HPLC

Principle & Instrumentations

V. Namasivayam

Spinco Biotech Pvt. Ltd.

Chennai
 Course Outline

• Concept and scope of HPLC

• Separation Mechanisms

• Instrumentation

2
 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

Gas
Chromatography
Chromatography

Supercritical-fluid
Chromatography

3
 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
 Scope of HPLC
Field Typical mixtures

Pharmaceuticals Antibiotics, sedatives, steroids, analgesics, crude drugs,

cosmetics
Biochemical Amino acids, proteins, peptides, carbohydrates, lipids,
enzymes, medicines, hormone
Food products Mycotoxins, additives, saccharides, amino acids, vitamins,
fatty acid, coloring agents, antibacterials
Industrial chemicals Condensed aromatics, surfactants, propellants, dyes,
polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics

Environmental field Inorganic ions, organic acids, agricultural chemicals,

pesticides, herbicides, phenols,
Clinical medicine Bile acids, drug metabolites, urine extracts, estrogens

5
 Flow Diagram of HPLC

Column
Detector
Injector
Pump Oven

Mobile Phase

6
 Chromatogram

tR
Peak tR : Retention time
Signal

h A : Area
A h : Height

Time

7
 Some Important Terms
• Chromatogram: A plot of detector signal output versus time or elution
volume.
• Mobile phase: The liquid that moves the solute through the column.
• Stationary phase: The packing material of the column, which is the
immobile phase involved in the chromatographic process.
• 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.
• Retention time: The time taken by the analyte peak to reach the detector
after sample injection.
• Qualitation: An analysis process which is designed to identify the
components of a substance or mixture.
• Quantitation: An analysis process which is designed to determine the
amounts or proportion of the components of a substance.

8
 History of chromatography
M. Tswett : first developer of chromatography

Petroleum ether

Chlorophylls

CaCO3

9
 Separation Mechanism

Compounds are separated because the molecules

move at different rates in the column.

1
2

column

10
 Separation Mechanism

Due to different interaction between stationary phase

and different sample, the molecules move at different
rate, therefore separation can be done.
Mobile Phase

1
2

Stronger Weaker
interaction interaction

Stationary Phase

11
 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 CH3
• C4 (butyl) type C18 H37
Si O Si
• Phenyl type
CH3
• TMS type
Non-polar
• Cyano type

Reversed phase HPLC

• Stationary phase: Non-polar property
• Mobile phase: Polar property

14
 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
 What Is the Interaction?
Hydrophobic
Interaction
A Less polar analyte

B More polar analyte

B B
A
A
B
A B
A
A B
A B

Support Nonpolar Interstitial area

particle bonded phase (mobile phase)

Less polar (more hydrophobic) analytes are more attracted and

spend more time associated with the hydrophobic bonded
phase, therefore, they are eluted last.
16
 Hydrophobicity

• If the sample has more

– CH3CH2CH2--- : Carbon chain

– : Aromatic group

– Hydrophobicity is stronger

• If the sample has more

– -COOH : Carboxyl group

– -NH2 : Amino group
– -OH : Hydroxyl group

Hydrophobicity is weaker
17
 Retention Time and Hydrophobicity

OH
1

C18 (ODS)
Weak

Strong
1 2
OH

18
 Increase of Solvent Polarity

H2O/MeOH=20/80 H2O/MeOH=30/70 H2O/MeOH=40/60

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
19
 Effect of Stationary Phase

C8

Medium
C18 (ODS)

sample
Strong
C4

sample
Weak

sample

20
 Choice of LC mode
Mode Solvent type used Compound type

Reversed H2O/Buffer, ACN, MeOH Neutral or non-ionised

Phase 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
Normal Phase Organic solvents Mixture of isomers and
compounds not soluble in
organic/water mixtures
Ion exchange H2O/Buffer Inorganic ions, proteins, nucleic
acids, organic acids.
SEC H2O, THF, CHCl3, DMF High molecular weight
compounds

