Nebulisin g gas Corona discharge 3-5 kV HPLC Drying gas Vacuum Ions 0.2 ml/min ESI probe Discharge to form primary ion: N 2  N2+ Gas phase ion – molecule reaction with charge or proton transfer Evaporate LC elute into gas phase by a heater (400oC)  Ionization in the gas phase by discharge.

APCI is chosen when its ionization effect is significantly better than ESI in certain analysis.Ionization diagram Molecular Weight 10.000 1. More reference data are available from open literature. “It is difficult to generalize which class of compounds can be ionized by which probe.” (Britt E. Erickson. because there are many exceptions.109 is chosen only when ESI and APCI could not ionize target compounds effectively. Today’s Chemist Feb 2001) . APPI . .000 100 EI (GCMS) Non-polar APPI ESI APCI polarity Very polar .ESI has been most widely used in various LC-MS systems.

Ion Detector Electron Multiplier 1. these secondary electron are then attracted to the next dynode where more secondary electrons are generated 4. A series of dynodes maintained at ever-increasing potentials 2. resulting in the emission of electrons. ultimately resulting in a cascade of electrons 110 . 3. Ions strike the dynode surface.

Ionization of Compounds in MS Detector • ESI – – – – drugs and their metabolites peptides proteins many kinds of natural product (-OH. SO2. PO3 etc. -NH2.-COOH.) • APCI – pesticides – steroids – drugs 111 .

What kind of benefits LC/MS users can get ? • Determination of MW • Qualitative capability • Selective quantitative capability • High sensitivity m/z=100 A B TIC A:100 B:100 D:150 C:150 m/z=150 C D 112 .

Comparison of Detectors Detectors UV-Vis & PDA* Fluorescence (RF) Refractive Index (RID) Conductivity (CDD) Electrochemical (ECD) Evaporative Light Scattering (ELSD) MS Gradient Compatibility Yes Yes No No No Yes Yes * The sensitivity of PDA Detector is slightly less than UV-Vis Detector 113 .

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THANK YOU & ALL THE BEST 115 .