"Innovations in capillary gas chromatography for improving reproducibility, reliability and ease-of-use in the gas chromatographic separation of complex samples" Jaap de Zeeuw Varian, Inc.. Middelburg, The Netherlands
Challenges in the field of fragrance analysis Types of samples in fragrance analysis tend to be more complex and deal with a variety of component types; Many methods use retention index for identification.. Looking for lower level of impurities Analysis must become faster (cost per analysis) Need for better reproducibility Reduced investment in knowledge (black box) GC-Systems, software and columns must become more users friendly
Areas of innovation to look at Improvement of: Area to optimize Reproducibility Column manufacture & testing Reliability Stationary phase technology Ease of use Design of capillary columns
Why reproducibility? Predictability in GC analysis When replacing a capillary column, the NEW capillary column must: produce the similar retention times (phase-ratio) separate the components of interest (plate number) must show similar peak elution sequence (selectivity) acceptable peak asymmetry and response have low background and acceptable life time
Types of stationary phases used Non polar 5% phenyl / 95% methyl PDMS or arylene equivalent Good for general screening usually higher activity for polar compounds can be prepared with tight specifications Polar PEG type or stabilized equivalent highest selectivity more issues with selectivity
Fragrance/allergenes analysis, non-polar phases Oven : 50 C (1 min), 12 C/min -> 250 C (11 min) Column : 100% PDMS 50m x 0.25 mm x 0.25 um carrier gas : He, 25 psi, 1 ml/min Injection : Split, 1:15, 0.5 ul Detection 3 6 1 4 2 5 7 : MS. Ion trap, Saturn 19 15 16 20 10 11 12 13 8 14 21 17 18 23 22 9 24 25 26 27 28 29 Peak Identification 1 Benzyl alcohol 2 Phenyl acetadehyde 3 Limolene 4 Linalool 5 2,3-dichlorotoluene 6 methyl heptin carbonate 7 methylchavicol 8 citronellol 9 neral 10 unknown 11 cinnamaldehyde 12 hydroxy citronellal 13 methyl octin carbonate 14 cinnamyl alcohol 15 eugenol 16 methyl eugenol 17 coumarine 18 iso-eugenol 19 methyl gamma ionone 20 lilial 21 amyl cinnamyl aldehyde 22 lyral 23 amyl cinnamic alcohol 24 farnesol isomer I + II 25 farmsol isomer III 26 hexyl cannamic aldehyde (trans) 27 benzyl benzoate 28 benzyl salicylate 29 benzyl cinnamate 7 20 [min]
Flavor/Aroma compounds CP-Wax 57 CB 60 C, 2min -> 200 C, 2 C/min
Flavor/aroma compounds CP-Wax 58 (FFAP) CB 50 C -> 250 C, 7 C/min PEG -nitro-terephthalic acid ester Ideal for acidic compounds..
Allergenes Column : CP-Wax 52 CB, 30 x 0.32 mm X 0.2 um Ref: University Eindhoven
Optimise GC column manufacture & testing
Fused silica tubing Drawn at 2100 C Surface characterization Flexibility test Winding on column cages
Capillary Fused Silica Column Production Preform Oven OD - diameter control Primary coatcup
Testing the strength of fused silica in -X and -Y direction Every meter is leaded through the bending-test Eliminates presence of micro cracks
Surface silanol groups dehydration 150 C >800 C Irreversible < 400 C Reversible
Total phase degradation Total Column Bleed = Σ surface Si-OH + phase Si-OH + hydrolization Si-OH surface Si-OH : depend on column length and ID phase Si-OH : depend on total amount of stationary phase hydrolization Si-OH : depend on system & carrier gas For thin films the fused silica surface becomes dominant
Column preparation GC capillary columns are prepared by length.. 10, 15, 25, 30, 50, 60,100 or 150 meters.. Shorter columns cannot be made reproducible by cutting long columns in shorter lengths If you cut a long column in smaller parts there will be higher deviation on selectivity, retention and efficiency For highest reproducibility, columns must be made by a defined length..
