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HPLC Principle & Instrumentations V. Namasivayam Spinco Biotech Pvt. Ltd. Chennai
HPLC
Principle & Instrumentations
V. Namasivayam
Spinco Biotech Pvt. Ltd.
Chennai
2
Course Outline
• Concept and scope of HPLC
• Separation Mechanisms
• Instrumentation
3
Concept of Chromatography
Chromatography
Supercritical-fluid
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
Gas
Chromatography
Liquid
Chromatography
4
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
5
Scope of HPLC
Field Typical mixtures
PharmaceuticalsAntibiotics, sedatives, steroids, analgesics, crude drugs, cosmetics
BiochemicalAmino acids, proteins, peptides, carbohydrates, lipids, enzymes, medicines, hormone
Food productsMycotoxins, additives, saccharides, amino acids, vitamins, fatty acid, coloring agents, antibacterials
Industrial chemicalsCondensed aromatics, surfactants, propellants, dyes, polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Environmental fieldInorganic ions, organic acids, agricultural chemicals, pesticides, herbicides, phenols,
Clinical medicine Bile acids, drug metabolites, urine extracts, estrogens
6
Flow Diagram of HPLC
Pump
InjectorColumn
Oven
Detector
Mobile Phase
7
Chromatogram
tR : Retention time
A : Area
h : Height
tR
Sig
nal
Time
Peak
hA
8
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.
9
History of chromatography
M. Tswett : first developer of chromatography
Petroleum ether
CaCO3
Chlorophylls
10
21
Separation Mechanism
Compounds are separated because the molecules
move at different rates in the column.
column
11
Separation Mechanism
Due to different interaction between stationary phase
and different sample, the molecules move at different
rate, therefore separation can be done.
Stationary Phase
Stronger interaction
Weaker interaction
Mobile Phase
21
12
Separation Modes
• Normal phase chromatography
• Reversed phase chromatography
• Ion chromatography
• Size exclusion chromatography
• Affinity chromatography
13
Reversed Phase Mode
Stationary phase: Non-polar property
Mobile phase : Polar property
This combination is defined as
Reversed Phase Mode
14
Stationary Phase in
Reversed Phase Column
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type
• Phenyl type
• TMS type
• Cyano type
C18H37Si O Si
CH3
CH3
Non-polar
Reversed phase HPLC
• Stationary phase: Non-polar property
• Mobile phase: Polar property
15
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
16
What Is the Interaction?
Hydrophobic Interaction
Less polar (more hydrophobic) analytes are more attracted and spend more time associated with the hydrophobic bonded phase, therefore, they are eluted last.
A
B
B
B
B
B
AA
A
AA
B
Support particle
Nonpolar bonded phase
Interstitial area(mobile phase)
Less polar analyte
More polar analyteB
A
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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
18
Retention Time and Hydrophobicity
OH
OH
C18 (ODS)
Strong
Weak
1
1
2
2
19
Increase of Solvent Polarity
1 : p-Hydoxymethylbenzoate2 : p-Hydoxyethylbenzoate3 : p-Hydoxypropylbenzoate4 : p-Hydoxybutylbenzoate
H2O/MeOH=20/80 H2O/MeOH=30/70 H2O/MeOH=40/60
20
Effect of Stationary Phase
C18 (ODS)
Strong
C8
sample
sample
sample
C4
Medium
Weak
21
Choice of LC mode
Mode Solvent type used Compound type
Reversed Phase
H2O/Buffer, ACN, MeOH
Neutral or non-ionised compounds which can be dissolved in water/organic mixtures
Ion-Pair RPSame as above with addition of ion-pair reagent
Ionic or ionizable compounds
Normal Phase
Organic solventsMixture 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
22
Scope of HPLC
Gel permeation
Gel filtration
Ion exchange
Size exclusion
Reversed phase
Normal phase
Water-insoluble Water-soluble
IonicNonpolar
Nonionic polar
Increasing polarity
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104
105
106M
olecu
lar we
igh
t
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Instrumentations
Modular HPLC
• Possible configurations
• Solvent delivery pumps
• Sample injectors
• Column ovens
• Detectors
Integrated HPLC
• LC-2010
24
Possible Configuration
Isocratic system
Low-pressure gradient system
High-pressure gradient system
Gradient
Time
B%
Isocratic
Time
B%
25
Elution Modes
Long Time Analysis
MeOH / H2O = 6 / 4
( Column : ODS )
Bad Separation MeOH / H2O = 8 / 2
Isocratic
Isocratic
MeOH%Volts
Time
Gradient
26
Isocratic System
Simple system with one pump and one solvent reservoir.
