2020/4/30

Objectives
Important Notice for Daily use of HPLC &
Moving from HPLC to UHPLC 1. Basic of HPLC

2. Part and Configuration of HPLC

3. Moving HPLC to UHPLC

Fakhru R.
PT Ditek Jaya
2020 4. Application of HPLC

1 2

Mobile Phase / Stationary Phase Three States of Matter and Chromatography

Types
 A site in which a moving phase (mobile phase) and a non-moving phase
(stationary phase) make contact via an interface that is set up. Mobile phase
 The affinity with the mobile phase and stationary phase varies with the
solute.  Separation occurs due to differences in the speed of motion. Gas Liquid Solid

Gas

Mobile Stationary
Liquid
phase
phase
Strong Weak Gas Liquid
chromatography chromatography
Solid
Stationary
phase
3 4

3 4

LC & HPLC Flow Channel Diagram for HPLC

 Liquid Chromatography (LC) is an analytical Labour-

Detector
method that the compounds are physically intensive
separated prior to measurement using an
appropriate detector Column
Pump(s) Column Oven
 High Performance (Pressure) Liquid
Chromatography (HPLC) is the analytical
technique to combine modern separation Sample injection unit Drain
science and high sensitivity detection Eluent (injector)
technology for analysis of trace level (mobile phase)
Data processor  Chromatogram
components in mixed samples Results Degasser
Reproducible?
 The progress of HPLC in recent years derive to Repeatable?
Ultra Fast LC (UFLC) and Ultra High Accurate ?
Performance Liquid chromatography Reliable ?
(UHPLC).
6

5 6

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HPLC Line-up Shimadzu HPLC System

7 8

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

Solvent Delivery Pump:

Schematic Diagram of Plunger Pump
Solvent Delivery System Pump(s)
& Degasser
Pump head
Motor and cam

Check
valves

Plunger
Plunger seal 10 -100µL

12

11 12

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Solvent Delivery Pump: Solvent Delivery Pump:

Single Plunger Type Dual Plunger Type

Check valves

Check valves

Plunger head
Plunger heads

Type Type
13 14

13 14

Gradient System Aim of Gradient System (1)

 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!!

Poor CH3OH / H2O = 8 / 2

separation!!

(Column: ODS type)

15 16

15 16

Aim of Gradient System (2) High- / Low-Pressure Gradient System

Low-pressure
 If the eluent composition is changed gradually during analysis... gradient unit

95%
Mixer
Mixer
Concentration of methanol in

High-pressure gradient Low-pressure gradient

30%
eluent

High-pressure gradient system Low-pressure gradient system

High gradient accuracy Simple system configuration
Complex system configuration Degasser required
17 (multiple pumps required) 18

17 18

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Introduction of Nexera Method Scouting

Smart Automation Kit

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

19

19 20

Advantages of Nexera Method Scouting High efficiency HPLC/UHPLC mixer

 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

Time Required Conventional Nexera Method

(UHPLC) Scouting
(1) Creating analysis 32 hours*1 5 minutes
methods Creation of 960 method files by hand 1 method file
(*Calculated assuming 2 minutes to create a method
file)

(2) Creating the batch file 8 hours*2 5 minutes

Created 1 line at a time by hand Batch creation using Method
(*Calculated assuming 30 seconds to create 1 line) Scouting Solution
MiRC mixer (20~180 uL), a high efficiency Traditional HPLC mixer with three options of
Total 40 hours 10 minutes mixer for Nexera UHPLC mixing volumes
21
21/14

21 22

Degasser Online Degasser

 Problems caused by dissolved air in the eluent

 Unstable delivery by pump Regulator Vacuum chamber
Helium Polymeric film tube
 More noise and large baseline drift in detector cell cylinder

To pump
To pump
To draft

Drain valve

In order to avoid these problems, the eluent must be

degassed.
Eluent container Eluent container

Helium purge method Gas-liquid separation membrane method

23 24

23 24

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Injector Unit

 Manual injector  Autosampler

Sample Injection Unit  Conventional injector, very low cost  High throughput and automatic
 Cannot use in batch run automatically operation
 Poorer accuracy and repeatability  More accurate and high repeatability
 Minimized injection amount (0.1 uL)

