Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (February 2020)
76 slides from Agilent Technologies's technical presentation, Feb 11, 2020
Slide 1: Techniques for Avoiding Unexpected Problems in LC and GC Analysis
Techniques for Avoiding Unexpected Problems in LC and GC Analysis Alexander Ucci Applications Engineer February 11, 2020 1 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 2: Agenda
Agenda • Strategies for sample cleanup • Chromatography problems caused by sample matrix – Physical effects – Chemical effects • Summary 2 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 3: Agenda
Agenda • Strategies for sample cleanup • Chromatography problems caused by sample matrix – Physical effects – Chemical effects • Summary 3 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 4: Dilute and Shoot
Dilute and Shoot Advantages • Fast and easy • High throughput GC inlet liner GC inlet seal Limitations • Interferences are not removed • Analyte concentration is reduced • Instrument and column contamination • Matrix interferences – ion suppression or poor peak shapes Image of salt buildup on an ESI
Slide 5: Importance of the Correct Sample Preparation/Cleanup
Importance of the Correct Sample Preparation/Cleanup Target analytes are the needle in the haystack of a matrix, sample preparation helps find the needle in the haystack. • Protect the instrument detection system from contamination • Improve the detection, method robustness, and reliability Sample w
Slide 6: Importance of the Correct Sample Preparation/Cleanup
Importance of the Correct Sample Preparation/Cleanup Captiva EMR-Lipid Protein precipitation RSD > 25% RSD < 3% Sample with the correct Sample without the correct sample preparation sample preparation 6 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 7: Striking the Right Balance in Sample Preparation
Striking the Right Balance in Sample Preparation 7 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 8: Offline Sample Preparation Options
Offline Sample Preparation Options More Specific ← Instrument Separation and Detection Specificity ← Less Specific Less Specific → Sample Preparation Specificity → More Specific Sample Preparation Protein Technique Dilute and Supported Liquid Protein Precipitation + Precipitation + Solid Phase Inter
Slide 9: Methods for Sample Preparation
Methods for Sample Preparation FLOW 1. Mechanical filtration a) Syringe filters Filters by particle size b) Syringeless filters 1. Filter vials 2. Spin filters FLOW Captiva products 2. Targeted filtration a) Matrix removal Filters by shape, charge b) LLE, SLE or hydrophobicity 3. Catch and release a
Slide 10: Techniques for Avoiding Unexpected Problems in LC and GC Analysis
Techniques for Avoiding Unexpected Problems in LC and GC Analysis Loss of Income Cost of More Cost of More Sample Due to More Matrix Frequent Frequent Technique Preparation Frequent Interference Column Instrument Cost/Time Instrument with Results Changes Maintenance Maintenance Yes, for some Direct
Slide 11: SIM and MRM – Remember the Matrix is Still There
SIM and MRM – Remember the Matrix is Still There 11 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 12: Filtration
Filtration Captiva premium syringe filters • Certified to be free of UV-detectable extractables on HPLC. PES and glass fiber also certified for LC/MS. • Color-coded boxes for easy identification • Comprehensive portfolio to meet all customers’ needs Premium Syringe Filters Membrane Diameter/Pore Siz
Slide 13: Filtration – Captiva Filter Vials
Filtration – Captiva Filter Vials Fill line Vent Part Number Description 5191-5933 PTFE filter vial, 0.45 µm, 100/pk 5191-5934 PTFE filter vial, 0.20 µm, 100/pk 5191-5935 Nylon filter vial, 0.45 µm, 100/pk 5191-5936 Nylon filter vial, 0.20 µm, 100/pk 5191-5939 RC filter vial, 0.45 µm, 100/pk 5191-59
Slide 14: Filtration
Filtration Captiva premium syringe filters Captiva syringe filters guide 5991-1230EN 14 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 15: Filtration – Targeted filtration
Filtration – Targeted filtration Captiva EMR-Lipid • One of the newest Agilent sample cleanup products with a 2-in-1 benefit of removing proteins and lipids. • It reduces ion suppression, increases analyte sensitivity, improves peak shape, and extends the lifetime of your analytical column. • Simple