Pretreatment of fused silica tubing Fused silica tubing often contains acid HNO3 and HCl are formed during drawing process Flushing with gas removes the acid Note: Flushing of non-deactivated fused silica surfaces can cause microcrack-formation
Fused silica surface characterization Intermediate test decanoic acid-methyl ester C10 25 m fused silica, untreated 130 C ca 0.2 ng on the column 2,6-DMA nonanal 1-octanol Surface = silica = adsorbent Retention factors on the surface reveil silanol interactions
Deactivation of silica surface Chemical process:reaction of Si-OH with reagent Octamethyl-tetrasiloxane (D4); Diphenyl-tetramethyl-disilane(DPTMDS), Hexamethyldisilane(HMDS), Hydrosilane Physical process: Carbowax Tri Ethanol Amine (TEA) covering activity of surface (shielding)
Static coating Vacuum Thermostrated Water-bath End-seal Static coating allows you to know exactly the amount stationary phase brought into the capillary..
Steps after coating the film of stationary phase.. Flushing/drying with high purity gas Polymerization (thermal, ideally 99+ %) Column rinsing (VERY clean solvents) Flushing/drying with high purity gas Conditioning at Tmax for > 10 hours, ultra pure gas Testing
Column testing Critical test mixture Isothermal analysis Efficiency Nth Inertness As Selectivity RI Retention k Stability bleed Permeability dp (PLOT) All these parameters must be specified
Column testing A testmixture is run isothermally 1 decane 2 octanol 3 dimethyl phenol 4 octanoic acid me-ester 5 2,6-dimethyl phenol 6 2,6-dimethyl aniline 7 dodecane 8 decanol 9 tridecane 10 decanoic acid me-ester Permeability Inertness Selectivity Retention Temperature setting is VERY important.. Efficiency
Test laboratory The test temperature must be adjusted very accurately Measurement of temperature in different ovens must be done at the same position in the oven (near the actual position of the test column) See chimneys on each GC.. An absolute necessity to calibrate each oven for absolute temperature using Pt-100 sensor
Retention Index non-polar phases octanoic acid me-ester RI values standard phase (CP-Sil 8 CB, Dimensions: 30 m x 0.25 mm x 0.25 µm RI values using new technologies (VF-5ms, 3500 columns) Deviation in selectivity has been decreased by factor 2
Test for PEG columns 1 2 3 Peak identification 1 5-nonanone 2 1-octanol 3 n-hexadecane 4 n-heptadecane 5 naphtalene 6 n-octadecane 7 2,6-dimethylaniline 4 5 6 7 Problem with PEG columns is that RI measurement is difficult..
Inertness Inertness asymmetrie As = b/a For good inertness measurement: needs to be a crital component (alcohol, amine, acid); component must have some retention (k >1); absolute amount injected < 2 nanograms; Columns: 30 m x 0.25 mm df = 0.25 µm 100 C ; 60 kpa H2 VF-200ms As=1.42 X -200ms As=2.94 Y -200 As=3.59 Asymmetry factor for 1-decanol is the lowest for the VF-200ms
Improve reliability.. Optimise stabilization of Stationary phases
Steps to optimize 1 Reduce Stationary Phase breakdown process by using polymer technology: Stabilizing blocks Ladder technology Purification methods Long polymer chains Surface bonding Non aggressive initiators 2 Optimization of the Fused silica surface using defined conditions for drawing 3 Optimizing the conditioning steps high temperature conditioning using high purity carrier gas
The stationary phase breakdown process Bleed product formation and ions found in MS spectrum Back - eating mechanism Spect 1 BP 207 (2075=100%) Col CP 0049.SMS 207 100% 17.241 min. Scan: 1954 Chan: 1 Ion: 25000 us RIC: 27828 75% 50% 281 341 429 25% 147 489 550 623 0% 100 200 300 400 500 600 m/z
In house synthesis of all stationary phases.. Apply newest stabilization technology High viscosity polymers for low-bleed and stable stationary phases