25 26

Injector Minimized Carryover

 Multi rinse function to minimize carryover

 Rinse of needle surface (2 rinsing solutions)
 Rinse of needle inner surface and needle port (3 rinse solutions)
 Support high-sensitivity LC/MS/MS analysis
From pump To column From pump To column

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
30

29 30

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Tubing

 Material O.D. (outer diameter)

Stainless steel (SUS)
Connector and Tubbing 
 PEEK (polyether ether ketone)
1.6 mm
I.D. (inner diameter)
 Fluororesin
0.1 mm
0.3 mm
0.5 mm
0.8 mm etc.

Stainless Steel (can be used on all connections)

1. Resistant to pressure thousands kg/cm2
2. Corrosion occurs in acidic conditions (especially at pH <2) or when used with a high concentration of salt

PEEK (can be used on all connections)

1. Hold up to 250 kg/cm2 pressure and all ranges of pH (pH 1-14)
2. Weak against organic solvents such as chloroform high solubility

Teflon (back pressure coil, reaction coil, drain tube)

1. Resistant to chemicals
2. Only resistant to the pressure of 5 kgf/cm2
32

31 32

Connectors Dead Volume

(Extra-column volume)
 Male nut (SUS)
Ferrule (SUS)  Dead volume can cause peaks broadening.
 Sealing possible up to 40 MPa Ferrule

 Male nut (PEEK) Male nut Male nut Dead volume

 Can be connected without any tools
 Resists pressures of up to approx. 25 MPa

Male nut (PEEK) Tube

Excellent connection Poor connection

33 34

33 34

Representative HPLC Detectors

Detectors No Detector Name Type

1 UV-Vis Detector SPD-20A SPD-20AV

2 Photo Diode Array SPD-M20 SPD-M30

3 Fluorescence RF-20A RF-20Axs

4 Refractive Index RID-20

5 Evaporating Light Scaterring ELSD-LT II

6 Conductivity CDD-10

7 Mass Spectrometry LCMS-2020

8 Mass Spectrometry TQ LCMS-8050 LCMS-8060

9 Mass Spectrometry QTOF LCMS-9030

10 Mass Spectrometry IT-TOF LCMS-IT-TOF

36

35 36

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Optical System of UV-VIS Absorbance

Detector

UV-Vis Detector
Grating
Sample cell
l Ein Eout Photodiode

Ein Ein Photodiode

Reference cell

D2 / W lamp

38

37 38

UV-VIS Detector

Optical: PDA / DAD Detector

 M1 for light selection D2, W or both
 Lights are filtered by filter and slit
 Grating to separate light into
spectrum
 Single wavelength beam to sample
cell and ref cell
 D1 and D2
 Mercury Lamp is used to check and
calibrate wavelengths (253.5 nm)

39

39 40

Optical PDA Detector Comparison UV vs PDA

Before Flow Cell After Flow Cell

Optical:

 Mixed lights from D2 & W enter sample cell by M1

 Lights after cell enter Grating to separate into spectrum

41 42

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Spectrum and Selection of Detection Advantages of Photodiode Array Detectors

Wavelength
 Peak Identification Using Spectra
 Complementation of identification based on retention time
 Library searches
 Evaluation of Peak Purity
 Purity evaluation performed by comparison of the shape of spectra from the peak
detection start point to the peak detection end point

The longer wavelength

is more selective.
3D Image
Contour Analysis
Spectrum Scanning
Purity Index
Library Editor

200 250 300 350

Wavelength [nm]
43 44

43 44

3D Image Contour Analysis

45 46

Intelligent Peak Deconvolution Analysis Intelligent Dynamic Range Extension

i-PDeA Calculation i-DReC
This is a new function for integration of co-eluted peaks or a small This is a new function that extends dynamic range and plots the
peak hiding in a main peak. This function reduces the time to points properly even though some points in the high concentration
develop a method and is a complement to quantitative integration. region deviate from the calibration curve.
Area Some points in the high
concentration region cannot be
Conventional method plotted properly because they
are saturated.
Vertical separation?
Without i-DReC
Area
Tailing peak? A
Able to extract a single peak
Baseline separation? With i-DReC off
How do we calculate peak Concentration
area? B

