Slide 1: Making a Grand Entrance: Techniques for Efficient Sample Introduction
Making a Grand Entrance: Techniques for Efficient Sample Introduction, Inlet Types, and Maintenance Mark Sinnott – Application Engineer Alexander Ucci - Application Engineer March 19, 2019 1 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 2: Sample Injection Goals
Sample Injection Goals •Introduce sample into the column •Reproducible •Minimize efficiency losses •Representative of sample 2 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 3: Influence of Injection Efficiency
Influence of Injection Efficiency Short concentrated Solute bands Long diffuse Same column, same chromatographic conditions 3 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 4: Inlet Choices
Inlet Choices Sample Sample to Inlet Column Mode Comments Concentration Column Split High Very little Most commonly Purged split High Very little Split/splitless Capillary used inlet. Very Splitless Low All flexible Purged splitless Low All Minimal Cool-on-column Capillary N/A Low or labile discrimi
Slide 5: Split/Splitless Inlet Schematic and Operation Modes
Split/Splitless Inlet Schematic and Operation Modes Modes • Split • Pulsed split (useful for small number of applications) • Splitless • Pulsed splitless 5 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 6: Split Injections – Considerations
Split Injections – Considerations Dirty samples are less problematic (compared to splitless) – backflushing Wide analyte boiling range Solvent properties • Wide boiling point range • Wide polarity range Discrimination can be due to liner or inlet temperature Most efficient sample transfer = nice sha
Slide 7: Split Injections – Inertness
Split Injections – Inertness More inert than splitless • Higher velocity through the inlet • Less exposure to inlet hardware/consumables – Shorter inlet residence time Glass wool is a compromise • Exhibits some activity (even if deactivated) – high surface area • Greatly improves fluidic performance
Slide 8: Split Liners: Recommended Liner
Split Liners: Recommended Liner Split/splitless liner with glass wool, low pressure drop Split injections have higher carrier gas flow through liner • Faster transfer onto column • Split liners have a smaller outer diameter than splitless liners to accommodate high flow to split vent If potential ex
Slide 9: Split flow path
Split flow path Smaller liner od with split Septum purge liners accommodates the Carrier in higher flow to the split vent Split vent 9 Agilent Restricted
Slide 10: What Does Mass Discrimination Look like?
What Does Mass Discrimination Look like? No glass wool C8 C40 10 20 30 10 20 30 C8 Glass wool added to liner C40 10 20 30 10 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 11: Split Injections – Maximizing Sensitivity
Split Injections – Maximizing Sensitivity Increase injection volume • Liner dependent (use the pressure-volume calculator) • 2 μL maximum Reduce split ratio • Go from 50:1 to 10:1 • 10:1 practical lower limit for liquid injections (for 250 – 320 μm id columns) • 1:1 possible for gas injections with
Slide 12: Split Injector: Injection Volumes
Split Injector: Injection Volumes Actual Expected 3 2 2 2 1 1 µL 3 µL 4 4 5 5 1 1 3 3 1 2 3 4 5 6 1 2 3 4 5 6 Time (min) Time (min) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 60 °C for 1 min, 60-180 °C at 20 °C/min; helium at 30 cm/s 1. n-heptane 2. toluene 3. n-decane 4. n-butylbenzene 5. n-tride
Slide 13: Higher flow rates
Minimum Recommended Split Ratio mm id Lowest ratio 0.10 1:50 - 1:75 Higher flow rates 0.18 - 0.25 1:10 - 1:20 0.32 1:8 - 1:15 0.53 1:2 - 1:5 13 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 14: Split Injections – Troubleshooting
Split Injections – Troubleshooting Column pressures <10 psi • The pressure pulse from evaporating solvent can cause discrimination and poor precision Liner residence times <0.5 s (>200 mL/min) • Poor mixing will cause discrimination No glass wool Solvents with high expansion volume Column position –