Stabilization of today s generation of ms phases Name Composition CH 3 VF-1ms 100 % methyl Si O CH 3 100% VF-5ms arylene/methyl modified O CH 3 Si CH 3 CH 3 Si O Si CH 3 CH 3 CH 3 X Y VF-Xms arylene/methyl modified CH 3 CH 3 CH 3 VF-35ms arylene/methyl modified O Si CH 3 O Si Si CH 3 CH 3 Si O 35% X Y VF-17ms arylene/methyl modified O CF 3 CH 2 CH 2 Si O CH 3 Si CH 3 CH 3 VF-200ms trifluoropropyl methyl modified C N (CH 2 ) 3 O Si VF-23ms high cyano modified (CH 2 ) 3 C N X
C Cyanopropyl/phenyl type phases have now also been developed showing improved stability.. O Si N (CH 2 ) 3 O CH 3 Si CH 2 86% Type 1701 Type 624 14%
Future development on column coating technology In-Situ deposition / preparation of phases Sol-gel technology: liquid Adsorbents: porous polymers, silica, carbon
Improving Ease of Use.. Simplify column Installation, operation and storage
Practical concerns with present GC columns Mounting of a pre-column, a retention gap or a transfer line is difficult Column coupling is difficult Column connectors cannot be fixated in the oven Inlet and outlet of column after usage, is difficult to recognize Multiple columns in a oven take a lot of space with risk of breakage The column dimensions and information is difficult to read Installation of column in injector/detector must be simplified
Simplification of Column Installation with EZ-GRIP Both ends are fixed I Aluminium EZ-grip allows writing with a pencil D Injector side Injector and detector sides fully identified Detector side 21
Improving Ease of Use.. Simplify Column coupling..
Problem areas of column coupling Guard columns/transfer lines have to be coupled Challenges: Do the right column cutting To get a leak-free connection To get this in an acceptable time Get the guard column on and off the column cage The connection must stay leak-free while in operation
Popular coupling system Quick-Seal/Press-fit Column Connector
Alternative connectors metal body & ferrule
Can we avoid Column Coupling? Yes.. The guard column and transfer lines can be integrated with the capillary using segment coating...
Segment Coating Segment Coating can be applied away from: The inlet section : Integrated Guard column The outlet section : Integrated Transfer- line There are different techniques to manufacture a segment coated capillary
Segment coated columns Removing stationary phase from existing coated capillary Only for non-bonded phases For CB phases must be done before polymerization Big risk for glogging impossible for thick films Very labour intensive Deposit stationary phase only in that part where we need it Can be done using existing coating technologies Principally for all phases and film thicknesses Relative fast to do Need to have good eyes (..)
Segment coating Static coating Vacuum Water-bath End-seal When coat front has reached this point, residue is pushed out.. Fill column and coating process starts here
Configuration of a segment-coated system Integrated guard or EZ-Guard, typically 5-10 meters Integrated transfer line outlet, approx. 50 cm
Having an (un)coated Inlet The integrated (or EZ-)guard column is realized by not-coating the stationary phase on the first meters of the capillary.. Advantages: There is no coupling involved and column cutting is not critical; No issues with dead volume, thermal capacity and fragility; No hassle in getting the guard column on and off the column cage; Absolute no risk of leakage;
Properties of EZ-Guard columns Low retention for optimal refocusing effect in the analytical column High inertness for good peak shape and recovery Organic solvent resistant Inertness can be an issue..
Comparison of Integrated-Guard Activity Mixture, 4 ng per compound on the column
Comparison of columns Traces Pesticide analysis
Segment coating: uncoated transfer lines That part of the capillary column that is positioned in the transfer line and/or in the detector, will not contribute to the chromatography as it s always at high temperature.. Stationary phase in this part of the capillary can only decompose and produce breakdown products: a potential source for increased background..