If more than one solvent is used, solvents should be premixed.
Data processor
Pump
InjectorColumn
Oven
Detector
Mobile Phase
27
Low-pressure Gradient System
•One pump used to control 4 reservoirs;
•Mixing is done before pump.
•On-line degasser is necessary.
low pressuregradient valve Data
processor
A B DC
Pump
InjectorColumn
Oven
Detector
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High-pressure Gradient System
Data processor
pump
pump
pump
A
B
C
InjectorColumn
Oven
Detector
Mixer
• Excellent gradient accuracy.
• 2-3 pumps required - one pump per solvent used.
• On-line degassing may not be critical.
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LC-10Avp Series Layout
Solvent delivery unit LC-10ATvp LC-10ADvp
LC Work Station CLASS-VP LCsolution
Low pressure GE Unit
Detector SPD-10A(V )vp SPD-M10Avp RF-10Axl RID-10A etc.
System Controller SCL-10Avp
Auto-injector SIL-10ADvp
Column Oven CTO-10ASvp CTO-10ACvp CTO-10Avp
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Shimadzu HPLC
LC-2010 LC-20A
Integrated LC Modular LC
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Outline of LC-2010
System Controller
UV detector
Auto sampler
Column Oven
Pump Unit
Reservior Tray
Degassing Unit
Low pressure gradient device
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LC-2010 Concept: HAVE
H : High Throughput
•High Speed Injection
– 15 seconds (when injecting 10L)
•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)
We HAVE it all!
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A : AutomationAutomated Analytical Operation
– Auto Start Up
– Auto Purge
– Auto Baseline Check
– Auto Shutdown
LC-2010 Concept: HAVE
We HAVE it all!
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V : Validation
Auto Validation(1) Approximately 1.5 hours with the Isocratic mode
(2) Approximately 3 hours with Gradient mode
Guarantee of system performance
We HAVE it all!LC-2010 Concept: HAVE
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Validation Support
･ Performance Check
－ Validation support through the Wizard
･ System Check
－ Validation report
･ IQ/OQ documents are attached as standard
We HAVE it all!
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E : Ease of Use
•Graphical Operation System
–GUI Capability
–Wizard Function
•Front access for maintenance
We HAVE it all!LC-2010 Concept: HAVE
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Prominence overview
Oven CTO-20A/C
Rack Changer
Fast autosampler SIL-20A/C
World' fastest!World' fastest!
Controller CBM-20A / CBM card
World first!World first!
Solvent delivery units -Low pulsation LC-20AD -General purpose LC-20AT -Binary LC-20AB
Low volume degasser DGU-20A
High Sensitive detector SPD-20A/20A Ｖ / M20A
World's highest sensitivity!World's highest sensitivity!
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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
….the future of HPLC
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Flexibility
Modular type provides excellent flexibility.
Isocratic system Fully automated gradient system
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LC-20ABLC-20ATLC-20AD
◆ Excellent solvent delivery performance
◆ Improved stability
◆ Improved durability
◆ Space saving design
LC-20AD/20AT/20AB Features
Low pressure GE valve
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Plunger Reciprocating Pump
motor and cam
plunger
plunger seal
check valvepump head
5 - 50µL
out
in
Mobile phase
check valve
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• 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
Plunger Reciprocating Pump
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Dual Plunger with Tandem Flow Line
check valve
Low pressure fluctuation UV / PDA detectorFluorescence detector
The number of maintenance parts is less. So this design is suitable for routine analysis.
