Useful GC/MS Sensitivity for Improved Environmental Analyses. Terry L. Sheehan, Ph.D. GC/MS Senior Product Manager Santa Clara, CA 7 February 2008
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1 Useful GC/MS Sensitivity for Improved Environmental Analyses Terry L. Sheehan, Ph.D. GC/MS Senior Product Manager Santa Clara, CA 7 February 2008
2 Definitions Sensitivity Analytical definition: slope of the plot of amount of analyte (x-axis) versus signal response (y-axis) Commonly used definition: minimum amount of analyte that gives an acceptable response (more correctly limit of detection ) Sensitivity Specification (GC/MS) S/N achieved for a very small amount of analyte injected under a carefully defined set of the instrument conditions using a matrix-free sample Useable Sensitivity Instrument and method parameters and tools that enhance the signal response and reduced noise when working with real sample matrices Page 2
3 eseminar Overview Technology Curve Where is GC/MS Today? Sample Preparation Hardware Evolution GC Capillary Flow Technologies (Backflush) MS Triple-Axis Detector, Gain Normalization and Fast Electronics Software Evolution MS Trace Ion Detection and Deconvolution GC Retention Time Locking and New Databases Summary Literature References Page 3
4 Technology Adoption Lifecycle Everett M. Rogers Diffusion of Innovation, 1962 Page 4
5 Positioning of Basic GC/MS Cumulative Use of Technology SIM MS Scan MS Page 5
6 Positioning of Newer GC/MS Technologies Cumulative Use of Technology Backflush Trace Ion Detection Deconvolution Detector Splitting Synchronous SIM/Scan SIM MS Scan MS Retention Time Locking Page 6
7 Technology Adoption Lifecycle Backflush Detector Splitting Retention Time Locking Synchronous SIM/Scan Trace Ion Detection Deconvolution Everett M. Rogers, 1962 Diffusion of Innovation Page 7
8 Technology Adoption Lifecycle Backflush Detector Splitting Retention Time Locking Synchronous SIM/Scan Trace Ion Detection Deconvolution chasm Geoffrey Moore, 1991 Crossing the Chasm Everett M. Rogers, 1962 Diffusion of Innovation Page 8
9 Reasons for Slow Adoption of Technologies Unaware of the technical and/or practical benefits Too complicated; too much work to setup Not robust enough for routine use Technology imperfections that impact performance Page 9
10 Reasons for Slow Adoption of Technologies Unaware of the technical and/or practical benefits Too complicated; too much work to setup Not robust enough for routine use Technology imperfections that impact performance Difficult to break long standing habits Time/effort required to change established methods Constraint of government regulations Page 10
11 eseminar Overview Technology Curve Where is GC/MS Today? Sample Preparation Hardware Evolution GC Capillary Flow Technologies MS Source, HED-EM Detector and SIM/Scan mode Software Evolution MS Trace Ion Detection and Deconvolution GC Retention Time Locking and New Databases Page 11
12 Honorable Mention for Sample Preparation No Sample Prep! The dream of many lab managers and analysts Sample Prep realities Limitations of the gas chromatograph Contamination and overload Limitations of the mass spectrometer Contamination and overload Limitations of the combined selectivity of GC + MS Page 12
13 eseminar Overview Technology Curve Where is GC/MS Today? Sample Preparation Hardware Evolution GC Capillary Flow Technologies (Backflush) MS HED-EM Detector and SIM/Scan mode Software Evolution MS Trace Ion Detection and Deconvolution GC Retention Time Locking and New Databases Page 13
14 Capillary Flow Technology a proprietary Agilent Technology Photolithographic chemical milling for low dead volume Diffusion bond two halves to form a single flow plate Small, thin profile provides fast thermal response Projection welded connections for leak tight fittings Deactivation of all internal surfaces for inertness SilTite (metal) ferrules Page 14
15 5 th Generation Electronic Pressure Control (EPC) 7890A GC 3 rd, 4 th Generation EPC psi -- Diffusion bonded plate (2D) -- one cable -- three gas connectors -- credit card size The only GC to regulate pressure to psi The only EPC regulating pressure to 1/1000 psi 6890N GC 1 st, 2 nd Generation EPC psi -- cables -- gas lines & connectors -- large size 5 th Generation EPC - Metal injection molded (3D) - Digital signal pathways 5890 GC improved reliability and precision Page 15