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

47 48

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Fluorescence Detector

Fluorescence Detector Excitation wavelength

+ hv1 *

* hv2 +
Fluorescence wavelength
Excited state
Quasi-excited state
hv1
hv2
Fluorescence
Ground state
50

49 50

Optical System of Fluorescence Detector

Refractive Index Detector

Xenon lamp
Fluorescence
grating
Photomultiplier tube

Fluorescence

Excitation
Excitation grating Sample cell
light

51

51 52

Differential Refractive Index Detector Optical System of Differential Refractive

(Deflection-Type) Index Detector (Deflection-Type)

Light-receiving unit Slit W lamp

Reference cell
Reference cell
Sample cell
The slit image moves if the
Light refractive index inside the
flow cell changes.

Sample cell Photodiode

53 54

53 54

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Evaporative Light Scattering Detector

Light-receiving unit

Evaporative Light Scattering Detector Drift tube

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.

Detection Principle Mobile phase: RP suitable

 Nebulization (N2 or Air gas)  Aqueous-MeOH, ACN, THF
 Evaporation of solvent  Evaporation of solvent
 Detection

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

Mass Spectrometer (LCMS)

MS and MS/MS Detector Schematic MS/MS

Diagram

Q1 for ion q2 for Q3 for fragment

Ion Source Ion Detector
selection fragmentation monitoring

 An instrument with only one quadrupole , such as the LCMS-2020 is referred to as a

single quadrupole mass spectrometer. “Triple quadrupole” indicates that three
quadrupole are connected in series. The major constituent parts, staring from the
ionization probe, are Q1, q2 and Q3
 Q1 selects the precusors ions, q2 at the centre is the collision cell that fragments the
ions, and Q3 select the produst ions. Since Q1 and Q3 are connected in series, they
are also called tandem quadrupoles.
 Both Q1 and Q3 are comprised of four rods, DC and RF voltages are applied to these
rods which provide a stable pathway for specificm/z values only, such that they pass
between the rods 58

57 58

Ion Source PDA vs MS

 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)

59 60

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Mobile Phase Mobile Phase Organic Solvent

 HPLC-grade solvent can be
used with confidence.
 Special-grade solvent is
 Water
 “Ultrapure water” can be
acceptable depending on the
used with confidence. detection conditions.
 Care is required regarding
 Commercial “distilled water
for HPLC” is also acceptable. solvents containing stabilizers
(e.g., tetrahydrofuran and
chloroform)

62

61 62

Water Used for HPLC Organic Solvent for HPLC

Mobile phase water :

Absorption

1. pure water
2. distilled water
3. deionized water Wavelength (nm)

Due to the presence of impurities,

Deionized water provide greater absorption
Methanol Acetonitrile Hexane

63 64

Replacement of Eluent

Aqueous solutions containing

Moving HPLC to UHPLC
 Mutually insoluble solvents must
not be exchanged directly. salt and organic solvents must
not be exchanged directly.

Water Buffer solution

2-Propanol Water

Hexane Water-soluble
organic solvent 65

65 66

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Successful Operation of Shimadzu

Development Concept of UHPLC
HPLC to UHPLC
To produce next generation UHPLC without compromise of performance

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

67 68

Successful Operation of Shimadzu Line-up HPLC Shimadzu

HPLC to UHPLC
Item Conventional LC Fast LC (High UHPLC (High Speed,
Speed) Resolution,
Sensitivity)
Partcile 5 um 2-3 um < 2 um
ID > 4.6 mmID 3 - 4.6 mmID < 3 mmID
Flow rate > 1 mL/min 0.7 – 1 mL/min < 0.7 mL/min
Pressure 20 – 30 MPa 30 – 60 MPa > 60 MPa
End time > 30 mins 10 – 30 mins < 10 mins