LC-20AT
Main plunger Sub
plunger
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Dual Plunger with Parallel Flow Line
Very low pressure fluctuation Refractive index detector Conductivity detectorElectrochemical detectorMS detector
The number of maintenance parts is more.
check valve
plungerplunger
check valve
LC-20AD/AB
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HPLC Manual Injector
Rheodyne 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.
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.
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6 Port Valve System
Typical sample loop volume is 5-200 µl.
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HPLC Auto Injectors
SIL-10ADvp
Inside of SIL-10Avp
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HPLC Auto Injectors
Inside of SIL-20AC
SIL-20AC
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Principle of Auto Injectors (1)
Sample Aspiration
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Principle of Auto Injectors (2)
Start of analysis
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SIL-20A/20AC Overview
◆ World's fastest sample injection
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.
Wow, so
fast!!
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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.
0.0007 % Not detected
Sample carryover test using Chlor-hexidine
Remained sample
SIL-HT SIL-20A
reduced to 1/4
Minimized touch area
SIL-20A provides good result even with adsorptive compound.
with HPLC from vendor A:
0.08 %
With rinsing pump, sample carryover is not detected.
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SIL-20A/20AC
High Throughput
World's fastest sample injection makes analysis cycle less than 1 minute possible.
0 1 2 3 min0.5 1.5 2.50 1 2 3 min0.5 1.5 2.5
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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
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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.
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High Throughput not High Pressure
As it tru
ly should be
Ultra Fast
Ultra Flexible
Ultra Fidelity
Ultra Quick Method Transfer
Ultra Durable XR-ODS Column Technology
Ultra Performance “Prominence” Proven Platform
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Shimadzu LCMS-2010EV system
MS detector
LCMSsolution
LC-20A
Probe holder with a
Source Window
Connector panelpilot lump
API probe
Ion inlet port Shutter
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Integrated and Modular HPLC
Integrated LC-2010 Modular Prominence
Simplicity and automation
Ease of operation
Ease of support
Routine analysis
Flexibility and expandability
Dependent on budget
Dependent on application
R&D, Multipurpose
HPLC
Detectors
V. Namasivayam
Spinco Biotech Pvt. Ltd.
Chennai
60
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
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Selection of Detectors
Detectors Type of compounds can be detected
UV-Vis & PDACompounds with chromophores, such as aromatic rings or multiple alternating double bonds.
RFFluorescent compounds, usually with fused rings or highly conjugated planar system.
CDDCharged compounds, such as inorganic ions and organic acid.
ECDFor easily oxidized compounds like quinones or amines.
RID & ELSDFor compounds that do not show characteristics usable by the other detectors, eg. polymers, sccharides.
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Ultraviolet / Visible Detector (1)
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Ultraviolet / Visible Detector (2)
A : absorbance
ε : molar absorptivity
C : analyte concentration
L : path length of the flow cell
E : energy
Lambert-Beer’s Law
A = ε C L = - log (Eout / Ein)
Ab
sorb
anc
e
Concentration
linea
r ran
ge
2.5
Backgroud absorbance
Actual
Ideal
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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
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Photodiode Array Detector (1)
Sample Cell
512 Elements Photodiode Array
Grating
D2 / W lampOne element detects one absorbance at one wavelength.
67W
avel
engt
h
Spectrum
Photodiode Array Detector
3-D Data
Time
Ab
so
rban
ce
Chromatogram
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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.
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Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds
S1
T1
S0
Light absorption
Non-radiation transition
Non-radiation transition
Fluorescence
Phosphorescence
Fluorescence of Compounds
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Relationship Between
Fluorescence Intensity & Concentration
F = 2.3 Φf I0εb c
F :Relative fluorescence intensityf :Quantum efficiency
I0 :Intensity of incident radiation
:Molar absorptivityb :Pathlength of flow cellc :Concentration
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Fluorescence Detector
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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
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Refractive Index Detector (1)
Sample
Reference
Photodiode
W Lamp
Refraction
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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
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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
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Conductivity Detector Principle
K (conductivity) = I [A] / E [V] =A [cm2] / L [cm] * k (k : specific conductivity)
k= (I/E)*(L/A)
I
V
L
A A
Electrodes
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Temperature Control of Conductivity Detector
• Conductivity is very affected by temperature.