Slide 15: Split Ratio Comparison
Split Ratio Comparison High Ratio Low Ratio Sample into column Low High Efficiency High Low Discrimination High Low Carrier gas use High Low 15 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 16: Splitless Injection Overview
Splitless Injection Overview •For trace level analysis •Use split/splitless injection port in the splitless mode (split vent temporarily closed) •The dilute sample is injected, the sample is volatilized, and majority of analytes transfer to the column •Later, the split vent is opened and residual so
Slide 17: Splitless Injections – Considerations
Splitless Injections – Considerations Dirty samples can be an issue – better if you have backflushing capabilities Analyte boiling range – wide Early eluters need bp difference versus solvent Solvent properties • Wide boiling point range – But consider BP of earliest eluting analyte • Wide polarity
Slide 18: Splitless Injections – Inertness
Splitless Injections – Inertness Less inert than cool on-column (cool on-column is most inert) • Liner and inlet interaction Less inert than split • Longer residence time in inlet and on glass wool • Used for trace analysis, so there’s a greater chance of analyte loss for actives 18 March 29, 2019 T
Slide 19: Splitless Liners
Splitless Liners Single taper with or without wool Splitless has lower flows through liner • Splitless liners are typically wider for a more snug fit – Ensures all available flow funnels through the liner, not around • You can do split injections with a split liner as long as split ratio is not too
Slide 20: Splitless Injections – Discrimination
Splitless Injections – Discrimination Improper purge time • Short purge times cause loss of late eluters (not enough time to vaporize) • Long purge times cause solvent tail interference with early eluters Improper initial oven temperature (solvent effect – refocusing) • Too high of a temperature pre
Slide 21: Splitless Injections – Splitless Time (Purge Time On)
Splitless Injections – Splitless Time (Purge Time On) Purge time too long results in large solvent tail 0.75 min purge time clips solvent tail 21 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 22: Splitless Injector: Injection Volume
Actual Splitless Injector: Injection Volume Expected 3 2 1 3 µL 1 2 3 1 µL 3 4 2 4 1 2 4 6 8 2 4 6 8 Time (min) Time (min) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 60 °C for 1 min, 60-180 °C at 20 °C/min; helium at 30 cm/s 1. n-decane 2. n-dodecane 3. n-tetradecane 4. n-hexadecane 22 March 29, 2
Slide 23: Splitless Injector: Injector Temperature
Splitless Injector: Injector Temperature 200 °C 250 °C 3 2 3 5 1 2 5 1 4 6 4 6 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Time (min) Time (min) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 50 °C for 0.5 min, 50-325 °C at 20 °C/min; helium at 30 cm/s Phthalates: 1. dimethyl 2. diethyl 3. dibutyl 4. benzyl
Slide 24: Splitless Injector: Sample Refocusing
Splitless Injector: Sample Refocusing Sample refocusing improves efficiency Use low column temperature to refocus solvent (10 – 30 °C less than solvent BP) • Called the solvent effect Use cold trapping 24 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 25: Splitless Sample Refocusing
Splitless Sample Refocusing • Sample refocusing • Also known as the “solvent effect” • Condenses sample as a thin film on the head of the column • Initial oven temperature must be at least 10 °C below the solvent BP – This results in better peak shape – Results in smaller injection band than would o
Slide 26: Splitless Injector: Solvent Effect
Splitless Injector: Solvent Effect 1. Solvent and Initial column temperature at solutes least 10°C below sample solvent boiling point Required to obtain good peak shapes unless cold trapping 2. Solvent film occurs Generally, if solute BP >150 °C above initial column temperature, the solute will 3. c
Slide 27: Splitless Injector
