The Tool Box Getting More Productivity from Your GC and GC/MSD

The Tool Box Getting More Productivity from Your GC and GC/MSD M Ramesh Application Engg GC/GCMS Agilent Technologies India Pvt. Ltd. Productivity Tool Box for GC & GC/MSOverview 5975C MSD Agilent Restricted Page 2

Defining Productivity More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 4

Productivity: the Sum of Many Technologies Low Thermal Mass GC Capillary Flow Technology - Backflush Hydrogen carrier gas for GC and GC/MS Retention Time Locking (RTL) Editor (& Methods) With more RTL Databases Trace Ion Detection Fast Electronic - Synchronous SIM/Scan Method Translator High signal-to-noise Triple-Axis Detector Gain Normalization of the EM EM Saver 2nd Generation Deconvolution Reporting Software (DRS) Return home (this slide) Next slide Return to section menu Page 5

Productivity: Low Thermal Mass GC More analyses per unit time... Faster and better separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 6

LTM (Low Thermal Mass) Technology Direct heating of fused silica GC columns Page 7

Rapid Cooldown LTM Cooldown Times (Standard Size) Temp (oc) 400 350 300 250 200 150 100 50 0 0 50 100 150 200 250 Time (sec) 2m LTM 5m LTM 10m LTM 7890 GC Additional Cooldown times available on website for LTM column modules up to 30m Page 8

Low Thermal Mass Technology for 7890/6890 GC Satisfies the need for high through-put, especially for low margin samples, where analytical cycle times are a critical element in making a profit Independent and simultaneous temperature programming of: 1-2 column modules (fastest cooldown), or 1-4 column modules (small format) LTM Column Modules (standard width shown) LTM Control System w/ Keypad User Interface Agilent LTM Control SW now available Note: Front thermal shield removed to show LTM column modules Entire thermal shield should always be in place during operation Page 9

LTM Also Greatly Reduces Cool-Down Times, 9x Faster Heating/Cooling Cycle Times GC Run Time Cool Down 40 min + 5.4 min 20 min + 5.4 min 3 min + 2 min LTM/7890 9x Faster Cycle Time (200 o C/min) Page 10

Obtaining Faster GC, GC/MS Analytical Cycle Times A Variety of Approaches to Fit Your Lab and Your Needs Pre-Run ALS Set-Up Chromatographic Run Post-Run Bake-Out Post-Run Cool-Down Included ALS Sample Overlap in in ChemStation SW HW/SW 7890 GC Faster Cool-down Faster GC Analyses Column Selection, Method Translation GC Add-Ons Signficantly Faster GC Analytical Cycle Times Capillary Flow Technology (Backflush) LTM Technology LTM Technology (Rapid Heating) (Rapid Cooling ) Page 11

PAH by LTM/GC/MS Fast Analysis - LTM 20 m x 0.18 mm ID x 0.18 µm DB-5MS Time--> 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Reference method Standard Oven 30 m x 0.25 mm ID x 0.25 µm DB-5MS Time--> 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Page 12

PAH by LTM/GC/MS Column contamination from just one prior run Contamination free when using backflush Page 13 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 Time (min)

LTM Productivity High efficiency temperature programming Fast cooling High throughput Fast method development High performance chromatography Utilize commercially available columns (typically < 30 m) Easily integrates with conventional GC instruments Utilize existing injectors, detectors, software Multicolumn temperature control Independent optimization Multidimensional GC High throughput parallel systems Page 14

Hydrocarbon Processing Industry (HPI) HPI Shorter analytical cycle times get data back to operations faster, or allow more samples/shift (eg. Simulated Distillation) Key Sales Tools 5990-3174EN Fast Hydrocarbon & Sulfur Simulated Distillation Using the Agilent Low Thermal Mass (LTM) System on the 7890A GC and 355 Sulfur Chemiluminescence Detector(Wang,Firor,Tripp - Fast Simdist (< 3 mins), 6x faster than conventional Simdist - Dual channel, simultaneous SimDist for hydrocarb s & sulfur Article (Fall 08) Page 15 Multidimensional Gas Chromatography with Capillary Flow Technology and LTM-GC J Luong (et. al) Dow Chemical, R Mustacich (RVM) Published:Journal of Separation Science, ISSN 0021-9665, Volume 31, pp. 3385-3394 http://www3.interscience.wiley.com/journal/121406899/abstract - HPI examples 2D LTM/CFT/GC using Deans Switch and LTM s simultaneous/independent temperature programming of multiple LTM column modules