Slide 16: Filtration – Targeted Filtration
Filtration – Targeted Filtration Captiva EMR-Lipid EMR-Lipid sorbent technology effectively traps lipids through two mechanisms: • Size exclusion – Unbranched hydrocarbon chains (lipids) enter the sorbent; bulky analytes do not • Sorbent chemistry – Lipid chains that enter the sorbent are trapped by
Slide 17: Captiva EMR-Lipid
Captiva EMR-Lipid Selective removal of lipids Removes lipids Does not remove target analytes 17 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 18: Captiva EMR-Lipid
Captiva EMR-Lipid General protocol for biological samples using 1 mL cartridge and 96-well plate Operating instructions Vacuum, positive pressure, or centrifuge can be used. Sample and crash solvent should contain 20% water. One drop every 3-5 seconds. Extra elution step with 80:20 acetonitrile: wat
Slide 19: Captiva EMR-Lipid
Captiva EMR-Lipid General protocol for food and food products using 3 mL and 6 mL cartridges Operating instructions Captiva EMR-Lipid method guide for 3 mL and 6 mL cartridges 19 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 20: Captiva EMR-Lipid Cleanup
Captiva EMR-Lipid Cleanup Efficient phospholipids removal from biological fluid matrices x107 184 precursor ion scan profile 5.5 5 Phospholipids profile for plasma blank use protein precipitation 4.5 only 4 Phospholipids profile for plasma 3.5 blank use protein precipitation + Captiva-EMR cleanup, w
Slide 21: Removal of Lipids Allows for Shorter LC Gradient Time
Removal of Lipids Allows for Shorter LC Gradient Time Target analytes TIC chromatogram 100 %Organic 80 x10 6 +ESI TIC MRM Frag=379.2V (** -> **) DOAinSer STD 7-2.d 1.5 60 1.4 40 1.3 1.2 1.1 1 0.9 0.8 20 0.7 0.6 0.5 0 0.4 0.3 0 2 4 6 8 10 12 0.2 0.1 0 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4
Slide 22: Captiva EMR-Lipid Cleanup
Captiva EMR-Lipid Cleanup Improved analyte response and reproducibility PPT only PPT with Captiva EMR-Lipid cleanup RSD >25% RSD <3% Lipids cause reproducibility problems resulting in high RSD values. Using Captiva EMR-Lipid enables low RSD values and higher peak areas. Higher peak area due to less
Slide 23: Application Note Examples
Application Note Examples • Determination of 14 Polycyclic Aromatic Hydrocarbon Compounds in Edible Oil (5994-1483EN) • Determination of UV Filters in Sunscreens Using Agilent Captiva EMR-Lipid Cleanup by HPLC (5994-1611EN) • A Fast Sample Preparation Workflow for Veterinary Drugs Analysis in Salmon
Slide 24: Innovative Lipid Products
Innovative Lipid Products Captiva EMR-Lipid – A pass through filtration FLOW Bond Elut Lipid Extraction – An SPE like lipid isolation for lipidomics “Captiva EMR like” Lipids collection Wash Lipid capture FLOW Coming soon 24 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.463
Slide 25: Liquid/Liquid Extraction (LLE)
Liquid/Liquid Extraction (LLE) • LLE has been successfully used as a method of sample preparation for many years. • It separates the more organic solvent soluble compounds from the more water-soluble compounds using water immiscible organic solvents. • It can remove many interfering substances like
Slide 26: Drawbacks of Liquid-Liquid Extraction
Drawbacks of Liquid-Liquid Extraction • LLE does have drawbacks – Inconsistent results from one analyst to another • Shaking time • Shaking motion • Determination of where to cut between layers – Emulsions – Labor intensive – Quite tedious with small sample sizes (<5 mL) – Challenging with large num
Slide 27: How Does SLE Work?
How Does SLE Work? Add Add aqueous solvent sample Hold 5 Load min Gravity elute 1. Add aqueous sample to SLE sorbent 2. Wait 5 minutes 3. Elute with water immiscible solvent 27 February February 11, 11, 20202020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 28: Supported Liquid Extraction (SLE)
Supported Liquid Extraction (SLE) Before Apply Extract with • A thin layer of aqueous extraction sample organic solvent sample is formed on the surface of SLE sorbent. Dry sorbent Aqueous Organic • When the organic solvent layer layer passes through the SLE bed, analytes are extracted under the same
Slide 29: What is SLE Sorbent?