Splitless Injector Initial column temperature Solvent effect Cold trapping Hexane solvent (BP = 68-69 °C) 70 °C 50 °C 2 3 4 3 4 2 1 1 2 4 6 8 2 4 6 8 Time (min) Time (min) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 50 °C or 70 °C for 0.5 min, to 210 °C at 20 °C/min; helium at 30 cm/s 1. n-decane 2
Slide 28: Splitless Injector
Splitless Injector Reverse solvent effect/polarity mismatch 4 Hexane 5 6 Methanol 1 4 2 5 6 2 3 3 1 0 1 2 3 4 5 6 1 2 3 4 5 6 Time (min) Time (min) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 50 °C for 1 min, 50-210 °C at 20 C°/min; helium at 30 cm/s 1. 1,3-DCP 2. 3-hexanol 3. butyl acetate 4. 1-he
Slide 29: Retention Gap
Retention Gap Also called a guard column Detector Injector Deactivated fused silica tubing Column Union Usually 2-10 m long and same diameter as the column (or larger if needed) 29 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 30: Splitless Injector
Splitless Injector 3 m x 0.25 mm id retention gap No retention gap With retention gap 4 Methanol Methanol 4 2 5 6 5 6 2 1 3 3 1 1 2 3 4 5 6 1 2 3 4 5 6 Time (min.) Time (min.) Agilent J&W DB-1, 15 m x 0.25 mm id, 0.25 µm 50 °C for 1 min, 50-210 °C at 20 °C/min; helium at 30 cm/s 1. 1,3-DCP 2. 3-hexa
Slide 31: EPC for Pulsed Splitless Injection
EPC for Pulsed Splitless Injection Pressure pulse contains sample expansion and transfers analytes to the column faster Pulsed splitless • Sample containment more critical than split injection • Sharper peaks than in traditional splitless injection • Two new parameters to set: pulse pressure and pul
Slide 32: Benefits of the Pulsed Splitless Mode
Benefits of the Pulsed Splitless Mode % Recovery of each labile pesticide relative to cool on- column injection 120 110 Methamidophos % Recovery (on-column=100) 100 90 Acephate 80 Azobenzene 70 Omethoate 60 High column flow Diazinon 50 Normal column flow Dimethoate 40 Chlorpyrifos 30 20 70 psi 22.5
Slide 33: Splitless Injections – Starting Parameters
Splitless Injections – Starting Parameters Injection volume = 1 µL (if water 0.5 µL) • Check the pressure-volume calculator Initial oven temp = 10 oC < solvent boiling point Purge flow = 20 to 60 mL/min Purge time = 0.75 min • Sweep with two liner volumes of carrier gas No pulse Try to avoid water a
Slide 34: Splitless Injections – Troubleshooting Tips
Splitless Injections – Troubleshooting Tips Injecting too much • Column overload = poor peak shape • Inlet overload = poor reproducibility (backflash) – Ghost peaks in subsequent blanks are possible No glass wool • Poor mixing • More matrix on column Glass wool • Has the potential to react with trac
Slide 35: Splitless Injections – Troubleshooting Tips
Splitless Injections – Troubleshooting Tips If you think you have an inlet issue related to splitless injections: • Run a 10:1 split injection • Make up a standard at 10x concentration and run a 10:1 split injection What if, when I changed from split to splitless, I don’t see an increase in response
Slide 36: MMI Inlet: Split/Splitless + PTV
MMI Inlet: Split/Splitless + PTV Split vent Carrier in Septum purge vent Hot split/splitless (also pulsed) • Similar to the S/SL inlet using the same liners • All previous S/SL discussions apply here Cold split/splitless • Significantly more inert than hot splitless (for thermally labile) • Can inje
Slide 37: Multi-Mode (MMI) Inlet Features
Multi-Mode (MMI) Inlet Features Temperature range of -160 to 450 oC Heating at 15 oC/s (900 oC/min) Septum/liner easily exchangeable using Turn Top inlet Injection modes: hot S/SL, cold S/SL, pulsed mode, solvent vent mode, residue removal mode Support for single stroke injections from 0.1 to 250 mL
Slide 38: Multimode Inlet Solves Many Problems
Multimode Inlet Solves Many Problems Performing large volume injection (LVI) of relatively clean samples? • Programmable injection slows solvent evaporation and maximizes analyte transfer into the column/detector • Decrease MDL by injecting more sample Injecting dirty samples? • Matrix vent, backflu
Slide 39: MultiMode GC Inlet – Cold Injections