16 QuickSwap MSD Interface Remove column w/o venting Air & H 2 O blocked Safe disconnection of column from inlet for inlet maintenance Column Effluent Reversed flow through column during inlet maintenance Backflushing Removes heavies from column Maintain constant flow to MSD MSD Transfer Line performance turbo recommended Aux EPC In Page 16
17 Backflush with QuickSwap During GC Run Split Vent Trap Aux EPC 4 psi S/S Inlet MSD 25 psi QuickSwap After GC Run Split Vent Trap Column Aux EPC 45 psi S/S Inlet MSD 1 psi Column QuickSwap performance turbo recommended Page 17
18 Benefits of Backflushing More samples per day per instrument Longer column life Lower operating costs Less frequent and faster GC & MSD maintenance Less chemical background Better quality data Page 18
19 Industry Standard: Low Bleed Stationary Phases Better signal to noise, higher upper temp limit, faster run time, improved spectral purity, and greater column inertness DB-35ms Primary Ion 498 DB-35ms S/N =10 DB-35ms, 320 C C m/z--> S/N =3 DB DB-35 DB-35, 300 C 300 C 78 Primary Ion CLP Pesticides Analysis 0 m/z--> July 2006
20 Benefits of Backflushing Problem of chemical noise No perfect injectors or columns Sample matrix bleed and column phase bleed Chemical noise increases with each injection in the sequence Higher temperature clean-up of the column after each analysis transfers more chemical noise into the source Increased probability of interfering response at target ions For trace analytes, chemical noise degrades search quality Backflush minimizes the column surface exposed to the higher boiling matrix components Backflush eliminates column bleed into the source during higher temperature cleanup of the column Page 20
21 Conventional Off-Axis Detector hyperbolic quartz transmission quadrupole analyzer ion beam Although not directly striking the HED- EM, excited helium neutrals can ionize other molecules and create secondary particles from collisions with surfaces (sputtered ions from surface contamination, ions from the surface, photons and electrons) high energy dynode (high voltage pulls the ions away from the neutrals) electron multiplier energetic neutrals Page 21
22 Triple-Axis Detector slide 1 of 3 steering rod attract the ions away from the neutrals This aperture is larger than the off-axis detector which means more ions and energetic neutrals pass to the detector ion beam Introduced 01 Feb 08 energetic neutrals Page 22
23 Triple-Axis Detector slide 2 of 3 shield blocking secondary ions formed by collisions with energetic neutrals in the detector region high energy dynode Introduced 01 Feb 08 Page 23 energetic neutrals triple channel electron multiplier
24 Triple-Axis Detector slide 3 of 3 hyperbolic quartz transmission quadrupole analyzer steering rod shield for secondary particles Offset Z high energy dynode Y ion beam Introduced 01 Feb 08 X triple channel electron multiplier Page 24
25 Triple Channel EM Exit of the triple channels Triple channels improve signal Triple channels increase life Collector removed to show exit passages Introduced 01 Feb 08 Collector Page 25
26 Triple-Axis Detector: Higher Signal Lower MDL Increased slope = higher sensitivity % confidence limits Improved detection limits! fg Page 26 Tech Overview EN
27 New Detector for Enhanced EI Sensitivity! Abundance--> Enhanced signal 250 fg New detector S/N-rms: 250 Standard detector S/N-rms: 35 New 1 pg OFN specification: 400:1 for turbo pumps 200:1 for diffusion pump 0 Time--> Reduced neutral noise Page 27 Tech Overview EN
28 What about Chemical Noise Limited Baselines? 10-ppb standard in Jin Ying Hua extract Abundance TIC: _00012std TIC: _00014std Larger signal Off-Axis Triple-Axis SIM s/n Time Chlorfenvinphos(Z) p,p -DDT Cyhalothrin (Gamma) Deltamethrin Comparable S/N in complex matrices.. Why? All ions (analytes and matrix peaks) will be amplified. Triple-Axis Detector eliminates neutral noise, not matrix chemical noise But increased signal will always improve ion statistics and integration Page 28
29 Gain Normalized Autotuning Ion source Quadrupole High Energy Dynode (10kV) i Electron Multiplier (EM) EMV EMV: 1kV to 3kV I I i GAIN Amplification of signal now, user-selectable!!! Page 29