21
 Scope of HPLC

106

Gel Size Gel

105
permeation exclusion filtration

Molecular weight
104

103
Normal Reversed Ion
phase phase exchange
102

Nonionic polar

Nonpolar Ionic
Water-insoluble Water-soluble

Increasing polarity
22
 Instrumentations

Modular HPLC
• Possible configurations
• Solvent delivery pumps
• Sample injectors
• Column ovens
• Detectors
Integrated HPLC
• LC-2010

23
 Possible Configuration

Isocratic system
Low-pressure gradient system
High-pressure gradient system

Isocratic Gradient

B% B%

Time
Time

24
 Elution Modes

MeOH / H2O = 6 / 4
Long Time Analysis Isocratic

Bad Separation MeOH / H2O = 8 / 2

Isocratic

Volts MeOH%
Gradient

Time
( Column : ODS )
25
 Isocratic System

Column
Detector
Injector
Pump Oven

Mobile Phase
Data
processor

Simple system with one pump and one solvent reservoir.

If more than one solvent is used, solvents should be premixed.

26
 Low-pressure Gradient System

Column Detector
Injector
Pump Oven
low pressure
gradient valve Data
processor

•One pump used to control 4 reservoirs;

•Mixing is done before pump.
•On-line degasser is necessary.
A B C D

27
 High-pressure Gradient System

pump

Mixer
B
Column Detector
pump Injector
Oven
Data
processor
C

pump • Excellent gradient accuracy.

• 2-3 pumps required - one pump per solvent used.
• On-line degassing may not be critical.

28
 LC-10Avp Series Layout

System Controller SCL-10Avp Detector

SPD-10A(V )vp
Auto-injector SPD-M10Avp
SIL-10ADvp RF-10Axl
RID-10A
etc.

LC Work Station
Solvent delivery unit CLASS-VP
LC-10ATvp LCsolution
LC-10ADvp
Column Oven
Low pressure GE Unit 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
UV detector
Degassing Unit

Pump Unit

Low pressure
gradient device
31
 We
LC-2010 Concept: HAVE 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 (1.5mL vial)
–100 x Samples (4 mL vial)
– 4 x Microtiter Plates
(96, 384well)

32
 We
HA
VE
LC-2010 Concept: HAVE it a
ll!

A : Automation
Automated Analytical Operation
– Auto Start Up
– Auto Purge
– Auto Baseline Check
– Auto Shutdown

33
 We
HA
VE
LC-2010 Concept: HAVE 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
 We
HA
Validation Support VE
it a
ll!

･ Performance Check
　－ Validation support through the Wizard
･ System Check
　－ Validation report
･ IQ/OQ documents are attached as standard

35
 We
HA
LC-2010 Concept: HAVE VE
it a
ll!

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

36
 Prominence overview
Low volume degasser
DGU-20A

Solvent delivery units

-Low pulsation LC-20AD World's
World'shighest
highestsensitivity!
sensitivity!
-General purpose LC-20AT
High Sensitive detector
-Binary LC-20AB
SPD-20A/20A Ｖ /
M20A

Rack Changer

Oven CTO-20A/C

Fast autosampler SIL-20A/C Controller CBM-20A / CBM card

World
World first!
first!
World'
World'fastest!
fastest!

37
 ….the future of HPLC

• 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

38
 Flexibility
 Modular type provides excellent flexibility.

Isocratic system Fully automated gradient system

39
 LC-20AD/20AT/20AB Features
Low pressure
GE valve
◆ Excellent solvent delivery performance

◆ Improved stability

◆ Improved durability

◆ Space saving design

LC-20AD LC-20AT LC-20AB

40
 Plunger Reciprocating Pump

out

pump head check valve

motor and cam

5 - 50µL

plunger
check valve
plunger seal

in
Mobile phase
41
 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
Main
plunger Sub
plunger The number of maintenance
parts is less. So this design is
check valve suitable for routine analysis.

43
 Dual Plunger
with Parallel Flow Line
LC-20AD/AB

Very low pressure fluctuation

check valve Refractive index detector
Conductivity detector
plunger plunger Electrochemical detector
MS detector
check valve The number of maintenance
parts is more.