Environmental/Food Safety/Forensics Need for high through-put, especially for low margin samples, where analytical cycle times are a critical element in making a profit Key Sales Tools 5990-3201EN Ultra-Fast Total Petroleum Hydrocarbons (TPH) Analysis with Agilent Low Thermal Mass (LTM) GC and Simultaneous Dual- Tower Injection (Wei & Szelewski) - 9x faster TPH throughput using LTM - 18x faster TPH using LTM and dual simultaneous injection Pub No. TBA Polyaromatic Hydrocarbons (PAH) by LTM/GC/MS (~end Jan 09) - 2x faster PAH runs using LTM, even faster with Backflush - Enviro and Food Safety samples can be dirty. LTM columns cannot be trimmed (not Agilent, not Thermo ) - LTM uses guard columns and now Backflush/CFT backflush delivers multiple benefits for LTM analyses including. reduced potential for carryover, longer column life, shorter analysis time Page 16

Food/Flavor/Fragrance and Pharmaceutical Chiral Separations Chiral GC is most effective at low temperatures, yet complex samples usually require a broad temperature program to separate target compounds from background and to elute highly retained sample components. LTM/GC/MS uses a traditional column in the GC oven to separate target compounds, then heart-cut of target compounds to a chiral column in LTM module at a lower temp. optimal chiral separations are achieved while decreasing analysis cycle times and increasing chiral column lifetime. Pub No. TBA (~end Jan 09) Article (Fall 08) Chiral Separations by LTM/GC/MS - 2D LTM/GC/MS, simultaneous/independent temperature programming of GC and LTM column module(s) Multidimensional Gas Chromatography with Capillary Flow Technology and LTM-GC, J Luong (et. al) Dow Chemical,R Mustacich Published:Journal of Separation Science, ISSN 0021-9665, Volume 31, pp. 3385-3394253-261 - Examples 2D LTM/CFT/GC using Deans Switch and LTM s simultaneous/independent temperature programming of multiple LTM column modules Page 17

Productivity: CFT Backflush More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 18

Backflush Complete Column During GC Run Split Vent Trap Aux EPC 4 psi S/S Inlet MSD 25 psi After GC Run Split Vent Trap Column QuickSwap or 3- way Splitter Aux EPC 45 psi S/S Inlet MSD 1 psi Column QuickSwap or 3- way Splitter performance turbo recommended Page 19

Backflush Mid-Column (Analysis Mode) Split/Splitless Injection Port Pressure / Flow Controller Z ml/min very low flow; set against column backpressure, not short restrictor as with QuickSwap 7890A GC Vent Z ml/min Capillary Flow Technology Device Y+Z ml/min 5975C MSD EI mode Y ml/min 15-m HP-5ms (0.25mm id x0.25um) 15-m HP-5ms (0.25mm id x0.25um) PCT available Oct 08 Column 1 and 2 could be different phases and different dimensions Page 20

Backflush Mid-Column (Backflush Mode) Split/Splitless Injection Port Pressure / Flow Controller 7890A GC Vent Decrease inlet pressure during backflush Pressure Controlled Tee 5975C MSD EI mode 15-m HP-5ms (0.25mm id x0.25um) 15-m HP-5ms (0.25mm id x0.25um) Page 21

Backflush Mid-Column (Backflush Mode) Split/Splitless Injection Port Pressure / Flow Controller 7890A GC Vent Decrease inlet pressure during backflush Pressure Controlled Tee 5975C MSD EI mode Purged Ultimate Union To 2 nd Column & MSD From 1 st column 15-m HP-5ms (0.25mm id x0.25um) 15-m HP-5ms (0.25mm id x0.25um) From P control Page 22

Backflush Mid-Column (Timing Considerations) Sample - no backflush 9.00 9.50 10.00 10.50 11.00 11.50 12.00 Sample - with backflush a b a = begin backflush (last peak exits column 1) b = increase flow 4 ml/min 9.00 9.50 10.00 10.50 11.00 11.50 12.00 Solvent blank - no backflush 9.00 9.50 10.00 10.50 11.00 11.50 12.00 Page 23

Comparison of Backflush Configurations QuickSwap (QS) Pressure Controlled Tee (PCT) 30 m column; restrictor (0.092mm*) Two 15 m columns PCT time Backflush (BF) BF flow 1 column vol MDS flow BF flow 1 column MSD flow savings Pressure (psi) (ml/min) (min) (ml/min) (ml/min) vol (min) (ml/min) (N x) 10.0 0.3 3.82 1.5 0.6 0.94 0.9 4.1 20.0 0.7 1.89 2.9 1.5 0.47 1.8 4.0 30.0 1.3 1.28 4.8 2.6 0.32 3.0 4.0 40.0 2.1 0.98 7.2 4.1 0.25 4.5 3.9 * smallest restrictor => best QS case 4x faster & lower flow! T=300 C; head pressure =0.5psi Page 24

Backflush Impact on Data Quality Late eluters are more than just a timing problem Bake-out may push the limits of phase stability Matrix may not bake out well even at the upper limit of the column For high boiling matrices, backflush offers: Improved retention time precision Improved spectra quality Consistent detector response Increased column lifetime Page 25