69 70

Comparison of HPLC, UFLC, UHPLC Method Transfer from HPLC to UHPLC

 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*

 Linear relation between RT (HPLC) and RT (UHPLC) if the column chemistry

factors are same (type, C content, end-capping etc)

71 72

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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

73 74

New Method Transfer Program

(SHIMADZU)

Application of HPLC

75 76

Portfolio of the New Nexera

On-line Dissolution – HPLC Series

• A UHPLC-like model up to • A new model up to 105MPa. • A high-end model up to

70MPa. • Isocratic, HPGE, LPGE… etc. 130MPa.
• Space-saving design for Any configuration is available. • Space-saving design for
HPGE with one pump unit. *Two pumps are needed for HPGE. HPGE with one pump unit.

All models have the AI functions.

77 78

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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

The gravimetry of the mobile phase is observed through smart devices

Automatically calculate mobile phase reservoir volume
An alert is sent in case of shortage

MPMChecker LabSolutions

Sends the data to PC or

smart devices

Bottle holder with

gravimetric sensor

System dry-up; column damage; sample wastage preventions

Real Mobile Phase Monitoring = Maximum Up-Time

79 80

FlowPilot Protects Column Auto-Diagnostics and Recovery Protects System

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

Without FlowPilot Flow With FlowPilot

Pressur ① Gradually increases flow to the half of set value.
Pressure e
Temp. ② Stays until the temp reaches to the set value. Detect the
jump ③ Gradually increases flow again to the set value. fluctuation

Prevent extra
pressure
1mL/min
1mL/min
③
Sample 1 Sample 2 Auto purge Sample 2
0.5 ②
(Failure) (Retry)
mL/min
①

Start Heating Stable (Time) Heating Stable

Start (Time)
temperature temperature Chemist does not need to be physical present when abnormal condition
Automated Column protection; extend life span of delicate HPLC column appears
Maximized return from cost of operation Nexera Series can perform auto-correction; ensure maximum system up-
time

81 82

Preparation Function in SIL Pretreatment Mode: Auto-Dilution

Dilution performance by autosampler is equal to the manual operation
2-100 times dilution is available
Autosampler: SIL-40 series
•Ultra-fast injection speed
•Ultra-low carryover Auto dilution Manual dilution operation
Area Area
•Air-circulation temperature control 面積 面積

•Automated sample preparation 700000

R2 = 0.99999
600000

600000
R2 = 0.99998
500000
preparation

Reagent addition Overlap injections

functions

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.
84

83 84

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Dual Injection Dual Injection

Flow path 1: Flow path 2:

Autosampler: SIL-40 series Organic acid
analysis
Amino acid
analysis
•Ultra-low carryover
•Ultra-fast injection speed
•Air-circulation temperature control
•Dual injection Multi Data Report
(MDR) function
Sample 1 Sample 2 summarizes the
Path 1
Conventional

quan results of
method

Analysis Analysis Analysis Analysis Analysis Analysis multiple

Organic acids

Switching
Flow path 1

1 1 1 2 2 2
substances in all
samples.

Path 1 Time-saving
New Nexera

Analysis Analysis Analysis

design
1 1 1
Amino acids
Flow path 2

Analysis Analysis Analysis

2 2 2

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

85 86

Application of HPLC Amino Acids Analysis

CONFIDENTIAL 87

87 88

Amino Acids ? Amino Acids Analysis (AAA)

Amino Acid Analysis (AAA) refers to the

methodology used to determine the amino
composition or content of proteins or peptide

89 90

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Method Amino Acids Comparison Pre/Post Derivatization

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

Not possible for simultaneous analysis Long analysis time

up to 38 amino acids, usually 20+ (50 min/ injection)
Disadvantage

92

91 92

Shimadzu Application Note for AAA

Solution for
Amino Acids Analysis

93 94

Shimadzu Application Note for AAA Manual derivatization process of AA

Vial Labour-
MPA 45 µL intensive
OPA 22 µL
Standard / Sample 7.5 µL

Mix

Wait 1.0 min

FMOC 10 µL
Mix

Results
Wait 2.0 min
Reproducible?
0.1 N HCl 5 µL Repeatable?
Inject 2 µL Accurate ?
Reliable ?