30 Disadvantage of ATUNE + nv Detectors age over use: the same EMV setting will not give the same signal! New detector Signal vs EMV Aged detector Signal Decreasing signal with age Page ATUNE+400V ATUNE (new) ATUNE (aged) ATUNE + 400V Tech Overview EN EM V
31 Sensitivity Advantage of Gain Normalization Detectors age over use: but the same GAIN setting will give the same signal! Signal vs GAIN Aged New Signal Gain Factor as GAIN x 10^5 Page 31 Tech Overview EN
32 As Experienced in a Laboratory Situation - New Aged 1000 GAIN Tune 1000 GAIN Tune ATUNE+ n V ATUNE+ n V Gain normalized methods maintain response over time! Page 32 Tech Overview EN
33 Using Gain Factor for Method Optimization Change (Atune + 200V) to (Atune V) How much increase? Change Gain Factor 1 to 11, a predictable x11 increase! 180k counts peak height RTIC of standard acquired at Gain Factor 1.0 Same standard reacquired with calculated Gain Factor of k counts peak height Page 33 Tech Overview EN
34 Fast Electronics Fast electronics allow SIM and Scan data in a single run SIM = maximum sensitivity for target compounds Scan = best identification of unknowns Scan data points SIM data points Chromatographic peak time Scan time SIM time more information from a single run Page 34 Tech Overview EN
35 Synchronous SIM/Scan Comparison of PAHs Triphenyl phosphate Chrysene Benz[a]anthracene SIM 5.55 cycles/s Scan u 5.55 cycles/s 0.2 ppm Page 35 Application EN
36 Synchronous SIM/Scan Under-sampling the peak (slow data rate) decreases area precision 6 samples/peak width for area; 8 samples/peak width for height Shorter dwell time decrease SIM S/N slightly Faster scan rates decrease scan S/N slightly Scan data points SIM data points Chromatographic peak time Scan time SIM time For many applications... the losses in precision and S/N are small compared to the gains in SIM sensitivity and scan library searches Page 36 Tech Overview EN
37 eseminar Overview Technology Curve Where is GC/MS Today? Sample Preparation Hardware Evolution GC Capillary Flow Technologies (Backflush) MS HED-EM Detector and SIM/Scan mode Software Evolution MS Trace Ion Detection and Deconvolution GC Retention Time Locking and New Databases Page 37
38 Trace Ion Detection Technology Agilent proprietary algorithm Reduced noise level Improved peak shape Especially under-sampled peaks Improved library match Without Trace Ion Detection S/N = 53 Default OFF in the ChemStation With Trace Ion Detection S/N = 92 better detection at trace levels Page 38
39 Improve Library Match Quality Fenthion in strawberry extract Trace Ion Detection off Match Quality 30 Trace Ion Detection on Match Quality 92 Page 39
40 NIST AMDIS Background AMDIS: Automated Mass Spectral Deconvolution and Identification Software G. Mallard, S. Stein, O. Toropov, NIST Deconvolution = disentanglement of multiple signatures Originally developed for detection of chemical weapons in complex mixtures (environmental samples, process streams) Designed to work without analyst input Agilent DRS Revision A.01, March 2004 AMDIS 2.64 released December 2005 (noise reduction) Agilent DRS Revision A.04, February 2008 Integrated into QEdit for qual, quant, manual integration and reports Page 40
41 AMDIS Purifies Spectra by True deconvolution Even if no available background for subtraction Detailed treatment of noise Complete noise analysis; used for component perception Correction for baseline drift Flat baselines not required; determines baselines for each m/z Corrects for spectral skewing in spectra Distortion generated by concentration change during scan period Extracts closely coeluting peaks Separates components that have peak apexes with a single scan Page 41
42 Trace Ion Detection with Deconvolution Peak detected with Trace Ion Detection TIC TIC & Spectrum Component 1 Component 2 Component 3 Deconvoluted peaks and spectra matrix interference Deconvolution target Page 42
43 AMDIS: Pulling a Useable Spectrum Out of a Mess Page 43
44 17 Surface Water Data Files: Pesticide Analysis Targets Found False Positives Processing Time *CDFA 37 1 ~ 8 hours Agilent DRS Same additional 0 32 minutes *CDFA is the California Department of Food and Agriculture Data files courtesy of Dr. Mark Lee and Steve Siegel Page 44