GC/MS Industry Standard: Low Bleed Stationary Phases Low bleed means less background (and background ions) Low bleed column DB-35ms Better detection limits S/N =10 DB-35ms, 320 C 320 C DB-35ms Primary Ion Better spectra 428 253 214 250 315 356 377 207 346 78 96 119 144 197 286 470 51 405 455 498 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 m/z--> 50 100 150 200 250 300 350 400 450 500 Standard column S/N =3 DB-35 207 253 DB-35 DB-35, 300 C 300 C 78 Primary Ion 498 315 331 377 8.00 10.00 12.00 14.00 16.00 18.00 20.00 CLP Pesticides Analysis Column bleed = chemical noise 0 m/z--> 135 156 428 197 281 356 470 52 96 119 405 249 451 50 100 150 200 250 300 350 400 450 500 Page 26

Typical GC/MS Separation Sequence Injection Filament ON after the solvent peak Separation and detection of the analytes Filament OFF after the last analyte peak and before bake-out In an effort to save the electron multiplier (EM), the operator is blind to the success of bakeout Bake-out may NOT be adequate for all samples This means the typical method will always have a potential for matrix carryover that the user does not expect. Page 27

Without Backflush: A Serious Problem Abundance 4.6e+07 4.4e+07 4.2e+07 4e+07 3.8e+07 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 0 Time A: TIC: lettuce_blank.d\data.ms B: TIC: lettuce_blank3.d\data.ms Data provided by MSD user in Almeria, Spain After only 3 samples, the background is significantly higher (increase chemical noise is every spectrum) 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 B A Overlay of two chromatograms of a blank extract injected BEFORE (A) and AFTER (B) three injections without backflush Page 28

Without Backflush: Changes in Retention Time Abundance 4.6e+07 4.4e+07 4.2e+07 4e+07 3.8e+07 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 0 Time After only 3 samples A: TIC: lettuce_blank.d\data.ms B: TIC: lettuce_blank3.d\data.ms Data provided by MSD user in Almeria, Spain Highly retained matrix is altering the column selectivity and changing the retention time 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 B A Overlay of two chromatograms of a blank extract injected BEFORE (A) and AFTER (B) three injections without backflush Page 29

Without Backflush: Increased Background Abundance 4.6e+07 4.4e+07 4.2e+07 4e+07 3.8e+07 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 0 Time After only 3 samples A: TIC: lettuce_blank.d\data.ms B: TIC: lettuce_blank3.d\data.ms Data provided by MSD user in Almeria, Spain 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Sample matrix = chemical noise Increased background due to carryover of concentrated matrix Overlay of two chromatograms of a blank extract injected BEFORE (A) and AFTER (B) three injections without backflush B A Page 30

With Backflush: Consistent Retention Times and Baselines (No Increase in Spectral Noise) Abundance 4.6e+07 4.4e+07 4.2e+07 4e+07 3.8e+07 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07 2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 0 Time TIC: lettuce_10_ppb.d\data.ms TIC: lettuce_100_ppb.d\data.ms TIC: lettuce_5_ppb.d\data.ms Data provided by user in Almeria, Spain Stable retention times and baseline... less chemical noise 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Overlay of three chromatograms of lettuce extract run with 2 min of back flush Page 31

What Real Value Does High Performance EPC Have If Matrix Carryover Destroys Rt Precision? N or m. 1 6 0 0 0 1 4 0 0 0 10 Runs with Backflushing 1 2 0 0 0 1 0 0 0 0 8 0 0 0 6 0 0 0 4 0 0 0 2 0 0 0 0 Fish oil N or m. analysis 1 4 0 0 0 1 2 0 0 0 1 0 0 0 0 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6. 1 10 Runs without Backflushing Retention times shift ~4-5 sec during 10 runs m in 8 0 0 0 6 0 0 0 4 0 0 0 2 0 0 0 5.3 5.4 5.5 5. 6 5.7 5.8 5.9 6 6.1 6.2 m in Page 32

Backflush with Purged Ultimate Union: Lipid Peroxidation Products in Blood 4-hydroxy-2,3-nonenal (HNE), an indicator of oxidative stress, and its metabolite, 1,4- dihydroxynonene (DHN) PCI with NH 3 App Note 5989-9359EN Late eluting peaks were quickly contaminating the source Page 33

Backflush with Purged Ultimate Union: Loss of Response without Backflush with backflush These were the first backflush results; the system is getting clearer without backflush App Note 5989-9359EN Page 34

Productivity Benefits of Backflush More samples per day per instrument Longer column life Lower operating costs Less frequent and faster GC & MSD maintenance Less chemical background More consistent retention times More consistent baselines Higher quality spectra (no increase in noise during analysis sequence) Higher quality quantitation (no increase in interfering ions during analysis sequence) Page 35

