Why Inertness Matters in
Gas Phase Analysis
Ken Lynam
Application Chemist, Agilent Technologies
1
What we will cover today
•
•
•
•
•
•
•
•
2
Inertness, what does it mean?
Where is it most important
Flow path diagram, activity focal areas
Five tips to optimize inertness
Inertness testing and it’s significance
Real life examples showing the importance
of inertness
Essential tools for your inert flow path
Brief summary-take home message
Some Basics
Inertness, what does it mean?
•
•
•
•
Lack of peak tailing
Lack of active compound adsorption
Consistent results for trace level active analytes
Better data integrity
How did this effort get started?
• Customer focused innovation
• Manufacturing process development
• Testing procedures
3
Where is inertness most important?
Trace level analysis of active analytes
Advanced GC techniques
•
•
•
•
•
GC/MS
GC/MS/MS
GC/MS TOF
GC/MS QTOF
GC x GC
Samples in difficult matrices
Critical samples
4
GC Flow Path Diagram
5
Tip Number 1
Maintain the inlet
Preventative maintenance helps ensure peak instrument performance
and productivity. Inspect and replace worn or dirty flow path supplies such as syringe needle, septa, ferrules, and inlet seals - to eliminate
leaks and minimize downtime. Using certified vials, caps, septa, ferrules,
and gold inlet seals also extends the flow path maintenance interval.
6
Tip Number 2
Prevent sample loss at injection
Inlet liners are a critical link in the sample flow path, and can be a
source of activity and analyte loss. Liner design and chemistry impact
the transfer of compounds into the column, so you should always use
a reliably deactivated liner suited to your injection technique. Change
the liner when there is visible discoloration indicating non-volatile
residue buildup from samples. This can be challenging to detect; so
when in doubt, change the liner. This will maximize sample transfer
and minimize sample loss.
7
Tip Number 3
Select a column with optimized inertness
Optimized column inertness minimizes compound loss and degradation
for more accurate quantitation of active analytes, especially at trace
levels. To ensure consistent column inertness, choose a column that
has been tested with a rigorous test probe mixture for in-depth
evaluation and certification of inertness. When installing the column,
start with high quality ferrules and examine column ends for chips or
burrs under magnification. Make sure the column is positioned the
recommended depth into the inlet and detector.
8
Tip Number 4
Remember your detector
To ensure accurate quantification and high sensitivity, the flow path
must be highly inert, including detector surfaces. This is especially true
of mass spectrometers, where an inert ion source is necessary to
prevent active compounds from attaching to metal surfaces. The best
inert sources are constructed of a solid inert material, as opposed to an
inert coating which can wear away over time.
9
Tip Number 5
Use gas purifiers
A clean, high quality gas supply that is free of oxygen and contaminants
reduces the risk of column damage, sensitivity loss, and downtime,
improving performance and increasing productivity.
10
Grob-type Test Mix Results Competitor’s Premium 5ms
2
pA
not probative
1. 1-Octanol
35
6
1
30
3
4
2. n-Undecane
5
3. 2,6-Dimethylphenol
4. 2,6-Dimethylaniline
25
5. n-Dodecane
8
20
6. Naphthalene
7
15
7. 1-Decanol
9
8. n-Tridecane
10
9. Methyl decanoate
5
0
11
1
2
3
4
5
6
7
8
9
min
Sampler:
Agilent 7683B, 5 µL syringe (Agilent part # 5181-1273), 1.0 µL split injection, 4 ng each component
Carrier:
Hydrogen constant pressure 37 cm/s
Inlet:
Split/splitless; 250 ºC, 1.4 ml/min. column flow, split flow 140 ml/min.