MultiMode GC Inlet – Cold Injections • No syringe-needle discrimination; minimal inlet discrimination • No special syringes, liners, or consumables • Large volume injection (5 to 250 µL) – lower detection limits – Sensitivity is better, but also introduces that much more matrix • Solvent vent/matrix
Slide 40: MMI Column Installation
MMI Column Installation Set to 12-15.5 mm Trim the column Thread the column Tighten the column nut • Graphite ferrules into the column adapter – with a ¼-inch wrench – are recommended stabilize the column adapter continue to hold the over Vespel with a 5/16-inch wrench column adapter with a 5/16-inc
Slide 41: Inlet Degradation and Maintenance
Inlet Degradation and Maintenance 41 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 42: Root Causes of Inlet Performance Degradation, and Consequences
Root Causes of Inlet Performance Degradation, and Consequences Accumulation of sample residues • Loss of response, tailing on active analytes, split vent trap fouling and inaccurate EPC flow control O-ring sealing surface Accumulation of consumables wear particles • Same as accumulation of sample re
Slide 43: Inlet Liner Troubleshooting
Inlet Liner Troubleshooting •Many chromatographic problems are blamed on the column •Often, a dirty liner is the culprit Evidence of a dirty liner: • Poor peak shape • Irregular baselines • Poor resolution • Poor response 43 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restric
Slide 44: Dirty Liners
Dirty Liners Silylated glass wool •Traps nonvolatile materials and mixes sample •Peak shape and discrimination affected by amount, location, and packing density 44 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 45: Liner Maintenance
Liner Maintenance •Liners become contaminated with use, collecting nonvolatiles, salts, excess reagents, and so on, or become damaged/cracked •Should inspect and replace liners often •Handle with gloves and forceps •Insert into or remove liners only from cool injection ports •Replacing with a new li
Slide 46: Leak in Septum
Leak in Septum Using septa beyond lifetime/temperature conditions • Use environments that decrease lifetime include manual injections, wrong syringe tip type, larger gauge syringes, non-Agilent autosamplers (Agilent autosamplers are precisely aligned) • Septum nut too tight • Septum type and syringe
Slide 47: Septum Maintenance: Septum Coring
Septum Maintenance: Septum Coring • After many injections, pieces of rubber from the septum may break off and fall into the inlet liner – This is called septa coring – Replace the inlet septa and liner frequently to prevent septa contamination – Use a cone tipped syringe to reduce the chance of tear
Slide 48: Septum Maintenance: Deconvoluted Inlet Septa Spectrum
Septum Maintenance: Deconvoluted Inlet Septa Spectrum 10 12 14 16 18 20 Decamethyl Dodecamethyl Tetradecamethyl Hexadecamethyl Octadecamethyl Eicosamethyl cyclopentasiloxane cyclohexasiloxane cycloheptasiloxane cyclooctasiloxane cyclononasiloxane cyclodecasiloxane 48 March 29, 2019 Techniques for Ef
Slide 49: Main Siloxane Peak Bandit
Main Siloxane Peak Bandit Multiple injections from same vial can dissolve silicone into the sample 49 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 50: Tips to Maximize Septum Life, Minimize Septum Leaks
Tips to Maximize Septum Life, Minimize Septum Leaks • Use Agilent Gold Standard, 23-26 gauge, HP point taper syringes - Point style cores septa significantly less when used with CenterGuide Septa - Taper minimizes septum coring/wear • Use Agilent CenterGuide Septa HP-point style - Molded hole minimi
Slide 51: Leaks Due to Septum Nut
Leaks Due to Septum Nut • With repeated use, conical needle guide gets worn (out of round) and needs replacement, as septum can begin to “bulge” out, especially with excessive tightening • Septa fail faster because needle is not guided with as much precision • Under or over tightening – tighten nut
Slide 52: Examples of Non-Optimized Operation