References for Backflush 5989-9359EN Capillary Flow Technology for GC/MS: Efficacy of the Simple Tee Configuration for Robust Analysis Using Rapid Backflushing for Matrix Elimination (Prest, Foucault, and Aubut, Aug 08) 5989-9341EN Screening for 430 Pesticide Residues in Traditional Chinese Medicine Using GC/MS: From Sample Preparation to Report Generation in One Hour (Luan, Xu, Sept08) 5989-8664EN Capillary Flow Technology for GC/MS: a Simple Tee Configuration for Analysis at trace Concentrations with Rapid Backflushing for Matrix Elimination (June 08, Prest) 5989-8588EN The Use of Automated Backflush on the 7890A/5975C GC- MS System 5989-8582EN Improved Forensic Toxicology Screening Using A GC/MS/NPD System with a 725-Compound DRS Database (Quimby, May08) 5989-7670EN, Replacing Multiple 50-Minute FPD/ELCD/SIM Analyses with One 15-Minute Full-Scan Analysis for 10x Productivity Gain (Meng/Szelewski, Nov07) Page 36

References for Backflush 5989-6460EN Analysis of Suspected Flavor and Fragrance Allergens in Cosmetics Using the 7890A GC and Capillary Column Backflush (March 2007) 5989-6095EN Direct Injection of Fish Oil for the GC-ECD Analysis of PCBs: Results Using a Dean Switch with Backflushing (Jan 2007) 5989-6066EN Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975C/DRS GC/MSD System (Jan 2007) 5989-6018EN Improving Productivity and Extending Column Life with Backflush (Dec 2006) 5989-5111EN Simplified Backflush Using Agilent 6890 GC Post Run Command (June 2006) 5989-1716EN New Tools for Rapid Pesticide Analysis in High Matrix Samples (October 2004) Page 37

Productivity: Retention Time Locking More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 38

Retention Time Locking What is Retention Time Locking (RTL)? The ability to precisely match chromatographic retention times in any systems to those in another chromatographic system with the same nominal method and column. How is Retention Time Locking Done? By adjusting column head pressure via EPC using interactive ChemStation software. Page 39

Pesticides on Two Different GCs Before RTL 23 23.5 24 24.5 25 25.5 23 23.5 24 24.5 25 25.5 Page 40

Pesticides on Two Different GCs After RTL 23 23.5 24 24.5 25 25.5 23 23.5 24 24.5 25 25.5 Page 41

RTL Calibration - Done Once for a Given Method Make 5 runs at 5 different inlet pressures: target -20% target -10% target target +10% target +20% Determine RT of your target compound in each chromatogram Enter values into the RTL Software Page 42

Acquire RTLock Calibration Data.. Page 43

RTL Calibration Runs Are Collected Automatically Target Compound for RTL Dichlorvos Methyl chlorpyrifos Mirex 34 psi 15.192 min 31 psi 15.771 min 28 psi 25 psi 16.433 min 17.201 min 22 psi 18.121 min 5 10 15 20 25 30 Page 44

RTL Calibration Runs Are Collected Automatically And times can be edit manually Page 45

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RTL Re-Locking Procedure for Trimmed Column 100 50 0 Original RTL Head pressure 16.00 psi 16.50 min. Trim front of column 16.34 min. 100 50 0 Re-lock method 100 50 0 New RTL Head pressure 15.71 psi 16.50 min. Page 47

Benefits of Locked Methods Faster method transfer & validation Faster startup after column maintenance Reduces / eliminates need to update RTs in calibration tables Easy set up of windows for SIM groups and MS/MS transitions Equivalent chromatography for all labs sharing a common method More efficient troubleshooting Easier review of data Faster identification of unknowns Page 48

Productivity: Trace Ion Detection More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 49

Trace Ion Detection Technology Proprietary algorithm Digital filtering Reduced noise level Improved peak shape Smoothes spikes on trace peaks Especially under-sampled peaks Improved library match Without Trace Ion Detection With Trace Ion Detection S/N = 53 S/N = 92 better detection at trace levels Page 50

Improve Library Match Quality Fenthion in strawberry extract Trace Ion Detection off Match Quality 30 Trace Ion Detection on Match Quality 92 Page 51

Trace Ion Detection Reduces False Negatives Page 52

Literature for Trace Ion Detection Technical Overview 5989-6425EN, The 5975C Series MSD: Method Optimization and Trace Ion Detection (Roushall/Prest, Mar 07) App Note 5989-7670EN, Replacing Multiple 50-Minute FPD/ELCD/SIM Analyses with One 15-Minute Full-Scan Analysis for 10x Productivity Gain (Meng/Szelewski, Nov07) Page 53

Productivity: Fast Electronic - SIM/Scan Mode More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 54

High Performance, Fast Electronics Higher percentage of time monitoring ions = more signal Higher data rate for narrow fast peaks = better area precision Page 55

High Performance Electronics for High Performance Synchronous SIM/Scan 5989-3108EN: Improving Productivity with Synchronous SIM/Scan Page 56

Fast Electronics: Synchronous SIM/Scan The best of both modes in a single run SIM = maximum sensitivity for target compounds Scan = best identification of non-targets (unknowns) High speed electronics is an Agilent strength Scan data points SIM data points Chromatographic peak time Scan time SIM time maximum information from a single run Tech Overview 5989-3108EN Page 57