Liner:
Deactivated single taper w glass wool (Agilent part # 5183-4647)
Oven:
120 ºC isothermal
Detection:
FID at 325 ºC, 450 ml/min. air, 40 ml/min. hydrogen, 45 ml/min. nitrogen makeup
Weak Probes
versus
Strong Probes
1-Propionic acid
2,6-Dimethylphenol
2,6-Dimethylaniline
12
4-Picoline
QC Testing for Today’s Demanding
Applications
Carefully selected probes designed to test inertness effectively
Probe
(ng on column)
1. 1-Propionic acid
2. 1-Octene
3. n-Octane
4. 4-Picoline
5. n-Nonane
6. Trimethyl phosphate
7. 1,2-Pentanediol
8. n-Propylbenzene
9. 1-Heptanol
10. 3-Octanone
11. n-Decane
1.0
0.5
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Column functional test
Basicity
Polarity
Hydrocarbon marker
Acidity
Hydrocarbon marker
Acidity
Silanol
Hydrocarbon marker
Silanol
Polarity
Hydrocarbon marker
5989-8665EN
13
UI Mix Results on a Competitor’s “Premium” 5ms Column
5
pA
20
2
fully probative
3
4
18
8
16
1
11
14
9
12
10
10
7
8
1.
1-Propionic acid
2.
1-Octene
3.
n-Octane
4
4-Picoline
5.
n-Nonane
6.
Trimethyl phosphate
7.
1,2-Pentanediol
8.
n-Propylbenzene
9.
1-Heptanol
10. 3-Octanone
6
11. n-Decane
6
0
2
4
6
8
Sampler:
Agilent 7683B, 0.5 µL syringe (Agilent part # 5188-5246), 0.02 µL split injection
Carrier:
Hydrogen constant pressure, 38 cm/s
Inlet:
Split/splitless; 250 ºC, 1.4 ml/min. column flow, split flow 900 ml/min., gas saver flow 75 ml/min. on
at 2.0 min.
Liner:
Deactivated single taper w glass wool (Agilent part # 5183-4647)
Oven:
65 ºC isothermal
Detection:
FID at 325 ºC, 450 ml/min. air, 40 ml/min. hydrogen, 45 ml/min., nitrogen makeup
14
10
min
UI Mix Results on an Agilent J&W DB-5ms Ultra Inert
pA
fully probative
2
3
1
18
16
1.
1-Propionic acid
2.
1-Octene
3.
n-Octane
4
4-Picoline
5.
n-Nonane
6.
Trimethyl phosphate
7.
1,2-Pentanediol
8.
n-Propylbenzene
9.
1-Heptanol
4
14
11
5
6
7
8
12
10
9
10
10. 3-Octanone
11. n-Decane
8
6
0
2
4
6
8
10
Sampler:
Agilent 7683B, 0.5 µL syringe (Agilent part # 5188-5246), 0.02 µL split injection
Carrier:
Hydrogen constant pressure, 38 cm/s
Inlet:
Split/splitless; 250 ºC, 1.4 ml/min. column flow, split flow 900 ml/min., gas saver flow 75 ml/min. on
at 2.0 min.
Liner:
Deactivated single taper w glass wool (Agilent part # 5183-4647)
Oven:
65 ºC isothermal
Detection:
FID at 325 ºC, 450 ml/min. air, 40 ml/min. hydrogen, 45 ml/min., nitrogen makeup
15
min
Agilent J&W DB-UI 8270D testing conditions and
probe significance
• Ultra low volume solvent test mix eliminates solvent masking of activity
• Low concentration test probe mixture (~5-10 ng on column)
• 45°C low test temp more stringent test for inertness by allowing us to test small molecule (stronger) acid probe, smaller molecule
(stronger) basic probe and smaller molecule (more demanding) primary alcohol plus a demanding –diol to measure silanol activity.
• m & p- xylene are surrogate representatives of aromatic isomers and optimized phase selectivity (RI measurement) for getting
highest resolution (resolution measurement) for semi-volatile aromatic isomers.
• Halogenated aromatic to measure for reproducible selectivity of the column for similar semivolatiles found in typical analyses.