What is SLE Sorbent? • There are two types of SLE media – Diatomaceous earth (DE) based products like our Chem Elut brand of SLE products • A mined fossil diatom material, which is heterogeneous and inconsistent from one mine to the next × Naturally occurring; mined Diatomaceous × Broad particle siz
Slide 30: Supported Liquid Extraction (SLE)
Supported Liquid Extraction (SLE) Chem Elut S • Same extraction mechanism as in traditional liquid-liquid extraction (LLE) • Cartridge and plate format, packed with proprietary synthetic sorbent– high surface area • Simple method, gravity flow • Smaller volume sample and solvent compared to LLE • No
Slide 31: Chem Elut S – 15 Minute Hold Time
Chem Elut S – 15 Minute Hold Time Large scale format comparison with aromatic amines using GC Recovery (30 µg/mL; n = 3), 87-119% Chem Elut S 5 mL Chem Elut S 10 mL Chem Elut S 20 mL 120 100 80 60 40 20 0 10.0 9.0 Reproducibility (30 µg/mL; n = 3), RSD 0.4-8.2% 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 59
Slide 32: SPME Fiber and Arrow Offering from Agilent
SPME Fiber and Arrow Offering from Agilent Solid Phase Microextraction (SPME) • Environmental analyses of water samples • Trace analysis in food • Odor analyses (ppt) • Drugs and pharmaceuticals • Flavor analyses of food products • Herbicides/pesticides • Forensic analyses of arson/explosives sample
Slide 33: Examination of Lower Molecular Weight PAHs in Drinking Water Using Agilent PDMS SPME Fibers
Examination of Lower Molecular Weight PAHs in Drinking Water Using Agilent PDMS SPME Fibers Polycyclic aromatic hydrocarbons (PAHs) are a large class of organic compounds containing two or more fused aromatic rings. PAHs are considered compounds of concern by environmental organizations; their conce
Slide 34: Agilent Bond Elut QuEChERS
Agilent Bond Elut QuEChERS Quick Easy Cheap Effective Rugged and Safe Initially developed for screening of pesticide residues in fruit and vegetables to make sample cleanup of food faster, simpler, less expensive, and greener. Now, QuEChERS is used with other matrices and compound classes as well. C
Slide 35: QuEChERS Workflow
QuEChERS Workflow QuEChERS extraction salts QuEChERS dispersive SPE sorbents 35 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 36: Bond Elut Dispersive SPE Kits
Bond Elut Dispersive SPE Kits Dispersive kit Centrifuge tubes containing preweighed SPE sorbent such as: • C18: Removes residual fats and lipids • PSA: ‘Primary/secondary amine’ for removal of organic acids and sugars • GCB: Graphitized carbon black, removes pigments • EMR-Lipid: Removes unbranched
Slide 37: Dispersive EMR-Lipid
Dispersive EMR-Lipid EMR-Lipid fits into current sample preparation workflows 37 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 38: Solid Phase Extraction (SPE)
Solid Phase Extraction (SPE) • Capabilities – Very selective – Highly clean samples – Concentrated samples – Wide range of applicability – Automation friendly • Types of SPE – Nonpolar (reversed phase) SPE – Polar (normal phase) SPE – Cation exchange SPE – Anion exchange SPE – Mixed mode SPE – Speci
Slide 39: Bond Elut Plexa
Bond Elut Plexa Advanced polymer architecture improves extraction performance 39 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 40: Bond Elut Plexa
Bond Elut Plexa • New generation of polymeric SPE • Divinylbenzene-based polymeric sorbent with hydrophilic exterior, hydrophobic interior, and advanced polymeric architecture. • Superior flow properties • Great for extraction of a wide range of acidic, neutral, and basic analytes from different mat
Slide 41: Agilent SPE Offering
Agilent SPE Offering • Reliable SPE with a 30-year history • Agilent offers the most comprehensive set of phases, sizes, and OMIX monolithic formats of any SPE provider (over 40 sorbent materials/phases silica tip SPE available) OMIX C18 OMIX MP1 • Easy adoption of methods due to high number of publ