96

95 96

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Automated Pre-column Derivatization Automated Pre-column Derivatization

of AA by Co-injection (Advanced) of AA by Co-injection (Advanced)
Load derivatization reagents, standards and samples Description of setting in LabSolutions software
 Temperature-controlled autosampler (4 ℃ – 35 ℃)
 Individual tray temperature setting Air gap
 Total sample loading of 216 vials (1.5 mL) ① MPA
② OPA
・
・
・ ③ Sample
・
・ Standard・/ 54
・
・ ・ ④ FMOC
・
・ Sample・・
・ ・
・ ⑤ HCl (0.1N)
・
・
・
・
・
・
Air gap

Mix
4 HCl
3 FMOC Injection
2 OPA
Prominence-i Plus Autosampler 1 MPA Tray 1

97 98

Pretreatment Mode: Co-Injection (Advanced) Amino Acids Analysis

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

Pipette 1 mL into a 1.5 mL vial.

mV
1200 Blow the sample to dryness using nitrogen gas
Met

Phe
Leu
ILe
Val
Glu

Pro

1000
Tyr

Transfer the sample to a hydrolysis tube and

vacuum for a few minutes.
Arg

800
Gly

Reconstitueit with 1 mL of 6 N HCl.

Ala
Ser
Asp

Thr

600
His

Cystine

400 Heat it at 115 ºC for 24 hours

Lys

200

0 Blow the sample to dryness using nitrogen gas

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min Continue with amino
acids derivatization
Pre-column derivatization amino acid analysis by OPA/FMOC process Reconstitute it to pH 8 with 1 mL of 0.1 N HCl.
99

99 100

Preparation Reagents for AAA

Results of Avastin Biosimilar Drug
Chromatogram of acid hydrolysed drug sample by fluorescence detection (2 µL injection)
mV
Detector B Ch1 Ex:350nm,Em:450nm mV mV
Preparation of derivatizing reagents
[Derivatizing reagents]
Ser

Val
Asp

350 7.5
Gln

1.5
Met

300

1. Mercapto propionic solution

Glu

Ser

Lys

1.0 5.0

250
Cys
Val

0.5

2. OPA solution
2.5
200
Gly

Ala

3.5 4.0 4.5min

12.75 13.00 13.25 min
150
Phe

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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Preparation Reagents for AAA

Mobile phase Application of HPLC

1. Solvent A : 20 mmol/L phosphate (potassium) buffer
(pH6.5)
2. Solvent B : 45/40/15 acetonitrile/methanol/water
3. Column cleaning solvent 50% Methanol contained 0.1%
formic acid

[Chemicals] AP Series
1. Potassium dihydrogen phosphate
Advanced Performance UniBloc Balances

2. Dipotassium hydrogen phosphate

3. Formic acid
4. Acetonitrile
5. Methanol CONFIDENTIAL 104

6. Phosphoric acid
103 104

Cannabis Analysis by HPLC Cannabis Analysis by HPLC

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.

CONFIDENTIAL 105 CONFIDENTIAL 106

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Simultaneous Analysis of Amino Acids Using

High Speed Analysis of a Commercial Automatic Pretreatment Function of
Cold Remedy ProminenceTM-i Integrated LC System

CONFIDENTIAL 107 CONFIDENTIAL 108

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 2020/4/30

Food-safety Market Background and Trends

Application of HPLC Background

• Food regulation is becoming strict, for food safety consumption
• There is a elevating demand on the mycotoxin test in food inspection department

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%)

Fast Screening Regulated Mycotoxins

• It is required the quickness for the imported foods inspection for import/export
• Demand for analyzing mycotoxins by 1 method

High accuracy than conventional method

• Conventional screening method (ELISA) has high risk of false positive
• Target compounds are identified in high accuracy in short time frame

CONFIDENTIAL 109 CONFIDENTIAL 110

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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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