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Important Notice for Daily use of HPLC &
Moving from HPLC to UHPLC 1. Basic of HPLC
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Gas
Mobile Stationary
Liquid
phase
phase
Strong Weak Gas Liquid
chromatography chromatography
Solid
Stationary
phase
3 4
3 4
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Chromatogram
Intensity of detector signal
tR
Peak tR : Retention time Part and Configuration of HPLC
t0 t0 : Non-retention time
h
A A : Peak area
h : Peak height
Time
9 10
Check
valves
Plunger
Plunger seal 10 -100µL
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Check valves
Check valves
Plunger head
Plunger heads
Type Type
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Isocratic system
Constant eluent composition
In isocratic mode
Gradient system
Varying eluent composition CH3OH / H2O = 6 / 4
HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)
Long analysis time!!
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95%
Mixer
Mixer
Concentration of methanol in
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A Method Scouting System Based on the Nexera (130 MPa Pressure Resistance) Smart Automation Kits
FCV-34AH
SPD-M20A Detector
MR180 L Detector
LC-30AD PDA
Manifold Manifold
SIL-30AC Column Column
Manifold
Column
4 Mobile Phase Smart Automation Kit 7 Mobile Phase Smart Automation Kit
LC-30AD CTO-20AC Mobile phase and (or) column switching can be operated automatically
Capable of searching conditions based on a maximum of 6 columns and 16 mobile phases
Can be used with basically all current UHPLC columns (100 MPa valve pressure resistance)
Time required for the method/analysis screening process is significantly reduced
Easily configured scouting conditions thanks to special software Screening of analysis methods can even be performed overnight; screening capacity is increased
LPGE Unit Automated control of everything from system checks to scouting, and then shut down tremendously
Quick evaluation of scouting results via Class Agent / Agent Report
Human error can also be minimized through batch processing via LabSolutions
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Requirement:
• Significantly reduced analysis preparation time! High mixing efficiency
• Human error reduced thanks to automated creation of method Smaller volume smaller delay volume of the HPLC system
files and batch files! o HPLC: 0.5, 1.7 and 2.6 mL
Ex. )
Mobile phase combinations: o UHPLC: 0.02, 0.04, 0.1, 0.18 mL
aqueous; 4 types organic; 4 types = 16 types in total Total number of condition combinations:
Columns: 6 16 6 10
Gradient patterns: 10 = 960
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To pump
To pump
To draft
Drain valve
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Injector Unit
25 26
Sample Loop
LOAD INJECT
27
27 28
Column Oven
Column Oven
Air circulation heating type
Block heating type
Functions of column oven
1. Holder of column
Aluminum block heater
Insulated column jacket type
2. Temperature controller
Water bath 3. Install column switching valve (2)
4. CMD (column management device)
CMD – a
memory chip to
record column
history of usage
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Tubing
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6 Conductivity CDD-10
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UV-Vis Detector
Grating
Sample cell
l Ein Eout Photodiode
Reference cell
D2 / W lamp
38
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UV-VIS Detector
39
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Optical:
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A
B With the i-DReC function on,
The i-PDeA eliminates one the points in the high
peak and integrates the concentration region can be With i-DReC on
other peak as a single peak plotted properly!
Nexera
High concentration chromatograms are saturated Concentration
Nexera
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Fluorescence Detector
+ hv1 *
* hv2 +
Fluorescence wavelength
Excited state
Quasi-excited state
hv1
hv2
Fluorescence
Ground state
50
49 50
Fluorescence
Excitation
Excitation grating Sample cell
light
51
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Nebulizer
Column eluate
Nebulizer gas
Drain Assist gas
The column eluate is evaporated and the light scattered by the particles of Light source
nonvolatile substances is detected.
Advantages in applications
Non and weakly-chromophoric compounds (e.g. carbohydrates, sugar alcohols, alcohols, terpenoids,
surfactants, macromolecules and polymers).
Gradient method (e.g. lipids, phospholipids, triglycerides, fatty acids, amino acids, natural macromolecules,
synthetic polymers). 56
55 56
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Analysis target: Areas differ according to ionization method PDA (photodiode array)
UV spectra
Molecular GC/MS (MS) (4) (2) 20 ppm AU
(4)
weight (Also if derivatization is required.)
