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![Page 1: Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry](https://reader035.fdocuments.net/reader035/viewer/2022062418/554e7929b4c905f66a8b4fad/html5/thumbnails/1.jpg)
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The world leader in serving science
HPLC 2014
Technical Seminar
New Orleans, LA
Easy One-Click Access to Powerful and Complete Workflows for LC and LC/MS
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Your Challenges
• Work faster with better results
• Produce more data with less injections
• Use fewer methods and less equipment
• Be always ready
• Someone is always happy to audit
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Agenda
1. Experimental Variability & Mistakes
2. Sample Preparation
3. System Control & Batch Setup
4. Very Fast & Data Rich Results
5. The Art of Tying it All Together
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1. Experimental Variability & Mistakes
sampling logging
preserving
weighing milling
pooling
dissolve dilute filter extract
spike transfer
prepareHPLC
UHPLC
run batchmonitor
(remotely)
preliminary report
submitreview
approve
report to project lead
(LIMS)
customeraction
batch release
![Page 6: Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry](https://reader035.fdocuments.net/reader035/viewer/2022062418/554e7929b4c905f66a8b4fad/html5/thumbnails/6.jpg)
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Experimental Variability & Mistakes
Data
Sam
ple
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2. Sample Preparation
sampling logging
preserving
weighing milling
pooling
dissolve dilute filter extract
spike transfer
prepareHPLC
UHPLC
run batchmonitor
(remotely)
preliminary report
submitreview
approve
report to project lead
(LIMS)
customeraction
batch release
![Page 8: Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry](https://reader035.fdocuments.net/reader035/viewer/2022062418/554e7929b4c905f66a8b4fad/html5/thumbnails/8.jpg)
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Sample Preparation
• material, labeling, preservation, protocol• clean, adequate, locationcontainer• calibration (drift)• electrode, temperature, contaminationpH/conductivity• temporary storage (alterations)• chain of custody, sample logging, recordstransfer to lab
• oxidation, volatiles, NSB homogeneity, extraction, pre-concentration, matrix, solventsextraction
• contamination, protection, product quality• pH adjustment, pooling, multiple assayshandling• chemistry, contaminant removal• (user) reproducibility, internal standardmatrix
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Sample Preparation
• transfer method, losses, labeling• correct sample positiontransfer• PP, PE, glass, modified glass• vial or well format, cover or septummaterial• cool until HPLC/UHPLC• light protectionstorage
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Application Example 1
• Sensitivity• Reproducibility • Low sample volume • Quality results
Bioanalysis - drug candidate from human plasma
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Reliability & Productivity – Low Volumes
New Thermo Scientific™ SOLAµ™ SPE Cartridges• Frit-less design removes conventional SPE issues• Enhanced flow through characteristics• Optimized droplet formation
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Same Workflow – Higher Sensitivity
Use SOLAµ cartridges and improve your workflow
sensitivity low(dilution)
sensitivity high(concentration up to 20x)
SOLAµ cartridge
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Same Workflow – Higher Sensitivity
SOLAµ cartridges
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Better Sample Identification and Tracking
New: Thermo Scientific™ Virtuoso™ Vial Identification System
• Less mistakes• Easy scanning• Facilitates more automation
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Faster Mass Transfer – Less Pressure
Thermo Scientific™ Accucore™ Solid Core HPLC Columns
Speed &Resolution
• Fast with less solvents• Higher throughput with lower cost• More analytes and flexibility
Selectivity• 14 phases• Easy method development• No compromise on selectivity
Rugged & Reproducibility
• Optimized production (QbD)• Made to be tough• More performance with less pressure
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3. System Control & Batch Setup
sampling logging
preserving
weighing milling
pooling
dissolve dilute filter extract
spike transfer
prepareHPLC
UHPLC
run batchmonitor
(remotely)
preliminary report
submitreview
approve
report to project lead
(LIMS)
customeraction
batch release
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System Control & Batch Setup