Slide 42: Manifolds for Processing Cartridges and 96-Well Plates
Manifolds for Processing Cartridges and 96-Well Plates 42 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 43: Agenda
Agenda • Strategies for sample cleanup • Chromatography problems caused by sample matrix – Physical effects – Chemical effects • Summary 43 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 44: Chromatography Problems Caused by Sample Matrix – Physical Effects
Chromatography Problems Caused by Sample Matrix – Physical Effects • Particulates in the sample can partially block the inlet frit of the column or guard, causing split/double peaks and high pressure. • Some components of the sample (proteins, salts) may precipitate as they come into contact with mo
Slide 45: Agenda
Agenda • Strategies for sample cleanup • Chromatography problems caused by sample matrix – Physical effects – Chemical effects • Summary 45 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 46: Chromatography Problems Caused by Sample Matrix – Chemical Effects
Chromatography Problems Caused by Sample Matrix – Chemical Effects • Chemical contamination/lipid build up can cause secondary interaction and result in retention time variability, peak shape variability/tailing, selectivity changes, and (in some cases) increased back pressure. • Lipids from the sam
Slide 47: Column Contamination from Sample Matrix Causing Split Peaks
Column Contamination from Sample Matrix Causing Split Peaks Injection 1 Injection 30 47 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 48: Column Contamination from Sample Matrix Causing Peak Tailing
Column Contamination from Sample Matrix Causing Peak Tailing 48 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 49: A Cleanup Step Improves Analytes S/N Ratio and Integration Accuracy on GC/MS(/MS)
A Cleanup Step Improves Analytes S/N Ratio and Integration Accuracy on GC/MS(/MS) Pesticide Captan Permethrin Deltamethrin EMR-Lipid cleanup Zirconia sorbent cleanup + MRM (151.0 -> 79.1) Post-S 50ppb Fatty dSPE C1-3.D Counts x10 5 1.4 1.2 1 C18/PSA 0.8 0.6 cleanup 0.4 0.2 *13.174 min. 0 12.8 12.9 1
Slide 50: Agenda
Agenda • Strategies for sample cleanup • Chromatography problems caused by sample matrix – Physical effects – Chemical effects • How to deal with unwanted matrix effects • Summary 50 February 11, 2020 Avoiding Unexpected Problems in LC and GC Analysis DE.4634722222
Slide 51: Summary
Summary • Many chromatography problems are due to the components present in the sample matrix. • In some cases, measures can be taken to temporarily overcome or mask the unwanted matrix effects. • Ultimately, sample preparation/cleanup is the most reliable way to address common chromatography data p
Slide 52: Offline Options for Sample Matrix Removal
Offline Options for Sample Matrix Removal Captiva EMR-Lipid filtration cartridges and plates QuEChERS Bond Elut Solid Phase Extraction cartridges and plates Chem Elut S SPME Filter vials Captiva syringe filters 14 52 February February 11, 11, 20202020 Avoiding Unexpected Problems in LC and GC Analys
Slide 53: Contact Agilent Chemistries and Supplies Technical Support
Contact Agilent Chemistries and Supplies Technical Support 1-800-227-9770 option 3, option 3: Option 1 for GC and GC/MS columns and supplies Option 2 for LC and LC/MS columns and supplies Option 3 for sample preparation, filtration, and QuEChERS Option 4 for spectroscopy supplies Option 5 for chemic
Slide 54: Appendix
Appendix 54 February 11, 2020 DE.4634722222
Slide 55: Other Sources of Unknown Peaks and Chromatography Problems