44
 HPLC Manual Injector

How to inject sample

• Insert a syringe at INJECT position.
• Turn the knob to the LOAD position.
• Load the sample.
• Turn the knob to the INJECT position.
• Remove the syringe.
• Wash the injection port. Rheodyne Manual injector

Cautions
• Do not use pointed or beveled needle tip.
– Must use square end type.
• Do not use more than pH 10 solution.
– Must change rotor seal.

45
 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
 SIL-20A/20AC Overview
Wow,
◆ World's fastest sample injection so
fast!
10µL injection -- 10 seconds !

◆ Near zero sample carry over

World's best low sample carry over performance is achieved.
Using optional rinsing kit, multi-liquid rinse is possible.

◆ Rack Changer for large number of sample processing

Switching max 12x MTP/DWP plates, continuous analysis is
possible.

◆ In MTP/DWP setting, vials can be used

Control rack to accommodate 10 x 1.5mL vials is available.

51
 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.

with HPLC from SIL-HT SIL-20A

vendor A:
Remained sample
0.08 %
reduced to 1/4

Minimized touch area

SIL-20A provides good result even with With rinsing pump, sample carryover is not
adsorptive compound. detected.

0.0007 % Not detected

52 Sample carryover test using Chlor-hexidine

 SIL-20A/20AC
High Throughput
World's fastest sample injection makes analysis cycle
less than 1 minute possible.

0 0.5 1 1.5 2 2.5 3 min

53
 Column Ovens
The temperature fluctuation of column will influence retention time
reproducibility.
Column temperature control devices are functioning to keep the
column temperature constant.

CTO-10ASvp CTO-10A/10ACvp CTO-20A/20AC

54
 CTO-20A/20AC Overview

◆ Wide temperature control range

Max 85ºC, applicable to sugar analysis
　　　 CTO-20A : (ambient +10ºC) - 85ºC
　 CTO-20AC : (ambient - 10ºC) - 85ºC

◆ Column management with CMD

Optional column management device automatically
records column usage history.

◆ Large inner space

Manual injector, flow switching valves, mixer, CDD detector cell
are accommodated for easy system expansion.

55
Ultra Fast High Throughput not High Pressure

Ultra Flexible
b e
ul d
Ultra Fidelity
h o
y s
u l
Ultra Quick Method Transfer
t r
it
s
A Technology
Ultra Durable XR-ODS Column

Ultra Performance “Prominence” Proven

Platform

56
 Shimadzu LCMS-2010EV system
LC-20A

MS LCMSsolution
detector

Probe holder with a

Source Window

Connector panel
pilot lump

API probe

Ion inlet port

Shutter
57
 Integrated and Modular HPLC

Integrated LC-2010 Modular Prominence

Simplicity and automation Flexibility and expandability

Ease of operation Dependent on budget

Ease of support Dependent on application

Routine analysis R&D, Multipurpose

58
 HPLC
Detectors

V. Namasivayam

Spinco Biotech Pvt. Ltd.

Chennai
 Detectors for HPLC

• UV-VIS Ultraviolet / Visible detector

• PDA Photodiode Array detector
• RF Fluorescence detector
• CDD Conductivity detector
• RID Refractive Index detector
• ECD Electrochemical detector
• ELSD 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, such as aromatic rings

or multiple alternating double bonds.
RF Fluorescent compounds, usually with fused rings or
highly conjugated planar system.
CDD Charged compounds, such as inorganic ions and organic
acid.
ECD For easily oxidized compounds like quinones or amines.

RID & ELSD For compounds that do not show characteristics usable
by the other detectors, eg. polymers, sccharides.

61
 Ultraviolet / Visible Detector (1)

62
 Ultraviolet / Visible Detector (2)
Lambert-Beer’s Law

A = ε C L = - log (Eout / Ein )

Ideal

Actual A : absorbance
2.5
e

ε : molar absorptivity
ng
ec nabr os b A

ra
r

C : analyte concentration
ea
lin

L : path length of the flow cell

E : energy
Backgroud absorbance
Concentration
63
64
 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.
Additional Functions
• Dual Wavelength mode
• Wavelength Time Program mode
• Wavelength Scan mode

65
 Photodiode Array Detector (1)

Sample Cell Grating

One element detects

D2 / W lamp one absorbance at
one wavelength.