performance •correct column installed and ready•performance check for precious samples
methods •multiple methods (switching)•equilibration, mobile phase compatibility
compatibility •pressure and flow limits•wetted system materials
startup/standby •system startup, standby or shutdown•correct sample positions
queue setup •sequence of standards, blanks, samples•compliance with SOP or official method
operation •sample cooling and light protection•progress monitoring and remote access
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System Control & Batch Setup
data •audit trail, system parameters, availability•all detector signals acquired, format
network •remote access to multiple result sets•data loss protection
repeat run •unexpected deviation•complete run or single injection repetition
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Application Example 2
One Click Workflow
Carbohydrates in Foods
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Carbohydrates in Fruit Juices
Column: Anion Exchange 4.1 x 250mmMobile phase: 80mM NaOHFlow rate: 1.5mL/minColumn Temp: 30 ºC Inj. Volume: 10 μL Detector: Thermo Scientific™ Dionex™ UltiMate™
ECD-3000RS EC detector with 6041RS cell and Au electrode
Sample Prep: Centrifuge with 0.2 µm centrifuge filter, 1:1000v/v dilution in DI water
Sample: A: Sunsweet Prune juiceB: Welch's apple juiceC: Welch’s orange juiceD: Welch’s grape juice
Peaks: 1. Sorbitol 2. Glucose 3. Fructose4. Sucrose
0.0 2.5 5.0 7.5 10.0-10002004006008001,0001,2001,4001,600
min
nC
A
B
C
D
1
2
3
4
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0.0 5.0 10.015.020.0-910
100200300400500600700800
min
nCColumn: Anion Exchange 4.1 x 250mmMobile phase: 20mM NaOHFlow rate: 1.5mL/minColumn Temp: 30 oC Inj. Volume: 25 μL Detector: UltiMate ECD-3000RS EC detector with
6041RS cell and Au electrode
Sample Prep: Protein precipitation with 1% perchloric acid (1:10 v/v), centrifuge, further dilution in DI water,
Sample: A: Lactose free milk, 1:500 v/v dilutionB: 10µg/mL standard mixC: Fat free milk, 1:1000 v/v dilutionD: Whole milk, 1:1000 v/v dilution
Peaks: 1. Galactose2. Glucose3. Unidentified4. Unidentified5. Lactose6. Lactulose7. Unidentified8. Unidentified
A
B
C
D
12
5
3 4
6
7 8
Lactose and Lactulose in Dairy
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Carbohydrates in Foods
• Selectivity reduces need for sample prep• Complete system setup incl. helium sparge• Choice for quaternary or isocratic pump• Plug & play electrochemical cells
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Application Example 4
One Click Workflow
Proteins and MAb
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Salt versus pH Gradient IEC of mAb Sample30 min gradient, Thermo Scientific™ MabPac™ SCX-10 column, 5 µm, 4 x 250 mm
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
5.0
10.0
15.0
min
%B: 10.0
30.0
Salt gradient
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
5.0
10.0
15.0
min
%B: 25.0
50.0
25.0
pH gradient
• Superior resolution• Information on pI
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Standard Phosphate Based pH Gradient
Separation of 4 proteins mix
30.0
40.0
50.0
60.0
70.0
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
pH
mAU
10.0
20.0
0 5 10 15 20 25 30 35 40-10.0 5.50
min
Retention Time (min)
pH trace
Lectin
0.06.00
12
Trypsinogen
Ribonuclease A
Cytochrome C
• Poor pH gradient linearity and limited buffer range• Poor chromatographic resolution and pI determination
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Thermo Scientific CX-1 pH Gradient Buffer
Separation of 4 proteins mix
pH trace
0 5 10 15 20 25 30 35 40-10
20
40
60
90
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Abs
orba
nce
(mA
U)
Retention Time (min)
1
Lectin-1 - 6.15 – pI 6.11
Lectin-2 - 7.25 – pI 6.28
Lectin-3 - 8.45 – pI 6.45
Trypsinogen - 16.41 - pI 7.75
Ribonuclease A - 22.38 – pI 8.72
Cytochrome C - 31.88 – pI 10.15
2
pH
• Perfect pH gradient linearity from pH 5.6 to pH 10.2• Excellent resolution and accurate pI determination
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0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0-100
-75
-50
-25
0
25
60
5.00
6.00
7.00
8.00
9.00
10.50
Run #300
Run #200
Run #100
Run #5
pH trace
pH
Ab
sorb
an
ce [
mA
U]
Retention Time [min]
Ribonuclease A Analysis Stability (300 Runs)
Retention time precision < 0.8 % RSD
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UltiMate 3000 BioRS Systems for Biopharma
• Innovative pH buffer kits and high resolution columns
• Seamless pH gradients for improved separation
• Complete bio-system solution• Extendable to 2D-LC• pH, conditions, UV/vis and MS data
