Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry
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The document discusses advancements in high-performance liquid chromatography (HPLC) showcased at a technical seminar, focusing on improving workflows, sample preparation, and data analysis. It highlights practical application examples using various workflows for analyzing compounds like carbohydrates and proteins, emphasizing the benefits of using innovative equipment and methodologies for better results. Additionally, it features solutions for troubleshooting and efficiency, aimed at enhancing laboratory practices in the pharmaceutical and food industries.
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Easy One-Click Access to Powerful and Complete Workflows for Liquid Chromatography and Liquid Chromatography/Mass Spectrometry
- 1. 1 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
- 2. 2 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
- 3. 3 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
- 4. 4 1. Experimental Variability & Mistakes sampling logging preserving weighing milling pooling dissolve dilute filter extract spike transfer prepare HPLC UHPLC run batch monitor (remotely) preliminary report submit review approve report to project lead (LIMS) customer action batch release
- 5. 5 Experimental Variability & Mistakes Anna Ebers aebers@syr.edu
- 6. 6 Experimental Variability & Mistakes DataSample
- 7. 7 2. Sample Preparation sampling logging preserving weighing milling pooling dissolve dilute filter extract spike transfer prepare HPLC UHPLC run batch monitor (remotely) preliminary report submit review approve report to project lead (LIMS) customer action batch release
- 8. 8 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, solvents extraction • contamination, protection, product quality • pH adjustment, pooling, multiple assayshandling • chemistry, contaminant removal • (user) reproducibility, internal standardmatrix
- 9. 9 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
- 10. 10 Application Example 1 • Sensitivity • Reproducibility • Low sample volume • Quality results Bioanalysis - drug candidate from human plasma
- 11. 11 Reliability & Productivity – Low Volumes New Thermo Scientific™ SOLAµ™ SPE Cartridges • Frit-less design removes conventional SPE issues • Enhanced flow through characteristics • Optimized droplet formation
- 12. 12 Same Workflow – Higher Sensitivity Use SOLAµ cartridges and improve your workflow sensitivity low (dilution) sensitivity high (concentration up to 20x) SOLAµ cartridge
- 13. 13 Same Workflow – Higher Sensitivity SOLAµ cartridges
- 14. 14 Better Sample Identification and Tracking New: Thermo Scientific™ Virtuoso™ Vial Identification System • Less mistakes • Easy scanning • Facilitates more automation
- 15. 15 Faster Mass Transfer – Less Pressure Thermo Scientific™ Accucore™ Solid Core HPLC Columns • Fast with less solvents • Higher throughput with lower cost • More analytes and flexibility Speed & Resolution • 14 phases • Easy method development • No compromise on selectivity Selectivity • Optimized production (QbD) • Made to be tough • More performance with less pressure Rugged & Reproducibility
- 16. 16 3. System Control & Batch Setup sampling logging preserving weighing milling pooling dissolve dilute filter extract spike transfer prepare HPLC UHPLC run batch monitor (remotely) preliminary report submit review approve report to project lead (LIMS) customer action batch release
- 17. 17 System Control & Batch Setup • correct column installed and ready • performance check for precious samplesperformance • multiple methods (switching) • equilibration, mobile phase compatibilitymethods • pressure and flow limits • wetted system materialscompatibility • system startup, standby or shutdown • correct sample positionsstartup/standby • sequence of standards, blanks, samples • compliance with SOP or official methodqueue setup • sample cooling and light protection • progress monitoring and remote accessoperation
- 18. 18 System Control & Batch Setup • audit trail, system parameters, availability • all detector signals acquired, formatdata • remote access to multiple result sets • data loss protectionnetwork • unexpected deviation • complete run or single injection repetitionrepeat run
- 19. 19 Application Example 2 One Click Workflow Carbohydrates in Foods
- 20. 20 Carbohydrates in Fruit Juices Column: Anion Exchange 4.1 x 250mm Mobile phase: 80mM NaOH Flow rate: 1.5mL/min Column 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, 1:1000 v/v dilution in DI water Sample: A: Sunsweet Prune juice B: Welch's apple juice C: Welch’s orange juice D: Welch’s grape juice Peaks: 1. Sorbitol 2. Glucose 3. Fructose 4. Sucrose A B C D 1 2 3 4
- 21. 21 Column: Anion Exchange 4.1 x 250mm Mobile phase: 20mM NaOH Flow rate: 1.5mL/min Column 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 dilution B: 10µg/mL standard mix C: Fat free milk, 1:1000 v/v dilution D: Whole milk, 1:1000 v/v dilution Peaks: 1. Galactose 2. Glucose 3. Unidentified 4. Unidentified 5. Lactose 6. Lactulose 7. Unidentified 8. Unidentified A B C D 1 2 5 3 4 6 7 8 Lactose and Lactulose in Dairy