45 Why are the Results so Different? CDFA Analyst was processing raw (dirty) spectra and relying on 1 Target ion + 3 Qualifier ions to confirm presence / absence of a target analyte DRS was processing deconvoluted (clean) spectra and using all the mass spectral information available to confirm the presence / absence of target analytes Page 45
46 Total ion chromatogram Targets are identified by comparison to locked retention times and 3 qualifying ion ratios, quantified using target ion area versus ISTD calibration table Quant Results AMDIS 32 deconvolutes component spectra and searches target MS database, locked RT used as a qualifier Confirmed AMDIS hits Deconvoluted Target spectra confirmed by AMDIS searched against NIST05 MS database Confirmed NIST05 hits Page 46 Combined quantitative and qualitative HTML Summary report
47 QEdit - before DRS importing Import Results to bring AMDIS deconvolved data into QEdit for quantitation. Page 47
48 QEdit - After DRS Import Result (butylbenzyl phthalate selected) Overlay of target(s) and Deconvoluted ion plots Target ion plot Deconvoluted ion plot Spectral review: Before AMDIS After AMDIS AMDIS Library Hits X = MSD A = AMDIS Areas & amounts from target ion and Deconvoluted ion Page 48
49 Enhanced DRS A.04 Capabilities ) DRS quant results will appear in the Summary Quant Report 2) AMDIS libraries, quant database and screener database can be created from a user selected PBM library 3) Retention times in user selected AMDIS libraries can be updated with retention times from the currently loaded method quant database Page 49
50 Summary Quant Report with both MSD Chemstation and AMDIS results partial report Page 50
51 Report- Spinach Example 1C Match against RTL Pesticide Library spectra "RI rejection" eliminates entries more than 20 seconds (user settable) from their expected R.T. The MSD ChemStation did not find these compounds, but AMDIS did and was confirmed by NIST05. Page 51 ISTD, used for targets amount Hit number of the top 100 hits from 163,000+ compounds
52 DRS A.04 Report with both MSD ChemStation and AMDIS results p,p -DDE target ion mismatch Page 52
53 Why 1/1000 psi Matters! -- Key to even better Retention Time Locking (RTL) Response Column 1 at psi psi psi 0.96 psi Agilent worries about the 3 rd decimal, so I don t have to worry about the 1 st or 2 nd decimal point - Pharma (USA) Time Page 53
54 Retention Time Locking Improve Confidence with Retention Time Locking Easy Quick min. Initial run 4.72 psi Repeatable Run-to-run Operator-to-operator min. Trim 1 meter 4.72 psi Instrument-to-instrument min. Relock 4.42 psi 0 Page 54
55 Industry Specific Retention Time Locked Libraries Hazardous Chemicals Organotins* VOCs* Pesticides and Endocrine Disruptors *free download Semi-Votatiles (coming soon) Indoor Air Toxics PCB Congeners* Japanese Positive List Pesticides or create your own Page 55
56 Industry Specific Retention Time Locked Libraries Part Number RTL Database/Library Number of Compounds G1671AA Hazardous Chemicals 730 G1672AA Pesticide 926 G1673AA Indoor Air Toxics 171 G1674AA Forensic Toxicology 723 G1675AA Japanese Positive List Pesticide 431 G1677AA Environmental Semi-Volatile one 8270 set of 273 ; two 525 sets of 120 Page 56
57 Summary of MSD-DRS-RTL Benefits Ease of Use Automation Speed Reproducibility No need to be an expert in deconvolution software, fully integrated into GC-MSD Chemstation software Part of a method or part of a sequence, quantitation based on Chemstation and Deconvoluted peak area / height A complex TIC containing hundreds of component spectra is processed and reported typically in less than 1 minute Program results are not subject to changes in mood / attention of Analyst Selectivity AMDIS will find data that an Analyst might miss Confidence DRS will report the fewest false positives / false negatives in the shortest possible time Page 57
58 Special Promotion for Capillary Flow Technologies and DRS-NIST-RTL Bundle Orders for the 5975C MSD with the Triple-Axis Detector DRS-NIST05-RTL bundle priced with a 50% discount Includes a choice of one RTL database With the standard or performance turbo only (diff pump systems excluded) Orders for the 7890A GC Selected Capillary Flow Technologies priced with a 50% discount 2-way and 3-way purged splitter and Deans switching Promotion active from February 1 until July 31, 2008 Page 58