in one chromatography data system (CDS) package
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4. Very Fast & Data Rich Results
sampling logging
preserving
weighing milling
pooling
dissolve dilute filter extract
spike transfer
prepareHPLC
UHPLC
run batchmonitor
(remotely)
preliminary report
submitreview
approve
report to project lead
(LIMS)
customeraction
batch release
![Page 30: Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry](https://reader035.fdocuments.net/reader035/viewer/2022062418/554e7929b4c905f66a8b4fad/html5/thumbnails/30.jpg)
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Data Processing, Review and Report
integration •review of integration•peak detection method (robustness)
resolution •unresolved peaks integration, purity review•multiple acquisition channels
calibration •linearity and reproducibility•standard concentration (compound purity)
reviewer •request and data transfer to reviewer•audit trail, electronic tools
process •review process, large and small labs•“freeze” results
approval •electronic tools or hardcopy•final results (location)
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Data Processing, Review and Report
time •manual processing vs. instant results•electronic vs. hardcopy
calculation •custom calculation (audit trail, controlled access)•formatted according to recipient
availability •electronic tools or hardcopy•final result location
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Application Example 5
One Click Workflow
System Setup, Data Review and Results
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Few Clicks for Complete Setup - eWorkflows
Select eWorkflow, instrument and launch
Set no. of samples and start position
Rack and sequence preview
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Intelligent Sequence Control
What if your RTs drift or is your %RSD is out of spec?
Thermo Scientific™ Dionex™ Chromeleon™ CDS’s Intelligent Run Control is the solution.
Allows decisions to be made during run:•Insert Injections•Re-inject•Perform arithmetic processes •Extract Channels or Optimum Integration from a PDA 3D Field
Performs conditionally or unconditionallyIncludes error handling!
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Easy Setup - Intelligent Run Control
Set pass and/or fail actions
Specify multiple actions
Built in error handling
Select required action
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Simplify Integration (Automation) - SmartPeaks
SmartPeaks Wizard
Select area of interest
Select integration you want
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Complex Data Review- Linking of Panes
Pin to overlay Overlaid
Injections
Link Panes
Only selected injections
shown
Only zoomed components
shown
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Complex Data or MS Data Processing
Overlay of all XIC’s
Quantitation and
confirmation ions
Component list
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Easy MS Library Search and Development
Library search results
NIST library search
Create your own library
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5. The Art of Tying it All Together
• Complete Solutions for different purposes and markets
• Packages include instrumentation, software, columns, chemicals, guidance documents, tubing kits, eWorkflows, etc.
• Examples include:• Thermo Scientific Xpert Troubleshooting Solution–
automated LC troubleshooting for pharma
• Thermo Scientific IonCount Solution— pharmaceutical counter ion and API analysis
• Thermo Scientific BeerNHop Solution— detailed profiling of bitters in beer and beer wort
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Complete Solution 1 – LC Auto Troubleshooting
One Click Workflow
XPertSolutionfor Pharma
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Questionable assay results?
Out-of control situations?
Want to avoid downtime while waiting for service?
XPert is a first-aid kit to easily find out:
Is the column or the instrument the source of deviation?
… and it assists: Preventive maintenance - column replacement - application fine tuning
Gain back full confidence in your analysis
What is the XPert Troubleshooting Solution?
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XPert Improvement Tips & Recommendation
Chromeleon CDS 7.2 Report with interactive tips
Example of gradient separation
Actual result
Target
ReferenceColumn
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Data Processing and Evaluation eWorkflow
• Reference Column Data Processing• Test parameter from processing method
Pump pressure limits, Number of peaks, Resolution, Peak asymmetry, Peak area RSD, Plate numbers, etc.