512 Elements Photodiode Array

66
 Photodiode Array Detector
3-D Data

Spectrum

Chromatogram
ec na br os b A

aW
Time ev
nel
ht g

67
68
69
70
71
72
73
74
75
76
77
 PDA Detector
Advantages:
• PDA Detector could analyze a sample simultaneously at many
different wavelengths.
• UV Visible spectra are useful for compound identification,
checking peak purity, as well as finding the optimum
absorbance for the compounds.
• UV Visible spectra of many compounds could be stored in the
spectrum libraries, which are useful for compound
identification.
• Relatively robust to temperature and flow rate fluctuations
• Compatible with gradient elution.

Disadvantages:
• Slightly less sensitive than UV-Visible detector.

78
 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
Non-radiation transition
T1

Light absorption

Fluorescence
Phosphorescence

S0

79
 Relationship Between
Fluorescence Intensity & Concentration

F = 2.3 Φf I0εb c

F :Relative fluorescence intensity

Φf :Quantum efficiency
I0 :Intensity of incident radiation
ε :Molar absorptivity
b :Pathlength of flow cell
c :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
83
 Refractive Index Detector (1)

Photodiode
Reference Refraction

W Lamp
Sample

84
85
 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
 Refractive Index Detector (4)
Application Example

• Analytical Conditions
– Column : Shim-pack CLC-NH2
– Mobile phase : Acetonitrile / water = 70/30
– Flow rate : 1.0 mL/min
– Temperature : Ambient
• Peaks
1. Glycerol
2. Xylose
3. Fructose
4. Glucose
5. Sucrose
6. Manose
7. Lactose

87
 Conductivity Detector Principle

V K (conductivity) = I [A] / E [V]

=A [cm2] / L [cm] * k
I (k : specific conductivity)

k= (I/E)*(L/A)

A A

Electrodes L

88
 Temperature Control of
Conductivity Detector

• Conductivity is very affected by temperature.

• Must keep the cell in the temperature control devise.

89
 Conductivity Detector

Advantages:
• Respond to ionic compounds and suitable for ion
chromatography.
• 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
 Application Example (Anions)

• Analytical Conditions
– Column : Shim-pack IC-A3
– Mobile phase :
8.0 mM p-hydroxybenzoic acid
3.2 mM Bis-Tris *
– Flow rate : 1.5 mL/min
– Temperature : 40ºC
– Injection Volume : 100 µL
• Peaks
– 1. F- (1.4 ppm)
– 2. Cl- (10200 ppm)
– 3. NO2- (10 ppm)
– 4. Br- (43 ppm)
– 5. NO3- (44 ppm)
– 6. SO42- (431 ppm)

Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane

91
 Application Example (Cations)

 Analytical Conditions
 Column : Shim-pack IC-C3
 Mobile phase : 2.0 mM Oxalic Acid
 Flow rate : 1.0 mL/min
 Temperature : 40ºC
 Injection volume : 100µL
 Peaks
 1. Na+ (8.25 ppm)
 2. NH4+ (0.01 ppm)
 3. K+ (1.66 ppm)
 4. Mg2+ (2.22 ppm)
 5. Ca2+ (11.85 ppm)

92
 Electrochemical Detector

Working
electrode
Reference
electrode

AUX electrode

93
 Principle of ECD Detection
Electrochemical detector responds to compounds that can
be oxidized or reduced, such as phenols, aromatic amines,
ketones, aldehydes.

R O + H+

e- Electrode
Glassy Carbon (GC)
Pt, Ag, Au
[ Applications ]
GC : phenol compounds
general use A
Pt : H2O2
Ag : halogen ion
Au : sugar analysis
94
 LC-EC Detection
(electro) chemical reaction detector
Reaction Principle potential (E) is driving force

• red ⇔ ox + n e-
– n determines signal
– potential sign determines
direction (ox: +, red: -)

• multiple steps:
– mass transport by diffusion
– electron transfer reaction
– follow-up reactions

95
 Electrochemical Detector

Advantages:
• Selective as relatively few compounds are electro-active.
• Excellent sensitivity for low concentration range.