Other Sources of Unknown Peaks and Chromatography Problems • Impurities and contamination of mobile phase components • Mobile phase is incompatible with LC system components, leaching out contaminants • Contaminants from air getting into the mobile phase bottle due to use of incorrect bottle cap • M
Slide 56: Solvent Contamination
Solvent Contamination Injections on Agilent 1100 Contamination of mobile phase component Various sources of acetonitrile tested 56 February 11, 2020 DE.4634722222
Slide 57: Retention Time Shifts and Peak Shape Problem
Retention Time Shifts and Peak Shape Problem Change in volatile buffer concentration – Incorrect solvent bottle caps used 57 February 11, 2020 DE.4634722222
Slide 58: What To Do
What To Do • Use high-purity solvents • Use appropriate solvent bottle caps (Agilent InfinityLab Stay Safe caps) • Use solvent compatible material for parts of LC that come in contact with mobile phase • Use freshly made HPLC grade solvent and filtered buffer • Replace solvent inlet filter as needed
Slide 59: InfinityLab Stay Safe Caps
InfinityLab Stay Safe Caps 59 February 11, 2020 DE.4634722222
Slide 60: Carry Over
Carry Over Carry over peaks can be caused by 1. Late eluting peaks from previous run 2. Contaminated sampling device components (rotor seal, needle, needle seat) 3. Contaminated/wrong solvent used for needle wash 4. Release of retained compounds on active sites of the system 5. Unswept areas in samp
Slide 61: InfinityLab Quick Connect and Quick Turn Fittings
InfinityLab Quick Connect and Quick Turn Fittings • Spring loaded design • Easy – no tools needed • Works for all column types • Reusable • Consistent ZDV connection 61 February 11, 2020 DE.4634722222
Slide 62: Column Cleaning
Column Cleaning 62 February 11, 2020 DE.4634722222
Slide 63: Column Cleaning – Protein/Peptide Removal
Column Cleaning – Protein/Peptide Removal Solubilization solvents for proteins/peptides, in the order of weakest to strongest: • Water/phosphate buffer • Dilute acid (TFA, HOAc or HCl) • Neutral pH 6-8 M guanidine-HCl or isothiocyanate • 5% HOAc/6 M urea • Dilute acid + aqueous/organic solvents (ACN
Slide 64: Post Column Infusion
Post Column Infusion Post column infusion setup for evaluation of ion suppression caused by the matrix 64 February 11, 2020 DE.4634722222
Slide 65: Bond Elut Plexa Method
Bond Elut Plexa Method 65 February 11, 2020 DE.4634722222
Slide 66: Bond Elut Plexa Method
Bond Elut Plexa Method 66 February 11, 2020 DE.4634722222
Slide 67: Online SPE (Trace Enrichment-SPE)
Online SPE (Trace Enrichment-SPE) 67 February 11, 2020 DE.4634722222
Slide 68: Step 1: Online SPE1
Step 1: Online SPE1 68 February 11, 2020 DE.4634722222
Slide 69: Step 2: Online SPE2
Step 2: Online SPE2 69 February 11, 2020 DE.4634722222
Slide 70: Agilent Online SPE System
Agilent Online SPE System 70 February 11, 2020 DE.4634722222
Slide 71: Agilent 1200 Infinity Series Online SPE Solution-Flex Cube
Agilent 1200 Infinity Series Online SPE Solution-Flex Cube 71 February 11, 2020 DE.4634722222
Slide 72: Online SPE with Flex Cube
Online SPE with Flex Cube 72 February 11, 2020 DE.4634722222
Slide 73: Flex Cube
Flex Cube System with alternating SPE loading: Load sample to SPE1 73 February 11, 2020 DE.4634722222
Slide 74: Flex Cube
Flex Cube System with alternating SPE loading: Elute sample from SPE1 to column/load sample 2 on SPE2 74 February 11, 2020 DE.4634722222
Slide 75: Online Options for Sample Matrix Removal
Online Options for Sample Matrix Removal Agilent RRLC in-line filter 0.2 μm pore size filter, max 600 bar - 4.6 mm ID, 5067-1553 - 2.1 mm ID, 5067-1551 Agilent 1290 Infinity II LC in-line filter 2.1 mm, 0.3 μm, 1300 bar, 5067-6189 Agilent Fast Guard, 3/pk RRHT, 600 bar RRHD, 1300bar One piece preass
Slide 76: February 11, 2020
76 February 11, 2020
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