Under-Sampled Peaks Acceptable for MS Acceptable precision can be achieved with fewer point per peak 6 samples/peak width for area; 8 samples/peak width for height Compromises to evaluate Affect of a slower data rate on quant precision Affect of shorter dwell time decrease SIM S/N Affect of faster scan rates decrease scan S/N For many applications... the decrease in precision and S/N are small compared to the benefits of SIM sensitivity and scan library searches Page 58

Literature for Synchronous SIM/Scan (slide 1 of 2) Tech Note 5989-3108EN, Improving Productivity with Synchronous SIM/Scan (Meng, May 05) Tech Note 5989-5669EN, Strategies for Developing Optimal Synchronous SIM-Scan Acquisition Methods AutoSIM/Scan Setup and Rapid SIM (Sept 06) App Note 5989-6066EN, Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975C/DRS GC/MSD System (Quimby, Jan 07) App Note 5989-4184EN, Synchronous SIM/Scan Low-Level PAH Analysis Using the Agilent Technologies 6890/5975 inert GC/MSD (Szelewski, Nov 05) App Note 5989-3571EN, Determination of Trace Levels of Aromatic and Chlorinated Hydrocarbons in Water Using the G1888 Headspace Sample/6890N and 5975 inert MSD (Firor, Oct 05) Page 59

Literature for Synchronous SIM/Scan (slide 2 of 2) App Note 5989-3196EN, The Determination of Residual Solvents in Pharmaceuticals Using the Agilent G1888 Headspace/6890N GC/5975 inert MSD system (Firor/Gudat, June 05) App Note 5989-5494EN, The Determination of Extractables and Leachables in Pharmaceutical Packaging Material Using Headspace GC/MS (Gudat/Firor, Aug 06) App Note 5989-4834EN, Screening for Hazardous Chemicals in Homeland Security and Environmental Samples Using a GC/MS/ECD/FPD with a 731 Compound DRS Database (Quimby/Szelewski, Feb 06) App Note 5989-3299EN, Identifying Pesticides with Full Scan, SIM, uecd, and FPD from a Single Injection (Meng/Quimby, July 05) Tech Note 5988-4188EN (AutoSIM), New Approaches to the Development of GC/MS Selected Ion Monitoring Acquisition and Quantitation Methods (Prest/Peterson, Nov 01) Page 60

Productivity Method Translator More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, MDL, linearity, consistent sensitivity Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 61

What is Method Translation? MTL is a freeware program that determines new method conditions such that relative elution order is constant, and relative run time is known. MTL works: g when changing column outlet pressure (e.g., from FID to MSD) g when changing carrier gas type (e.g., from He to H 2 ) or flow rate g when changing column dimensions (same phase, different diameter, length, or phase ratio) Page 62

Method Translator Screen capture showing the Method Translation (MTL) Software data entry used in a 4X speed gain translation. Page 63

Method Translations to 100 um Column Column id. Speed Gain Length (m) I.D. (µm) D.F. (µm) Inlet P (psig) Outlet P (psia) Flow (ml/min) Init Temp ( C) Init Hold (min) Ramp 1 ( C/min) Final Temp1 ( C) Final Time 1 (min) Ramp 2 ( C/min) Final Temp 2 ( C) Final Time 2 (min) Ramp 3 ( C/min) Final Temp 3 ( C) Final Time 3 (min) 250 µm (X1) 30 250 0.250 18.0 0 1.9 70 2.00 25.0 150 0.00 3.0 200 0.00 8.0 280 10.00 100 µm (X2) 10 100 0.100 36.55 0 0.4 70 1.00 50.0 150 0.00 6.0 200 0.00 16.0 280 5.00 100 µm (X3) 10 100 0.100 63.17 0 0.8 70 0.67 75.0 150 0.00 9.0 200 0.00 24.0 280 3.30 100 µm (X4) 10 100 0.100 90.00 0 1.5 70 0.5 100 150 0.00 12.0 200 0.00 32.0 280 2.50 Page 64

Method Translations to 15 m Column Column Length (m) Speed Gain Part Number I.D. (µm) D.F. (µm) Inlet P (psig) Outlet P (psia) Flow (ml/min) Init Temp ( C) Init Hold (min) Ramp 1 ( C/min) Final Temp1 ( C) Final Time 1 (min) Ramp 2 ( C/min) Final Temp 2 ( C) Final Time 2 (min) Ramp 3 ( C/min) Final Temp 3 ( C) Final Time 3 (min) 30 m (X1) 19091S-433 250 0.25 18.0 0 1.9 70 2.00 25.0 150 0.00 3.0 200 0.00 8.0 280 10.00 15 m (X2.5) 19091S-431 250 0.25 5.74 0 1.49 70 0.80 62.5 150 0.00 7.5 200 0.00 20 280 4.00 15 m (X4) 19091S-431 250 0.25 18.0 0 3.8 70 0.50 100 150 0.00 12 200 0.00 32 280 2.5 Page 65