Propanoic acid
Pyridine
Selectivity and resolution of
m&p xylene
1-Chloro-2fuorobenzene
16
Resolution of benzo-b & k fluoranthene isomers
Abundance
Positional isomers
Benzo-k-fluoranthene
1500000
Benzo-b-fluoranthene
1000000
Resolution of 1.2
500000
100000
0
19.00
19.50
Time-->
Column:
Liner:
Inlet:
Carrier:
Septum purge flow:
Oven program:
GC/MSD:
Sampler:
17
Agilent DB-UI 8270D, 30 m x 0.25 mm, 0.25 µm (p/n 122-9732)
Dual taper direct connect liner (p/n G1544-80700)
MMI in non-pulsed splitless mode 1 µL at 275 oC
He, 1.2 mL/min, constant flow
3 mL/min, purge time on 0.7 min 50 mL/min
30 oC (1.0 min), 15 oC/min to 100 oC, 20 oC/min to 240 oC (0.5 min),
15 oC/min to 325 oC (6.7 min) Gas saver Off
Agilent 7890/5975C, 325 oC transfer line, 280 oC source,
150 oC quad, 35-500 AMU range
Agilent 7693, 10.0 µL syringe (p/n G4513-80216)
Ultra Inert Forensics and Food Examples using
the Agilent J&W DB-35ms Ultra Inert GC columns
Drugs of Abuse
–
Key opiate derivative separation
Challenging Pesticide analyses
18
–
Organo-phosphorus pesticides
–
Analysis in a red snapper fish tissue matrix
Agilent J&W DB-35ms Ultra Inert Fast
Toxicology Analyzer Checkout
9
10
8
4
12
11
7
1
6
17
16 18 21
15
14
22
19
25 26
2
3
20
5
23
27
24
28
13
1
2
3
1 Amphetamine
15 Codeine
2 Phentermine
16 Lorazepam
3 Methamphetamine
17 Diazepam
4 Nicotine (highlighted area)
18 Hydrocodone
5 Methylenedioxyamphetamine(MDA)
19 Oxycodone
4
5
6
7
Instrument Conditions
Carrier:
Inlet:
Helium fixed pressure 35.0 PSI
splitless 1 µl 280 °C, total flow 56.4 ml/min, 3 ml/min switched septum
purge, gas saver off, 50 ml/min after 0.4 minutes
Agilent GC/MS Toxicology Checkout Mixture (Agilent p/n 5190-04710
dual taper deactivated (Agilent p/n 5181-3315)
Agilent J&W DB-35msUI 15m x 0.25mm x 0.25µm (Agilent p/n 12235 PSI constant pressure mode
post run: 1 min. 1 psi inlet, 75 psi aux EPC
100 °C (0.25 min) to 345 °C (40 °C/min, 2.25 min hold)
transfer line 300 °C, source 300 °C, quadrapole 180 °C scan mode
Blos bead 300 °C H2 3 ml/min, 60 ml/min air, 11 ml/min makeup + col
12 SKF-525a (RTL Compound)
Sample:
Inlet Liner:
20 Temazepam
Column:
21 Diacetylmorphine
3812UI)
22 Flunitrazepam
Back-flush:
Oven:
23 Nitrazepam
MSD:
24 Clonazepam
NPD:
25 Alprazolam
flow
26 Verapamil
CFT Device: 2-Way splitter with solvent venting between MSD and NPD
13 Oxazepam
27 Strychnine
14 Tetrahydrocannabinol
28 Trazodone
6 Methylenedioxymethamphetamine(MDMA)
7 Methylenedioxyethylamphetamine
8 Meperidine
9 Phencyclidine
10 Methadone
11 Cocaine
19
8
Application note 5990-6577EN
Oxymorphone (OMOR) and Oxycodone (OCOD)
derivatives unresolved on 5 % Phenyl Column
474 OCOD
533 OMOR
465 d6 OCOD
462 d3 OMOR
17.62
17.64
17.66
17.68
17.70
17.72
17.74
17.76
17.78
17.80
17.82
17.84
17.86
Data courtesy of Christine Giffin of the Delaware Office of the Chief Medical Examiner
20
Oxymorphone (OMOR) and Oxycodone (OCOD)
derivatives resolved on DB-35msUI
536 d3 OMOR
533 OMOR
459 OCOD common ion
462 d3 OMOR
459 OMOR common ion
18.10
21
18.20
18.30
18.40
18.50
18.60
18.70
18.80
18.90
19.00
19.10
19.20
19.30
19.40
Separation of Pesticide Analyzer Checkout Solution
with an Agilent J&W DB-35msUI Column