59 What is Your Technology Adoption Lifecycle? Backflush Deconvolution Synchronous SIM/Scan Detector Splitting Retention Time Locking Trace Ion Detection Everett M. Rogers, 1962 Diffusion of Innovation Page 59
60 Upcoming GC eseminars SIMPLIFY Discover New Proteins Using Immunodepletion February 19, :00am EST and 1:00pm EST Introduction to Capillary GC Series 2 February 20, :00pm EST Selection of a Capillary GC Column Series 3 March 13, :00pm EST
61 Appendix A: Literature References Page 61
62 References for Backflush EN Analysis of Suspected Flavor and Fragrance Allergens in Cosmetics Using the 7890A GC and Capillary Column Backflush (March 2007) EN Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975C/DRS GC/MSD System (Jan 2007) EN Direct Injection of Fish Oil for the GC-ECD Analysis of PCBs: Results Using a Dean Switch with Backflushing (Jan 2007) EN Improving Productivity and Extending Column Life with Backflush (Dec 2006) EN Simplified Backflush Using Agilent 6890 GC Post Run Command (June 2006) EN New Tools for Rapid Pesticide Analysis in High Matrix Samples (October 2004) Page 62
63 References for Fast Electronics and Synchronous SIM/Scan EN New Approaches to the Development of GC/MS Selected Ion Monitoring Acquisition and Quantitation Methods EN 5973 Inert Performance Electronics: Considerations for GC/MS Methods in Scan and Selected Ion Monitoring Modes EN Strategies for Developing Optimal Synchronous SIM- Scan Acquisition Methods AutoSIM/Scan Setup and Rapid SIM EN Improving Productivity with Synchronous SIM/Scan EN Synchronous SIM/Scan Low-Level PAH Analysis Using the Agilent Technologies 6890/5975 inert GC/MSD Page 63
64 Supporting Literature for DRS App Note EN, Replacing Multiple 50-Minute FPD/ELCD/SIM Analyses with One 15-Minute Full-Scan Analysis for 10x Productivity Gain (Meng/Szelewski, Nov07) Capillary flow technology for 3-way splitter Simultaneously monitor multiple detector Backflush to shorten analysis time and increase column lifetime Trace Ion Detection to reduce noise before DRS Next generation of DRS with AMDIS results in QEdit Real results from US Food and Drug Administration/Center for Food Safety and Applied Nutrition (FDA/CFSAN) Better, more accurate identification and quantification of trace compounds in complex matrices with less operator experience and time Page 64
65 Supporting Literature for DRS App Note EN, Screening for Pesticides in Food Using the Japanese Positive List Pesticide Method: Benefits of Using GC/MS with Deconvolution Reporting Software and a Retention Time Locked Mass Spectral Database (Wylie, Sept07) App Note EN, Reducing Analysis Time Using GC/MSD and Deconvolution Reporting Software (May07) App Note EN, Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975/DRS GC/MSD System (Quimby, Jan07) App Note EN, Screening for 171 Volatile Organic Air Pollutants Using GC/MS with Deconvolution Reporting Software and a New Indoor Air Toxics Library (Wylie, Aug06) Page 65
66 Supporting Literature for DRS App Note EN, Screening for 926 Pesticides and Endocrine Disruptors by GC/Ms with Deconvolution Reporting Software and a New Pesticide Library (Wylie, Apr06) App Note EN, Screening for Hazardous Chemicals in Homeland Security and Environmental Samples Using a GC/MS/ECD/FPD with a 731 Compound DRS Database (Quimby, Szelewski, Feb06) App Note EN, New Tools for Rapid Pesticide Analysis in High Matrix Samples, (Szelewski, Quimby, Oct04) App Note EN, A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit Extracts Using Deconvolution Reporting Software App Note EN, Comprehensive Pesticide Screening by GC/MSD using Deconvolution Reporting Software (Wylie/Szelewski/Meng, May04) Page 66
67 Appendix B: Supplemental Information Page 67
68 Detector Splitting for PAHs and PCBs In one run the customer can acquire SIM, Scan and ECD data! Page 68
69 Page 69
70 High Temperature Ion Source 350 C Programmable to 350ºC Improved response for many compounds Combined with heated quartz quadrupole More robust operation for dirty samples Rapid conditioning of analyzer for analysis 325 C 300 C 275 C 230 C Up to 1050 u for all models Polyaromatic Hydrocarbon (PAH) more performance for a wide range of applications Page 70
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