Examples of Non-Optimized Operation Typical cause: re-use and misinstallation • Leak from O-ring, gold seal, ferrules, column nuts • O-rings are elastomer compression fittings, designed for one use, not perfectly elastic • Gold seals are designed for one use; knife edge cuts into gold layer giving l
Slide 53: Split Vent Trap
Split Vent Trap What is it? Where is it? 53 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 54: Split Vent Trap Changed (Column Bleed)
Split Vent Trap Changed (Column Bleed) Before After 54 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 55: Example Of Gross Contamination
Example Of Gross Contamination 1.3e4 This is not a normal 1.2e4 column bleed 1.1e4 1.0e4 9000 8000 7000 6000 5000 0 10 20 30 40 50 60 70 Time (min) Agilent J&W DB-624, 30 m megabore Temperature program: 35 °C, hold 1.50 min, 30 °C/min to 65 °C, hold 15 min, 20 °C/min to 260 °C, hold 50 min 55 March
Slide 56: Cleaning the Split/Splitless Injector
Cleaning the Split/Splitless Injector Cotton swab Carrier gas flow off Disconnect split vent line and replace split vent trap GC Off Injector body Remove column, reducing nut, gold seal, washer and liner Ace MeCl2 tone 56 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 57: Same Column After Inlet and Column Maintenance
Same Column After Inlet and Column Maintenance 1.3e4 2.04 1.2e4 1.1e4 1.0e4 7.19 10.50 9000 Normal column bleed 16.69 12.08 8000 19.94 7000 6000 5000 0 10 20 30 Time (min) *Temperature program: 35 °C, hold for 1.50 min; 30 °C/min to 65 °C, hold 15 min; 20 °C/min to 260 °C for 5 min 57 March 29, 2019
Slide 58: Agilent Bond Elut Sample Cleanup Products
Agilent Bond Elut Sample Cleanup Products Filtration cartridges and plates Solid Phase Extraction cartridges and plates Captiva EMR Lipid 58 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 59: Enhanced Matrix Removal: Agilent Captiva EMR-Lipid
Enhanced Matrix Removal: Agilent 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 EMR are trappe
Slide 60: Application Case – Pesticides in Edible Oil by GC/MS/MS
Application Case – Pesticides in Edible Oil by GC/MS/MS Classification Pesticides Classification Pesticides Classification Pesticides Dichlorvos Lindane Dichlorfluanid Sulphamide Trichlorfon Aldrin Tolylfluanid Sulfotep Endrin Captan Organochlorine Endosulfan Diazinon Phthalimide Folpet Chlorpyripho
Slide 61: Pesticides in Edible Oil by GC/MS/MS
Pesticides in Edible Oil by GC/MS/MS Sample preparation procedure visual Olive oil Olive oil extract Captiva Transfer Mix w/ 20% EMR-Lipid LLE x2 water cleanup supernatant Olive oil Supernatant for extract MgSO4 salt GC/MS/MS EMR-Lipid analysis partition eluent 61 March 29, 2019 Techniques for Effic
Slide 62: Captiva EMR-Lipid Cleanup Improves Analytes S/N Ratio and Integration Accuracy
Captiva EMR-Lipid Cleanup 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
Slide 63: A New Portfolio of GC Consumables
A New Portfolio of GC Consumables UI inlet liners Guard Chip Smart keys Intuvo Flow Chips No-trim column Tools and accessories 63 March 29, 2019 Techniques for Efficient Sample Introduction Agilent Restricted
Slide 64: When Do I Change Each Part?
When Do I Change Each Part? Item Typical Schedule Comments Septum nut can get warn and shed metal particle into Septum nut 3-6 months the liner. Replace to minimize activity in the inlet/liner. Check movement of plunger and replace if it does not Syringe Every 3 months move freely and cannot be clea
Slide 65: Conclusions
Conclusions • Start off with good inlet parameters • Develop a maintenance schedule that fits your application and sample load • Don’t skimp out on replacing your inlet consumables • Use the same type of liner for the same type of application • Trim more than 2 in from the front of the column (back
Slide 66: 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 or GC/MS columns and supplies Option 2 for LC or LC/MS columns and supplies Option 3 for Sample preparation, filtration and QuEChERS Option 4 for Spectroscopy supplies Available in the U.S.