Abundance
340000
320000
300000
280000
260000
240000
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Dichlorvos
Mevinphos
Ethafluralin
Trifluralin
Atrazine
Lindane
Heptachlor
Chlorpyrifos-methyl
Malathion
Chlorpyrifos
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
p,p’-DDE
Dieldrin
Propargite isomers
Hexazinone
Leptophos
Mirex
Fenarimol
Coumaphos
Ethofenprox
Deltamethrin
5 6
220000
11
10
12
7
15
9
180000
160000
19
8
200000
14
17
4
140000
120000
16
3
1
13
2
18
100000
80000
20
60000
40000
20000
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
GC/MS Chromatogram of 1 ng on column loading of pesticides
Time
Sample:
1 µg/mL Pesticide Analyzer Checkout solution (Agilent part
#5190-0468)
GC/MSD: 7890/5975B with purged ultimate union
Column:
DB-35ms UI 20 m 0.18 mm 0.18 µm (Agilent part #1213822UI)
MMInlet: 1µL, splitless, 50°C (0.02 min), 400°C/min to 250°C
purge flow 50mL/min at 1.5 min
gas saver 30mL/min at 2.25 min
Carrier:
Helium, 1.3 mL/min cnst flow
Oven:
50°C (1.3 min) to 135°C (50°C/min),
15°C/min to 200°C, 20°C/min to 310°C (2.35 min)
Restrictor: 0.7m x 0.15mm ID Deactivated capillary column tubing
PCM 1:
3.8 psi constant pressure
Backflush: Post column, Postrun backflush 5 min @310°C
70 psi Backflush Pressure
2 psi Inlet Pressure during Backflush
MSD:
Transfer line 320°C Source 320°C Quad 150°C
Application note 5990-6595EN
22
Separation of 11 Pesticides with an
Agilent J&W DB-35ms UI Column
Abundance
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
200000
180000
1
160000
140000
120000
5
1,3-Dimethyl-2-nitrobenzene
Mevinphos
Terbufos
Pentachlorophenol
Aldrin
Bromacil
Endrin
Carboxin
p,p’-DDT
Bis(2-ethylhexyl)phthalate
Triphenylphosphate
9
3
10
7
100000
11
8
80000
6
2
60000
4
40000
20000
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
Time
GC/MS Chromatogram of 1 ng on column loading of pesticides
Sample:
1µg/mL Custom Standard (Ultra Scientific)
GC/MSD: 7890/5975B with purged ultimate union
Column:
DB-35ms UI 20 m 0.18 mm 0.18 µm (Agilent part #1213822UI)
MMInlet: 1µL, splitless, 50°C (0.02 min), 400°C/min to 250°C
purge flow 50mL/min at 1.5 min
gas saver 30mL/min at 2.25 min
Carrier:
Helium, 1.3 mL/min cnst flow
Oven:
50°C (1.3 min) to 135°C (50°C/min),
15°C/min to 200°C, 20°C/min to 310°C (2.35 min)
Restrictor: 0.7m x 0.15mm ID Deactivated capillary column tubing
PCM 1:
3.8 psi constant pressure
Backflush: Post column, Postrun backflush 5 min @310°C
70 psi Backflush Pressure
2 psi Inlet Pressure during Backflush
MSD:
Transfer line 320°C Source 320°C Quad 150°C
Application note 5990-6595EN
23
GC/MS Chromatogram of Red Snapper fish extracts
Blank Relative Vs. Spiked Sample (26 components)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Abundance
70000
65000
60000
55000
Dichlorvos
Vernolate
Mevinphos
Ethafluralin
Trifluralin
Naled
Prometon
Atrazine
Lindane
β-BHC
Heptachlor
Chlorpyrifos-methyl
Chlorothalonil
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Methyl parathion
Malathion
Chlorpyrifos
Bromacil
p,p’-DDE
Dieldrin
Propargite isomers
Hexazinone
Leptophos
Mirex
Cypermethrin isomers
Fluvalinate isomers
Fenvalerate isomers
18
9
10
23
19
50000