10,000
Non-polar nm
(2) l 580 nm AU
(5)
Volatile (3) l 210 nm
Thermally stable (3) (5) 感度低く,バックグラウンドも
time nm
10,000
LC/MS 高い
ESI
(High LC/MS(MS) LC/MS MS spectra
LC/MS polarity) (Simple, requiring almost no (2) (1) init.
pretreatment.) 10 ppb (4)
1,000 APCI Contents: (3)
polyvalent ions
GC/MS (Low/mediu High polarity (1) TIC m/z
m polarity) (2) m/z 582 init.
Non-volatile (5)
(3) m/z 193
Large mass numbers 高感度だが,バックグラウンドが高
Non- Medium High polarity い
time m/z
polar polarity Thermally unstable (4)
(5)
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1. pure water
2. distilled water
3. deionized water Wavelength (nm)
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Replacement of Eluent
2-Propanol Water
Hexane Water-soluble
organic solvent 65
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Maximizing Performance
Key features of HPLC: Key features of UHPLC :
•Highest accuracy and precision in ultra fast analysis
Performance
•Fastest injection with minimized carryover High pressure/durable mobile Ultra High pressure/durable
phase delivery pumps (< 250 mobile phase delivery pumps
Bar) (> 600 Bar)
High throughput (with auto- Ultra High Speed & throughput
sampler) (with auto-sampler)
High resolution column (5 um Ultra High resolution column (<
HPLC Expandability
particles) 2 um particles)
UHPLC
UFLC
High efficiency mixers (1 mL) Ultra High efficiency mixers (<
Maximizing Throughput High sensitivity detectors, 0.2 mL)
Maximizing Expandability
•Highest pressure range with •Versatile system configuration multiple detectors (10 Hz Ultra High speed & high
widest flow rate range. •Pretreatment, multi-dimensional LC with valve
control, etc.
speed) sensitivity detectors, multiple
•Fastest cycle time for maximization of sample detectors (up to 100 Hz)
throughput
•Solvent consumption reduction for environment
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The retention time relationship between the UHPLC (UFLC) and conventional HPLC
can be described by the following equation based on a simplified model which
assumes a zero dead volume of piping system*
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Ultra-High Speed Analysis of USP Methods Conforming Linear Method Transfer between HPLC
to Permissible Limits in New USP General Chapter 621
and UHPLC
Linear relationship RTs of 8 phenolic antioxidants on UFLC and
HPLC under gradient conditions*
CONFIDENTIAL 73
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Application of HPLC
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P r e ve n t i o n of m o b i l e p h a s e
Advanced Real-Time Mobile Phase Monitoring (MPM)
runni ng out Intelligenc e-1
MPMChecker LabSolutions
79 80
FlowPilot ramps the mobile phase flowrate gradually to the set point
automatically Air bubbles formation may occur by mixing of solvents
Avoid sudden surge of excessive pressure applied on the column, to protect the The air bubble interfusion brings for a drop in the flow rate and disturb
column in the automated start-up. analysis flow
Prevent extra
pressure
1mL/min
1mL/min
③
Sample 1 Sample 2 Auto purge Sample 2
0.5 ②
(Failure) (Retry)
mL/min
①
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R2 = 0.99999
600000
600000
R2 = 0.99998
500000
preparation
500000
Sampler
400000
Dilution Stack injections 400000
300000
Co-injection Mixing 300000
200000
Online-dissolution Derivatization 200000
100000 100000
The patented technology designed Nexera Series Autosampler and
user-friendly LabSolutions graphical user interface offers simple 0 0
0 50 100 150 200 濃度 0 50 100 150 200 濃度
operation as it eliminates the need for complicated programming of Conc. Conc.
pretreatment methods.
Calibration curves of caffeine diluted by the auto preparation function (left), and
manual operation (right). The linearity is represented in R2 value.
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quan results of
method
Switching
Flow path 1
1 1 1 2 2 2
substances in all
samples.
Path 1 Time-saving
New Nexera
Patj 2
Integrated two analytical systems into the single platform; enables analysis of two
independent samples through independent flow channels resulting in twice the
amount of samples processed in the same time
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CONFIDENTIAL 87
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89 90
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Pre-Column Post-Column
Application Derivatization is performed before Derivatization is performed
separation by reversed phase. after separation by ion-
exchange.