Disadvantages:
• Sensitive to temperature and flow rate fluctuations
• Not compatible with gradient elution.
• Aqueous or other polar solvents containing dissolved
electrolytes are required and they must be rigorously free
from oxygen.

96
 Application Example
(catecolamines)
 Analytical Conditions
 Column : Shim-pack CLC-ODS
 Mobile phase :

80 mM phosphate buffer (pH=2.7)

100 mM NaNO3, 200 mg/l SOS
5 mg/l EDTA, 4 % acetonitrile
 Flow rate : 1.0 mL/min
 Applied Potential : + 0.8 V
 Temperature : 40 C
 Injection volume : 10 uL
 Peaks
 1. Noradrenalin ( 5 ppb)
 2. Adrenalin (5 ppb)
 3. Dopamine (5 ppb)

97
 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, fatty
acids ）
　
Chemical Industry Pharmaceutical
（ Polymers, surfactants ） （ Impurities ）

100
 Mobile Phase & Nebulizing Gas

Mobile Phase Nebulizing Gas

• Water • Nitrogen
• Methanol • Compressed air
• Acetonitrile • etc
• THF
• etc

101
 Evaporative Light Scattering Detector

Advantages:
• Most compounds can be detected (universal detector)
• Compatible with gradient elution

Disadvantages:
• Mobile phase must be volatile.
• Nebulizing gas is required.

102
 　 Single Quadrupole LC/MS
　
System
HPLC Interface MS
Ionization probe Q-array Octopole Quadrupole Detector

Atmospheric
Pressure 10-3 ～ 10-4 Pa
80 ～ 150
Pa
TMP １ TMP ２

Rotary Pump

103
 Interface of LC-MS

Key Technology

HPLC Interface MS

 Aqueous/organic  To Remove solvent  High vacuum

solvent with buffers
 To Ionize analyte
 Non-volatile compounds molecules  Analyze ions, m/z

 Research on interfacing HPLC to MS began in the 1970s; API (atmospheric

pressure ionization) sources were commercialized in 1987.
 API interfaces: electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI)

104
105
106
 Principles of ESI
Electro Spray Ionization

Vacuum
Drying
Nebulizing gas
gas

3-5 kV
Ions
HPLC
0.001-1 ml/min

ESI probe
+ ++ +
+ + ++++ ++
+ + + + +
++ + [M+H]
++ +++ ++ +

 Ionization in liquid phase

107  Ionization at room temperature
 Principles of APCI
Atmospheric Pressure Chemical Ionization
Heater (400oC)
Drying
Vacuum
Nebulisin gas
g gas
Corona
discharge
3-5 kV
HPLC
Ions
0.05 - 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)

108  Ionization in the gas phase by discharge, ion-molecule reaction
 Ionization diagram

Molecular
Weight

10,000 ESI

1,000 APCI

APPI
100 EI (GCMS)

Non-polar Very polar

polarity

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

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

110
 Ionization of Compounds in MS Detector

• ESI
– drugs and their metabolites
– peptides
– proteins
– many kinds of natural product
(-OH, -NH2,-COOH, SO2, PO3 etc.)

• APCI
– pesticides
– steroids
– drugs

111
 What kind of benefits LC/MS users can get ?

• Determination of MW
• Qualitative capability
• Selective quantitative capability m/z=100
• High sensitivity
A
B
TIC
A:100
B:100
D:150
C:150
m/z=150

C D

112
 Comparison of Detectors

Detectors Gradient Compatibility

UV-Vis & PDA* Yes

Fluorescence (RF) Yes

Refractive Index (RID) No

Conductivity (CDD) No

Electrochemical (ECD) No

Evaporative Light Scattering Yes

(ELSD)
MS Yes

* The sensitivity of PDA Detector is slightly less than UV-Vis Detector

113
114
 THANK YOU & ALL THE
BEST
115