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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

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Experimental Variability & Mistakes

Anna Ebersaebers@syr.edu

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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

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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

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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

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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)

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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

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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

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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!