- 22. 22 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
- 23. 23 Application Example 4 One Click Workflow Proteins and MAb
- 24. 24 Salt versus pH Gradient IEC of mAb Sample 30 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
- 25. 25 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 0.0 6.00 1 2 Trypsinogen Ribonuclease A Cytochrome C • Poor pH gradient linearity and limited buffer range • Poor chromatographic resolution and pI determination
- 26. 26 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 Absorbance(mAU) 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
- 27. 27 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 Absorbance[mAU] Retention Time [min] Ribonuclease A Analysis Stability (300 Runs) Retention time precision < 0.8 % RSD
- 28. 28 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
- 29. 29 4. Very Fast & Data Rich Results sampling logging preserving weighing milling pooling dissolve dilute filter extract spike transfer prepare HPLC UHPLC run batch monitor (remotely) preliminary report submit review approve report to project lead (LIMS) customer action batch release
- 30. 30 Data Processing, Review and Report • review of integration • peak detection method (robustness)integration • unresolved peaks integration, purity review • multiple acquisition channelsresolution • linearity and reproducibility • standard concentration (compound purity)calibration • request and data transfer to reviewer • audit trail, electronic toolsreviewer • review process, large and small labs • “freeze” resultsprocess • electronic tools or hardcopy • final results (location)approval
- 31. 31 Data Processing, Review and Report • manual processing vs. instant results • electronic vs. hardcopytime • custom calculation (audit trail, controlled access) • formatted according to recipientcalculation • electronic tools or hardcopy • final result locationavailability
- 32. 32 Application Example 5 One Click Workflow System Setup, Data Review and Results
- 33. 33 Few Clicks for Complete Setup - eWorkflows Select eWorkflow, instrument and launch Set no. of samples and start position Rack and sequence preview
- 34. 34 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 unconditionally Includes error handling!
- 35. 35 Easy Setup - Intelligent Run Control Set pass and/or fail actions Specify multiple actions Built in error handling Select required action
- 36. 36 Simplify Integration (Automation) - SmartPeaks SmartPeaks Wizard Select area of interest Select integration you want
- 37. 37 Complex Data Review- Linking of Panes Pin to overlay Overlaid Injections Link Panes Only selected injections shown Only zoomed components shown
- 38. 38 Complex Data or MS Data Processing Overlay of all XIC’s Quantitation and confirmation ions Component list
- 39. 39 Easy MS Library Search and Development Library search results NIST library search Create your own library
- 40. 40 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
- 41. 41 Complete Solution 1 – LC Auto Troubleshooting One Click Workflow XPert Solution for Pharma
- 42. 42 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?
- 43. 43 XPert Improvement Tips & Recommendation Chromeleon CDS 7.2 Report with interactive tips Example of gradient separation Actual result Target Reference Column
- 44. 44 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)
- 45. 45 Complete Solution 2 – Counter Ions and APIs IonCount Solution for Pharma One Click Workflow
- 46. 46 Components of the IonCount Solution UltiMate 3000 SD System Thermo Scientific™ Dionex™ Corona™ Veo™ Charged Aerosol Detector System package Chrom starterkit Thermo Scientific™ Acclaim™ Trinity™ P1 and P2 Columns Eluents and Buffers Test Standards eWorkflows GuidanceCD eWorkflows for different API and Counter Ion Analyses Installation and Operation Guides + + + +
- 47. 47 Cation/Anion Assay for Advanced Formulations
- 48. 48 More Applications with the IonCount Solution 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
- 49. 49 Complete Solution 3 – Bitter Acids in Beer One Click Workflow BeerNHop Solution For Brewers
- 50. 50 BeerNHop – Fast & Easy for Innovative Brewers Brewers can create new innovative brands and flavors
- 51. 51 Direct Injection vs. On-Line SPE: Results Injection # 501 8 min Automated On-Line SPE Retention times shift (no time offset) Resolution adulterates rapidly Only about 160–180 injections possible Incrementsof50injections Injection # 160 7 min Incrementsof40injections Trans-isoadhumulone
- 52. 52 Complete BeerNHop Solution Kit On-Line SPE
- 53. 53 Your Partner For All Analytes & All Applications
- 54. 54 • iPhone App (App Store) • File based on website • Chromeleon 7 CDS with automated method builder http://dionex-uhplc.com/uhplc-focused/go-faster/ http://www.separatedbyexperience.com/products/ GradientMethod.aspx Method Speed-Up Calculators
- 55. 55 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
- 56. 56 Thank You!