Productivity Triple Axis Detector with Gain Normalization and EM Saver More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, MDL, linearity, consistent sensitivity Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 66

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 67

Triple-Axis Detector With a combination of three axes and appropriate shielding, noise from metastable helium, photons, etc. can be reduced to a minimum Offset Z This aperture is larger than the off-axis detector which means more ions and energetic neutrals pass to the detector Y ion beam X shield blocking secondary ions formed by metastable helium after the analyzer Page 68

Triple-Axis Detector: Higher Signal Lower MDL Dashed line show 95% confidence limits Increased slope = higher sensitivity Lower detection limits! Tech Overview 5989-7655EN fg Page 69

Triple Channel EM Exit of the triple channels Triple channels improve signal amplification Lower initial EM voltage required Triple channels increase life Lower cost of operation Collector removed to show exit passages Collector Page 70

Tuning Optimization of Multiple Components Tuning ion transmission characteristics Optimize potentials on lens and analyzer Effects the relative abundance of ions WITHIN a spectrum For example, the intensity ratio of m/z 100 to 200 or 100 to 300 Effects the absolute number of ions entering the EM Improves spectra comparisons between instruments US EPA standards made this a critical issue in many areas of the world Gain Normalization of ion amplification (EM only) Effect the ion count or absolute abundance of each ion Does NOT affect the number of ions entering the EM Changes the amplification of charge passing through the EM Page 71

Gain Normalized EM Adjustment for Sensitivity Ion source Quadrupole High Energy Dynode (10kV) i Electron Multiplier (EM) EMV EMV: 1kV to 3kV I I i GAIN Tuning EM voltage changes sensitivity for all ions -- NOT the relative abundances Amplification of signal now, user-selectable!!! Tech Overview 5989-7654EN Page 72

Sensitivity Limitation of ATUNE + nv During aging, the same EMV setting will not give the same signal! 300000 New detector S ig nal vs E M V Aged detector 250000 Page 73 200000 Sig n al 150000 100000 50000 0 1000 1200 1400 1600 1800 2000 2200 2400 ATUNE+400V ATUNE + 400V ATUNE (new) ATUNE (aged) Tech Overview 5989-7654EN Decreasing signal with age As signal decreases, it appears the MS has lost sensitivity In reality, the MS was OK, but the EMV was too low + 600V EM V

Sensitivity Advantage of Gain Normalization During aging, the same GAIN setting will give the same signal! S ig n al v s G AIN 40000 35000 Aged 30000 25000 New Signal 20000 15000 10000 5000 With Gain Normalization, the operation does not have to think about aging, sensitivity is the same for a new or aged EM at any Gain setting simple! 0 0 2 4 6 8 10 12 14 16 18 20 G ain Factor as G A IN x 10^ 5 Tech Overview 5989-7654EN Page 74

ATune + nv versus Gain Normalized Methods New Aged 1000 GAIN Tune ATUNE+ n V 1000 New triple channel EM ages more slowly GAIN Tune ATUNE+ n V 9.80 9.90 10.00 10.10 10.20 10.30 9.80 9.90 10.00 10.10 10.20 10.30 Consistent sensitivity over the life of the electron multiplier! Consistent sensitivity from MSD to MSD and lab to lab! Tech Overview 5989-7654EN Page 75

Benefits of Gain Normalization (EM Optimization) New Option: GAIN Normalized methods Gain Factors are set & saved as a method parameter! Finer Gain Adjustment for better optimization Greater reproducibility over time Greater consistency -for any instrument, or -between a group of instruments No linearity errors with new EM Easier tuning & system troubleshooting Six excellent reasons to use Gain Factors!! Page 76

Literature for Gain Normalization Technical Overview 5989-7654EN, Enhancements to Gain Normalized Instrument Tuning: Understanding the Benefits and Features Technical Overview 5989-7655EN, The Triple-Axis Detector: Attributes and Operating Advice Technical Overview 5989-6050EN, The 5975C Series MSD: Normalized Instrument Tuning Page 77

EM Saver When too many ions are striking the EM, the response saturates and the overload can damage the EM EM Saver and fast electronics quickly alters the analyzer voltages to decrease ion transmission A simple concept with real benefits to EM life Highly concentrated peaks cannot damage the Agilent EM Page 78

Productivity Hydrogen Carrier Gas More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 79

Helium Shortage Means Higher Operating Costs www.cen-online.org Pg 31 October 8, 2007 Page 80

H 2 Requires Different Setup and Extra Care Lower column head pressure experienced with H 2 H 2 is upwards of 100% faster than with helium; the column internal diameter may need to decrease to have optimal pressure control There is a downloadable Method Translation Software to guide the customer H 2 is reactive towards some compounds. This can be an advantage or a disadvantage depending on the application Sometimes very reactive compounds can be measured as their hydrogenated counterparts Diffusion is much faster in H 2 The van Deemter curve will shift and the HETP (height equivalent of a theoretical plate; column efficiency) will be different compared to helium GC resolution can be improved under optimal conditions H 2 is explosive Auto-ignition temperature is 400ºC and the LEL/UEL are very wide Special cautions may be required for safety Hydrogen generators can greatly reduce safety concerns Page 81