45000
40000
8
11
16
35000
2
30000
25000
1
4
3
20000
7
5
12 15
14
20
21 22
24
17
26
25
13
Spiked Fish Matrix
15000
10000
Fish Matrix Blank
5000
0
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
Time
GC/MS Chromatogram of 0.5 ng on column loading of pesticides in Red Snapper
GC/MSD: 7890/5975B fish
with purged
ultimate union
matrix
Column:
DB-35ms UI 20 m 0.18 mm 0.18 µm (Agilent part
#121-3822UI)
MMInlet: 1µL, splitless, 50°C (0.02 min), 400°C/min to 250°C
purge flow 50mL/min at 1.5 min
gas saver 30mL/min at 2.25 min
Carrier:
Helium, 1.3 mL/min cnst flow
Oven:
50°C (1.3 min) to 135°C (50°C/min),
15°C/min to 200°C, 20°C/min to 310°C (2.5 min)
Restrictor: 0.7m x 0.15mm ID Deactivated capillary column tubing
PCM 1:
3.8 psi constant pressure
Backflush: Post column, Postrun backflush 5 min @310°C
70 psi Backflush Pressure
2 psi Inlet Pressure during Backflush
MSD:
Transfer line 320°C Source 320°C Quad 150°C
Application Note 5990-6595EN
24
Agilent J&W DB-624 UI vs. Competitor 624ms
Organic acid performance at 200 ppm
pA
Octanoic acid
butanoic acid
18
propionic acid
16
14
12
Severe tailing on
competitor’s 624ms
Acetic acid
10
No detection on
Competitor’s 624ms
8
6
4
2
10
12
Column:
Oven:
Carrier :
Inlet:
Inlet liner:
Detector:
25
14
16
18
20
22
24
26
Agilent J&W DB-624UI 30 m x 0.32 mm x 1.8 um vs Rxi-624Sil MS
35C 7.45 min hold, 6.72/min to100C (2.23min hold),10.08 /min to 220C(4.47 min hold),
16.79 /min (4.17 min hold)
Helium 39.6 cm/s (approx. 2.6 mL/min) set at 35C, EPC-Constant Flow
Split, 20:1 at 250C (total flow approx 51 mL/min, and 11.2 psi)
Ultra Inert with wool
FID at 280C, H2 @ 40 mL/min, Air @ 400 mL/min, N2 makeup @ 30 mL/min
min
Agilent J&W DB-624 UI-Organic Acid
Proof of Performance 25 to 200 ppm
pA
Butanoic acid
Acetic acid
Octanoic acid
18
Propionic acid
16
14
12
10
all four acids observed
at each level
8
6
4
10
12
14
Column:
Oven:
Carrier :
Inlet:
Inlet liner:
Detector:
26
16
18
20
22
24
Agilent J&W DB-624UI 30 m x 0.32 mm x 1.8 um (p/n 123-1334UI)
35C 7.45 min hold, 6.72/min to100C(2.23min hold),10.08 /min to 220C(4.47 min hold),
16.79 /min (4.17 min hold)
Helium 39.6 cm/s (approx. 2.6 mL/min) set at 35C, EPC-Constant Flow
Split, 20:1 at 250C (total flow approx 51 mL/min, and 11.2 psi)
Ultra Inert with wool
FID at 280C, H2 @ 40 mL/min, Air @ 400 mL/min, N2 makeup @ 30 mL/min
26
min
Signal to Noise Comparison Residual Solvents
Class 1 Standard at Target Limit
DB-Select 624UI <467>
Vendor R G43
Vendor P G43
Benzene/1,2 dichloroethane
R= 1.82
R= 1.38
R= 1.59
S/N = 6.85
S/N=3.78
Carbon Tetrachloride
27
Carbon Tetrachloride
S/N=2.65
Below 3.0
Carbon Tetrachloride
Acetonitrile (2)/ Dichloromethane (3) Resolution
Residual Solvents Class 2A Standard at Limit
DB-Select 624UI <467>
Vendor R G43
Rs = 2.13
Vendor P G43
Rs = 2.02
Rs = 1.20
3
3
3
2
2
2
3.50
4.5
5.0
28
5.5
4.00
4.50
3.50
4.00
4.50
Pyridine Peak Shape Comparison Residual
Solvents Class 2 B Standard at Target Limit
DB-624UI Select<467>
Vendor R G43
Vendor P G43
USP Tailing = ND
USP Tailing = 2.5
USP Tailing = 1.3
Pyridine
Pyridine
Pyridine
16.00
17.00
29
18.00
19.00
18.00
20.00
22.00
20.00
21.50
23.00
24.50