• Reference values to test against are embedded in sequence table
• Evaluate and Report Results• Control Chart for quick overview
• Report on possible error causes and recommended solutions
• Call To Action• System maintenance
• Check application column
=> Run Xpert solution on application column
(use CM method transfer tool)
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Complete Solution 2 – Counter Ions and APIs
IonCountSolutionfor Pharma One Click
Workflow
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Components of the IonCount Solution
UltiMate 3000 SD System
Thermo Scientific™ Dionex™
Corona™ Veo™ Charged Aerosol Detector
Syst
em
pack
age
Chro
m
star
ter k
it Thermo Scientific™ Acclaim™ Trinity™ P1 and P2 Columns
Eluents and Buffers
Test Standards
eW
orkf
lows
Gui
danc
e CD eWorkflows for different API and
Counter Ion Analyses
Installation and Operation Guides
+
+ +
+
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Cation/Anion Assay for Advanced Formulations
0,00 2,00 4,00 6,00 8,00Time [min]
Sodium
Chlorid
e
Tosy
late
IonCount-Reference-P1-gradient-demo-data #4gradient run #3CAD_1
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More Applications with the IonCount Solution
20140226-P2-ion-calibration-pfv1p15-repeat #16100 µg/mL CAD_1 Column: Acclaim Trinity P2, 3 x 50mm
System: UltiMate 3000 Quaternary SD LC
Mobile phase: A - H2O
B - CH3CN
C -100 mM NH4HCO2, pH=3.65
Gradient: 0-1 min 10% C, 1-11 min 10-100% C, 11-20 min 100% C, 20-21 100-10% C,
21-29 min 10% C
Flow rate: 600 L/min
Power Func.: 1.5
Temperature: 30 ºC
Injection: 1 µL
Detection: Corona Veo Charged Aerosol Detector
Analytes: All 100 µg/mL
Counter Ion Screening
Column: Acclaim Trinity P1, 3x50mm
System: UltiMate 3000 Quaternary SD LC
Mobile phase: A - H2O
B - CH3CN
C -100 mM NH4HCO2, pH=3.65
Gradient: 0-14 min 5-80% B,
14-18 min 80% B, 18-19 80-5% B, 19-25 min 5% B 0-25 min 15% C
Flow rate: 700 L/min
Power Func.: 1.3
Temperature: 30 ºC
Injection: 1 µL
Detection: Corona Veo Charged Aerosol Detector
Simultaneous API and Counter Ion Analysis
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Complete Solution 3 – Bitter Acids in Beer
One Click Workflow
BeerNHopSolutionFor Brewers
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BeerNHop – Fast & Easy for Innovative Brewers
Brewers can create new innovative brands and flavors
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Direct Injection vs. On-Line SPE: Results
Injection # 501
8 min
Automated On-Line SPE
Retention times shift (no time offset)Resolution adulterates rapidlyOnly about 160–180 injections possible
Incr
emen
ts o
f 50
inje
ctio
ns
Injection # 160
7 min
Incr
emen
ts o
f 40
inje
ctio
ns
Trans-isoadhumulone
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Complete BeerNHop Solution Kit
On-Line SPE
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Your Partner For All Analytes & All Applications
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• iPhone App (App Store)
• File based on website
• Chromeleon 7 CDSwith automated method builder
http://dionex-uhplc.com/uhplc-focused/go-faster/
http://www.separatedbyexperience.com/products/GradientMethod.aspx
Method Speed-Up Calculators
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Useful Links:
Method Development Application Search
Revolutionary Next Generation SPE
Accucore Column Technology
Accurately Linear pH Gradients
Chromatography Resource Center - Separated by Experience
All About Mass Spectrometry - Planet Orbitrap
Smart Phone Method Speed-Up Calculator
iPhone HPLC Trouble Shooter
Thermo Scientific Dionex UltiMate 3000 XRS
Thermo Scientific Dionex UltiMate 3000 BioRS
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Thank You!