Principle and Instrumentation
Principle and Instrumentation
Principle and Instrumentation
V. Namasivayam
• Separation Mechanisms
• Instrumentation
2
Concept of Chromatography
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
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
1
2
column
10
Separation Mechanism
1
2
Stronger Weaker
interaction interaction
Stationary Phase
11
Separation Modes
• Ion chromatography
• Affinity chromatography
12
Reversed Phase Mode
13
Stationary Phase in
Reversed Phase Column
14
Mobile Phase for Reversed Phase HPLC
15
What Is the Interaction?
Hydrophobic
Interaction
A Less polar analyte
– Hydrophobicity is stronger
Hydrophobicity is weaker
17
Retention Time and Hydrophobicity
OH
1
C18 (ODS)
Weak
Strong
1 2
OH
18
Increase of Solvent Polarity
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
21
Scope of HPLC
106
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
Volts MeOH%
Gradient
Time
( Column : ODS )
25
Isocratic System
Column
Detector
Injector
Pump Oven
Mobile Phase
Data
processor
26
Low-pressure Gradient System
Column Detector
Injector
Pump Oven
low pressure
gradient valve Data
processor
27
High-pressure Gradient System
pump
Mixer
B
Column Detector
pump Injector
Oven
Data
processor
C
28
LC-10Avp Series Layout
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
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
Rack Changer
Oven CTO-20A/C
37
….the future of HPLC
38
Flexibility
Modular type provides excellent flexibility.
39
LC-20AD/20AT/20AB Features
Low pressure
GE valve
◆ Excellent solvent delivery performance
◆ Improved stability
◆ Improved durability
40
Plunger Reciprocating Pump
out
5 - 50µL
plunger
check valve
plunger seal
in
Mobile phase
41
Plunger Reciprocating Pump
• Advantage
– Low pressure fluctuation
– Very easy to replace other solvent
• Disadvantage
– Change the plunger seal
42
Dual Plunger
with Tandem Flow Line
LC-20AT
43
Dual Plunger
with Parallel Flow Line
LC-20AD/AB
44
HPLC 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
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 !
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.
SIL-20A provides good result even with With rinsing pump, sample carryover is not
adsorptive compound. detected.
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.
54
CTO-20A/20AC Overview
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
56
Shimadzu LCMS-2010EV system
LC-20A
MS LCMSsolution
detector
Connector panel
pilot lump
API probe
58
HPLC
Detectors
V. Namasivayam
60
Selection of Detectors
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
Actual A : absorbance
2.5
e
ε : molar absorptivity
ng
ec nabr os b A
ra
r
C : analyte concentration
ea
lin
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)
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
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
k= (I/E)*(L/A)
A A
Electrodes L
88
Temperature Control of
Conductivity Detector
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)
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 :
97
Evaporative Light Scattering Detector
Detection Pinciple
Three steps
• Nebulization
• Evaporation
• Detection
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
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
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]
++ +++ ++ +
ESI
probe Discharge to form primary ion:
N 2 N2+
Gas phase ion – molecule reaction
with charge or proton transfer
Molecular
Weight
10,000 ESI
1,000 APCI
APPI
100 EI (GCMS)
- 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
Conductivity (CDD) No
Electrochemical (ECD) No
113
114
THANK YOU & ALL THE
BEST
115