Robustness Endrin decomposition Test :
5
5
Agilent Ultra Inert single taper liner (p/n 5190-2292)
Peak identification:
1. DDE*
2. Endrin
3. DDD*
4. Endrin aldehyde*
5. DDT
6. Endrin ketone*
2
2
* Breakdown products
3
1
101st injection
1st injection
1
4
6
3
6
5
5
%Breakdown
Competitor’s single taper deactivated liner
Agilent UI liner
2
2
Competitor’s liner
4
101st injection
1st injection
3
DDT
2.5
3.0
1st injection:
1.1
101st injection: 33.8
1.7
2.2
6
3
1
Endrin
1st injection:
1.2
101st injection: 12.2
6
Agilent Ultra Inert deactivation passes Endrin/DDT decomposition test after 100 injections
due to better stability and inertness than competitor’s deactivated liner
30
Semi-Volatiles Suitability - Ultra Inert
Agilent Ultra Inert single taper liner with wool (p/n 5190-2293)
IS 1
IS2
4
Higher responses
1
2
3
5
IS1
IS2
4
2
1
3
Peaks:
1. 2,4-Dinitrophenol
2. 4-Nitrophenol
3. 4,6-Dinitro-2-methylphenol
4. 4-Aminobiphenyl
5. Pentachlorophenol
IS1. Acenaphthene-d10
IS 2. Phenanthrene-d10
5
Competitor’s deactivated gooseneck liner with deactivated wool
Even with glass wool, Agilent Ultra Inert deactivated liners provide high responses
for sensitive semivolatile acidic compounds. Competitor’s deactivated liners show
activity and adsorption
31
Basic Drug Suitability : Ultra Inert
4
3
Agilent UI single taper liner with wool (p/n 5190-2293)
Higher response
1
2
5
6
Better
peak
shape
7
Peaks:
1. Oxycodone
2. Temazepam
3. Flunitrazepam
4. Heroin
5. Nitrazepam
6. Clonazepam
7. Alprazolam
2
1
5
3
4
6
7
Competitor’s deactivated gooseneck liner with deactivated
wool
Drug of abuse are shown on GC/MS SIM chromatograms 5 ng of checkout standards on
column
32
Agilent Ultra Inert Inlet Liners
Get a robust, reproducible, and reliable inert flow path with Agilent Ultra Inert
Inlet liners
•
•
•
•
•
33
Higher sensitivity, accuracy and reproducibility
Exceptional batch-to-batch uniformity
Low to no bleed or background contamination
Exclusive Agilent touchless packaging removes the risk of contamination
Available in economical bulk packs; 5, 25 and 100 packs
Industry Leading Agilent J&W Ultra Inert
GC Columns
• Leading the industry standards for consistent column inertness and
exceptionally low column bleed
• Lower detection limits and more accurate data for difficult analytes
• Tested with demanding Ultra Inert test probe mixtures
• UI Columns available:
DB-1ms UI, HP-1ms UI
DB-5ms UI, HP-5ms UI
DB-35ms UI
DB-624UI, DB-Select 624UI for <467>
DB-UI 8270D
34
Additional productivity enhancing tools
 Free method translation software
 Gas Clean filters
 CFT fittings and devices
 Turn-key analyzer solutions
Purged
Ultimate
Union
35
Take Away Message
•
Be aware of potential active sites in the flow path and how to minimize
their impact
•
Rigorous inertness testing is necessary to assure that flow path
components are inert
•
Use Ultra Inert liners and columns for critical and trace-level applications
•
Ultra Inert liners and columns deliver excellent performance over a range
of applications and challenging analyte sets
•
Stay tuned for future flow path inertness innovations
To learn more please visit www.agilent.com/chem/ultrainert
36
Questions?
37