Having an (un)coated Outlet The segment coating also allows the possibility to leave the last meters of the column without stationary phase: Integrated transfer line Advantages: There is no coupling involved and column cutting is not critical; Its fast operate; No issues with dead volume, thermal capacity and fragility; Absolute no risk of leakage; Faster stabilization in ms and other detection systems Lower background
Uncoated transfer line Especially important in: Sensitive detection systems (MS, PDD, ECD,..) Higher detector temperatures Oxygen & moisture sensitive phases When applying THICKER films with GC and GC/MS
Impact of detector temperature Oven : 100ºC iso thermally Column : VF-1ms with integrated transfer line Detector : FID, Temperature increased with steps of 30 ºC 360 ºC 330 ºC Without integrated transfer line 300 ºC With integrated transfer line 270 ºC Especially > 300C the impact becomes visible; As velocity at the end of the column is high, stabilization time (conditioning) will be relatively short..
To show the impact of the transfer line visible we tested this also with ms and took a capillary with a thick film: Column : 60 m x 0.32mm VF-5ms, df= 3 µm Detector: Saturn 2000 ion trap One column was WITH - and one column WITHOUT integrated transferline.. Both columns were treated exactly the same way and tested in the same instrumentation..
Effect of uncoated transferline [kcounts] 500 s Column : 60m X 0.32mm VF-5ms, df=3.0um Column flow : 1.5 ml/min, helium Temp Injector: 250 C Detector : Saturn 2000; Trap: 150 C; Manifold: 40 C Transferline : 180 C Temp prog : 50 C(5min) to 250 C(5min), 25 C/min RIC all 2000.40021.sms 400 WITHOUT Integrated transferline Lower offset after temperature ramp 300 200 s R I C a ll 2 0 0 0.4 0 0 1 8. s m s WITH Integrated transferline 100 Lower offset Lower background
Ionization times Ionization time in ion trap ms is a measure for the number of ions present High value few ions (clean) Low value many ions (dirty/overload)
Ionization Times 50 C 25 C/min 250 C 50 C
Stabilization profile Oven Column : 50 ºC 250 ºC, 10 ºC/min : 30 m x 0.32mm VF-1ms, 3.0 um Without integrated transfer line With integrated transfer line Stable in approx. 40 minutes Still stabilizing 0 30 70 140 [min]
Transfer line in MS Transfer lines can be activated by exposure to oxygen and water: Upon maintenance in inlet section (vacuum sucks air through) when injecting sample and introducing an air plug with the sample headspace liquid injection How much stationary phase is present in this small section of the capillary?
Example A capillary column of 30 m x 0.25 mm x 0.25 µm Transfer line for ms = 20 cm long In this transfer line we have approx. 40 µg of stationary phase.. If all this phase is decomposed due to oxydation/hydrolization it will bleed into the detector, resulting in background, drift and contamination.. How long will this process take and what signal can such a small amount of stationary phase produce?
Reference values are available using calibrated FID as this detector is used for bleed measurements.. Decomposing 40 µg PDMS will produce a signal of 1 pa for 1400 hours Decomposing 40 µg PDMS will produce a signal of 10 pa for 140 hours Decomposing 40 µg PDMS will produce a signal of 100 pa for 14 hours The impact of a coated transfer line can therefore be significant Uncoated transfer line is preferred
Factors that potentially can initiate stationary phase breakdown at higher temperature: water oxygen aggressive compounds derivatization agents acidic / basic compounds reactive chemicals
Impact of water in carrier gas on phase degradation Carrier gas is contaminated with 200 ppm water; Column repeatedly programmed from: 50 C (10min), 20 C/min to 325 C(60min). After water exposure it takes a long time before the signal stabilizes.. 4 hours!!
Limitations of segment coating Diameter of uncoated section is the same as the analytical column No different deactivations can be used Upon cutting a part of the EZ-guard column, retention times will change and integration window has to be adjusted
Segment coating technologies will become more important for future generation of optimized GCcolumns: using thicker films transfer lines for direct heating hardware designs making miniaturized & integrated solutions, like wafers
Conclusions New technologies has resulted in more stable stationary phases that also show high reproducibility Reproducible GC columns can only be realized using accurate testing The use of segment coating allows integration of guard columns/transferlines offering many advantages No Leaks (no coupling) Protects analytical column EZ to install
Thank you for your attention