Agilent Tests for Hydrogen Safety Page 82

Hydrogen Generators Make the Cost Very Low and Ensure High Quality Gas Page 83

Literature for Hydrogen Carrier Gas Manual G3170-90010, 5975C MSD Hydrogen Safety (Oct 05) Technical Overview 5988-4971EN, A Complete Solution for Chlorinated Pesticides and Herbicides Using DB-35ms and DB- XLB Columns (hydrogen and helium comparison) (Dec 01) Page 84

Productivity Deconvolution Reporting Software More analyses per unit time... Faster and more efficient separations Shorter analysis cycle time (time from injection to injection) Reduced requirement for sample preparation with consistently better results... Quantitative: accuracy, precision, detection limits, linear range Qualitative: response ratios, confirmatory retention times, library searches (eliminate false negatives and false positives) while using less operator time for each analysis. Less time performing maintenance Extensive computer control and data processing Simplified operator review/approval Page 85

What is Deconvolution Reporting Software (DRS)? A second opportunity for accurate result without another analysis A post-run calculation based on the NIST algorithm A solution to the unpredictable matrix interferences DRS makes optimal use of both GC and MS information Combines peak profiles and spectra Extract pure component information from the coeluting peaks Uses RTL database spectra to filter analytes of interest from vast number of matrix peaks Confirms against complete NIST library spectra ChemStation QEdit summary of standard and deconvoluted data Page 86

How Does AMDIS Work? Uses Difference in Spectra, Retention Time and Peak Shape Peak detected with Trace Ion Detection TIC TIC & Spectrum Component 1 Component 2 Deconvoluted peaks and spectra Mathematical Separation matrix Component 3 interference Deconvolution Some m/z values pure Some m/z values mixed target m/z Page 87

Pulling a Useable Spectrum Out of a Mess Analyte ions are buried in the matrix ions!! Cleaned analyte spectrum is easy to match! Page 88

What is the Agilent DRS? ChemStation for Quant + AMDIS 32 Deconvolute and ID + CONSOLIDATED QUANTITATIVE and QUALITATIVE HTML REPORT NIST Confirm Page 89

Reduced Errors and Less Faster Results Summary of Pesticide Analysis in 17 Surface Water Samples Targets Found False Positives Processing Time *CDFA 37 1 ~ 8 hours Agilent DRS Same 37 + 99 additional 0 32 minutes *CDFA is the California Department of Food and Agriculture Data files courtesy of Dr. Mark Lee and Steve Siegel Save about 7.5 hours to do other jobs Page 90

QEdit Summary of Qual and Quant (DRS A.04) 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 91

Summary Report with MSD and AMDIS Results partial report Page 92

Graphics Report MSD and AMDIS 5- ion overlay Raw, deconvoluted and library spectra MSD and AMDIS areas and amounts Page 93

Supporting Literature for DRS (slide 1 of 3) App Note 5989-8582EN, Improved Forensic Toxicology Screening Using a GC/MS/NPD System with a 725-Compound DRS Database (Quimby, May08) Capillary Flow Technology for solvent vent and NPD Backflush to shorten analysis time and increase column lifetime Synchronous SIM/scan (max sensitivity and max information) Trace Ion Detection to reduce noise before DRS Next generation of DRS with AMDIS results in QEdit and Agilent database Real blood extracts from NMS Lab, Willow Grove, PA, USA App Note 5989-7670EN, 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 multiple detectors (3-way splitter) 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 and Agilent database Real results from US Food and Drug Administration/Center for Food Safety and Applied Nutrition (FDA/CFSAN) Page 94

Supporting Literature for DRS (slide 2 of 3) App Note 5989-7436EN, 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 5989-6677EN, Reducing Analysis Time Using GC/MSD and Deconvolution Reporting Software (May07) App Note 5989-6066EN, Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975/DRS GC/MSD System (Quimby, Jan07) App Note 5989-5435EN, Screening for 171 Volatile Organic Air Pollutants Using GC/MS with Deconvolution Reporting Software and a New Indoor Air Toxics Library (Wylie, Aug06) App Note 5989-5076EN, Screening for 926 Pesticides and Endocrine Disruptors by GC/Ms with Deconvolution Reporting Software and a New Pesticide Library (Wylie, Apr06) Page 95

Supporting Literature for DRS (slide 3 of 3) App Note 5989-4834EN, 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 5989-1716EN, New Tools for Rapid Pesticide Analysis in High Matrix Samples, (Szelewski, Quimby, Oct04) App Note 5989-1654EN, A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit Extracts Using Deconvolution Reporting Software App Note 5989-1157EN, Comprehensive Pesticide Screening by GC/MSD using Deconvolution Reporting Software (Wylie/Szelewski/Meng, May04) Page 96