• Must keep the cell in the temperature control devise.
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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.
Conductivity Detector
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• 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. SO4
2- (431 ppm)
Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane
Application Example (Anions)
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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)
Application Example (Cations)
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Electrochemical Detector
Working electrode
AUX electrode
Reference electrode
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Principle of ECD Detection
e-
A
R O + H+
ElectrodeGlassy Carbon (GC)Pt, Ag, Au
[ Applications ]GC : phenol compounds general usePt : H2O2
Ag : halogen ionAu : sugar analysis
Electrochemical detector responds to compounds that can be oxidized or reduced, such as phenols, aromatic amines, ketones, aldehydes.
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LC-EC Detection
Reaction Principle
• 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
(electro) chemical reaction detectorpotential (E) is driving force
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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.
Electrochemical Detector
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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)
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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
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Evaporative Light Scattering Detector
Nebulization
Mobile phase
Detection
Evaporation
Gas
LightPC
100
Applications of ELSD
Food
（ Saccharides, fatty acids）
Chemical Industry
（ Polymers, surfactants ）
Pharmaceutical
（ Impurities ）
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Mobile Phase
• Water
• Methanol
• Acetonitrile
• THF
• etc
Mobile Phase & Nebulizing Gas
Nebulizing Gas
• Nitrogen
• Compressed air
• etc
102
Advantages:
• Most compounds can be detected (universal detector)
• Compatible with gradient elution
Disadvantages:
• Mobile phase must be volatile.
• Nebulizing gas is required.
Evaporative Light Scattering Detector
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Single Quadrupole LC/MS System
Single Quadrupole LC/MS System
MSInterface
TMP １ TMP ２
Rotary Pump
Atmospheric
Pressure 10-3 ～ 10-4 Pa80 ～ 150 Pa
Q-array Octopole Quadrupole DetectorIonization probe
HPLC
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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)
Interface of LC-MS
HPLC MSInterface
Aqueous/organic solvent with buffers
Non-volatile compounds
High vacuum
Analyze ions, m/z
To Remove solvent
To Ionize analyte molecules
Key Technology
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ESI probe
HPLC
Nebulizing gas
3-5 kVIons
VacuumDrying
gas
+++++++++
++++++
+++
++++++
+++
[M+H]+
0.001-1 ml/min
Ionization in liquid phase Ionization at room temperature
Principles of ESIElectro Spray Ionization
108
ESI probe
HPLC
Nebulising gas
Corona discharge
3-5 kV
Ions
VacuumDrying
gas
Heater (400oC)
0.05 - 2 ml/min
Discharge to form primary ion:
N2 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, ion-molecule reaction
Principles of APCIAtmospheric Pressure Chemical Ionization
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Ionization diagram
ESI
APCI
APPI
polarityVery polarNon-polar
Molecular Weight
10,000
1,000
100
- 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)
- APPI is chosen only when ESI and APCI could not ionize target compounds effectively.
EI (GCMS)
110
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
Ion Detector
111
• ESI– drugs and their metabolites– peptides – proteins– many kinds of natural product (-OH, -NH2,-COOH, SO2, PO3 etc.)
• APCI– pesticides– steroids– drugs
Ionization of Compounds in MS Detector
112
What kind of benefits LC/MS users can get ?
• Determination of MW
• Qualitative capability
• Selective quantitative capability
• High sensitivity
A:100
D:150B:100C:150 m/z=150
TIC
m/z=100
A
B
C D
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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 (ELSD)
Yes
MS Yes
* The sensitivity of PDA Detector is slightly less than UV-Vis Detector
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THANK YOU & ALL THE BEST