Fully automated derivatization Small sample matrix effect
Easy operation Possible of simultaneous
Shorter analysis time (25 min) analysis up to 38 amino acids
Advantage High selectivity
Better sensitivity
Excellent repeatability of peak area
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Solution for
Amino Acids Analysis
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Vial Labour-
MPA 45 µL intensive
OPA 22 µL
Standard / Sample 7.5 µL
Mix
Results
Wait 2.0 min
Reproducible?
0.1 N HCl 5 µL Repeatable?
Inject 2 µL Accurate ?
Reliable ?
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Mix
4 HCl
3 FMOC Injection
2 OPA
Prominence-i Plus Autosampler 1 MPA Tray 1
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Multiple solutions are mixed in sampling needle and injected Acid Hydrolysis of sample – Total Amino Acids
For the dedicated application that requires auto-preparation, e.g. the pre-column
derivatization amino acid analysis Dilute the given sample from 25 mg/mL to 0.1 mg/mL with ultra pure water
Phe
Leu
ILe
Val
Glu
Pro
1000
Tyr
800
Gly
Thr
600
His
Cystine
200
99 100
Val
Asp
350 7.5
Gln
1.5
Met
300
Ser
Lys
1.0 5.0
250
Cys
Val
0.5
2. OPA solution
2.5
200
Gly
Ala
3. FMOC solution
His
Arg
Ile
100
Thr
Tyr
50
Met
Cys
Gln
0
0.0
mV 1.0 2.0 3.0
Detector B Ch2 Ex:266nm,Em:305nm
4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 min
[Chemicals for preparing derivatizing reagents]
Derivatization Reagent
1250
1. Boric acid
1000
Pro
2. Sodium hydroxide
750
500
3. 3-Mercapto propionic acid
250
4. o-phthalaldehyde
0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min
5. 9-fluorenylmethyl chloroformate
For Internal Use Only
101 6. Ethanol
7. Acetonitrile
101 102
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[Chemicals] AP Series
1. Potassium dihydrogen phosphate
Advanced Performance UniBloc Balances
6. Phosphoric acid
103 104
shows chromatograms of extracts from a THC-rich and CBD-rich flower samples overlaid
with a 10 mg/L standard mixture.
Cannabis analysis has gained new importance in
the USA in light of the legalization of marijuana in
several states. Cannabis contains a number of
chemical alkaloids known as cannabinoids.
Primary cannabinoids of interest to most
laboratories are tetrahydrocannabinol (THC),
cannabidiol (CBD) and cannabinol (CBN). In
extracts from the plant, THC and CBD exist as the
native acid forms, tetrahydro-cannabinolic acid
(THCA) and cannabinolic acid (CBDA). These
gradually decarboxylate to THC and CBD through
exposure to heat and light.
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Global
• Food safety testing market was 10.5 billion USD in 2014 and is expected to be 13.6 billion USD in 2019
(Growth rate is 5.3%)
109 110
Mycotoxins Analysis
High sensitivity
The combination of PDA detector on i-
SHIMADZU –
TODAY & TOMORROW
Series and RF-20Axs that provides the
world-highest sensitivity enables to
detect the mycotoxin at the EU
directive level (*) without fluorescence
derivatization. * Baby food is not included
Quick screening
10 mycotoxins are monitored in 14-
BEST PROVEN TOTAL
minutes analytical cycle time.
<Contents of Mycotoxin Screening
System >
Analytical column
Method file
HARDWARE SOFTWARE SOLUTIONS
Spectrum library
Instruction manual
i-Series(LC-2040C 3D)
RF-20Axs(+Optional Detector
Installation Kit)
High-throughput
Results and reports are
“True Solutions Vendor”
returned immediately after
the end of measurement. CONFIDENTIAL 111
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Thank you
Brand statement “Excellence in Science”
W e in the Shimadzu Group have delivered products and services to enable our customers around the world to develop a diverse range of
new products, to protect and improve the environment, and to improve the health and lives of mankind. This brand statement represents our
sense of pride in this endeavor. It is our commitment to society and ourselves that Shimadzu remains dedicated in our pursuit of technology
and accumulation of knowledge, so that we can offer even more outstanding technologies, products, and services, so as to be recognised
for excellence in the field of science.
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