RTL is more flexible now GC/MSD user can manually edit these times Page 97

Largest Selection of Industry Specific Retention Time Locked Databases Product No. RTL DBL Description Compounds G1671AA Hazardous Chemical 730 G1672AA Pesticide 926 G1673AA Indoor Air Toxics 171 G1674AA Forensic Toxicology 723 G1675AA JPN Positive List Pesticide 431 G1676AA Fiehn Metabolomics 665 G1677AA Environmental Semi-voas 273 Library include GC method details, Getting Started manual, application notes, and HELP files Free Volatile Organic Compounds 65 Free PCB Congeners 209 Free Forensic Toxicology 277 Free Fatty Acid Methyl Ester 37 Free Flavors 409 Free Organotin Derivatives Methyl, Ethyl, Pentyl modify a library to meet your need or create your own Page 98

Supporting Literature for RTL (slide 1 of 4) App Note 5989-7875EN, Semivolatiles Retention Time Locked (RTL) Deconvolution Databases for Agilent GC/MSD Systems (Szelewski, Feb 08) G1677AA App Note 5989-7670EN, Replacing Multiple 50-Minute FPD/ELCD/SIM Analyses with One 15-Minute Full-Scan Analysis for 10x Productivity Gain (Meng/Szelewski, Nov07) G1672AA App Note 5989-7436EN, 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) G1675AA App Note 5989-6677EN, Reducing Analysis Time Using GC/MSD and Deconvolution Reporting Software (May07) App Note 5989-6066EN, Rapid Forensic Toxicology Screening Using an Agilent 7890A/NPD/5975/DRS GC/MSD System (Quimby, Jan07) free DBL Page 99

Supporting Literature for RTL (slide 2 of 4) App Note 5989-5435EN, Screening for 171 Volatile Organic Air Pollutants Using GC/MS with Deconvolution Reporting Software and a New Indoor Air Toxics Library (Wylie, Aug06) G1673AA App Note 5989-5076EN, Screening for 926 Pesticides and Endocrine Disruptors by GC/Ms with Deconvolution Reporting Software and a New Pesticide Library (Wylie, Apr06) G1672AA App Note 5989-4834EN, 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) G1671AA App Note 5989-3347EN, Developing an emethod for the Analysis of Volatile Organic Compounds in Water Using Purge and Trap/GC with Agilent s New 5973 inert Mass Spectrometer (Wylie, Jul 05) App Note 5989-2850EN, Determination of Polybrominated Diphenyl Ethers in Polymeric Materials Using the 6890 GC/5973N inert MSD with Electron Impact Ionization (Tu/Prest, Apr05) Page 100

Supporting Literature for RTL (slide 3 of 4) App Note 5989-1716EN, New Tools for Rapid Pesticide Analysis in High Matrix Samples, (Szelewski/Quimby, Oct04) App Note 5989-1654EN, A Blind Study of Pesticide Residues in Spiked and Unspiked Fruit Extracts Using Deconvolution Reporting Software (Sandy, Oct04) Tech Overview 5989-0916EN, Building and Editing RTL Screener/Quant Databases and Libraries (Szelewski/Weiner/Meng, June04) App Note 5989-1157EN, Comprehensive Pesticide Screening by GC/MSD using Deconvolution Reporting Software (Wylie/Szelewski/Meng, May04) App Note 5968-5871EN, Improving the Analysis of Fatty Acid Methyl Esters Using Retention Time Locked Methods and Retention Time Databases (David/Sandra/Wylie, Sept 03) App Note 5988-9256EN, Improving the Analysis of Organtin Compounds Using Retention Time Locked Methods and Retention Time Databases (David/Sandra/Wylie, Apr03) Page 101

Supporting Literature for RTL (slide 4 of 4) App Note 5988-7150EN, Solid-phase Extraction and Retention Time Locked GC/MS Analysis of Selected Polycyclic Aromatic Hydrocarbons (PAHs) (Prest, July 02) App Note 5988-3934EN, Retention Time Locked GC-MS Analysis of Phenols (Reese/Prest, Apr02) App Note 5988-4392EN, Identification and Quantitation of Pesticides in the Partper-Trillion Range Using Retention Time Locking and GC/MS (Meng, Nov 01) Tech Note 5988-4188EN (AutoSIM), New Approaches to the Development of GC/MS Selected Ion Monitoring Acquisition and Quantitation Methods (Prest/Peterson, Nov 01) App Note 5988-2244EN, A New Approach to the Analysis of Phthalate Esters by GC/MS (George/Prest, Mar 01) App Note 5988-9455EN, Ambient Headspace GC and GC-MSD Analysis of Non- Polar Volatiles in Water (Szelewski/Quimby, Feb 00) App Note 5968-8657E, Retention Time Locking: Creating Custom Retention Time Locked Screener Libraries (Weiner/Prest, Dec 99 ) Page 102