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Developing Large Volume Injection (LVI) in Split / Splitless Inlets
Philip J. Koerner Phenomenex Inc.
NEMC 2014
Goal To produce a method that provides the largest response of analytes that will produce the most sensitive method possible (S/N). The most sensitive method will mean that less sample needs to be processed best meeting the initial reduction of sample volume requirements.
Typical Analysis 1.0 µL Splitless 500 g soil S/N = 25
Larger Injection 5.0 µL Splitless 500 g soil S/N = 125
Larger Injection / Smaller Sample 5.0 µL Splitless 100 g soil S/N = 25
Plan
• Getting more analyte on-column will increase response – Liner selection (linearity of injection volumes (backflash
considerations) 0.5 µL to 12.5 µL) • Direct connect liner with top taper (bottom hole) • Direct connect liner (top hole) • Cup liner • Single taper with wool at bottom • Single taper with wool at middle
– Pressure Pulse • Optimize separation
– Guard Column – Column Dimensions
Objective: Get the highest response from the system to obtain the best sensitivity
What to Watch Out For!
• 1.0 µL Dichloromethane @ 250 ºC & 14 psi = 336 µL • Liner Volume ≈ 990 µL • 3.0 µL = 1008 µL; exceeds liner volume!
– Flashback – Ghost peaks – Loss in sensitivity / linearity
Preliminary
Assumption: Previous work has shown that Pressure Pulsed injections have been beneficial to improve analyte responses by aiding the transfer of analytes onto the column. We therefore started with Pressure Pulsed conditions and tested our assumption during our work. Analytes: We also started this process with a relatively simple list of compounds, the EPA list of PAHs. This mixture contains compounds with a wide range of retention times so we can gauge response of early eluting as well as late eluting compounds. Later work would include other compounds for other specific LVI Methods.
Initial Conditions Column: Zebron ZB-SemiVolatiles Column Dimension: 30 m x 0.25 mm ID x 0.25 µm Column Flow: 1.4 mL/min Helium (constant flow) Oven Program: 100 ºC for 0.5 to 260 ºC @ 30 ºC/min to 295 ºC @ 6 ºC/min to 325 ºC @ 25 ºC/min for 2 min Inlet Liner: Direct Connect Liner with top taper and bottom hole Injection: Pressure Pulse @ 30 psi for 0.66 min, splitless for 0.60 min Detector: MSD; 46-450 amu, transfer line = 320 ºC
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
2000000
4000000
6000000
8000000
1e+07
1.2e+07
1.4e+07
1.6e+07
Time-->
Abundance
TIC: APAH3.D
Liner Performance DirectConnect Dual Taper, bottom hole
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Injection Volume (uL)Area
Fluorene
Chrysene
Benzo[g,h,i]perylene
• Maximum Injection volume = 10 µL • Maximum intensity = 1.67e7 • Better response for higher MW • Response decreases after 10 µL!
DirectConnect Top Hole
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1.0E+07
1.2E+07
1.4E+07
1.6E+07
1.8E+07
2.0E+07
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Injection Volume (uL)
Area
Fluorene
Chrysene
Benzo[g,h,i]perylene
• Maximum Injection volume = 12.5 µL+ • Maximum intensity = 1.40e7 • Better response for lower MW
Liner Performance Cup Liner
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
3.0E+07
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Injection Volume (uL)
Area
Fluorene
Chrysene
Benzo[g,h,i]perylene
Single Taper With Wool
0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
2.5E+07
3.0E+07
3.5E+07
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
INjectin Volume (uL)
Area
Fluorene
Chrysene
Benzo[g,h,i]perylene
• Maximum Injection volume = 10 µL • Maximum intensity = 2.07e7 • Consistent response, except at larger
volumes which benefit lower MW • Response decreases after 10 µL!
• Maximum Injection volume = 7.5 µL • Maximum intensity = 2.71e7 • Slightly better response for higher MW • Response decreases after 7.5 µL
Liner Performance
FocusLiner (single taper with wool in middle)
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
7.0E+06
8.0E+06
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Injection Volume
Area
Fluorene
chrysene
Benzo[g,h,i]perylene
• Maximum Injection volume = 2.5 µL • Maximum intensity = 6.23e6 • Much better response for lower MW • Possible cooling of glass wool
Average Liner Performance Average Liner Responses
0.E+00
5.E+06
1.E+07
2.E+07
2.E+07
3.E+07
3.E+07
0 2 4 6 8 10 12 14
Injection Volume
Area
(ave
rage
)
Direct Connect BottomHole dual taper
Direcdt Connect top hole
Cup Liner
Single Taper Wool Botom
Focusliner
Single Taper with Wool at bottom provides highest response at 7.5 µL injection volume
Analytes 1. Pentachlorophenol 2. DFTPP 3. Hexadecanoic acid 4. Octadecanoic acid 5. Benzidine 6. Hexanedioic acid, bis(2-ethylhexyl)ester 7. DDT
Relative Responses 1-taper Wool-middle Wool-bottom straight Cup Wool-bottom 2 PCP 0.44 0.70 0.60 0.43 0.80 0.32
Benzidine 0.87 1.17 1.11 0.90 1.22 1.34 DDT 1.23 1.24 1.19 0.99 1.17 1.59
Liner Activity
7.00 7.50 8.00 8.50 9.00 9.50 10.00
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
Time-->
Abundance
TIC: AG8499_2.D
7.00 7.50 8.00 8.50 9.00 9.50 10.00
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
Time-->
Abundance
TIC: AG4657_2.D
Normal Liner
Competing Liner
1
2
3 4
5
6
7
1
2 5 7
Inlet Temperature = 275 ºC
Injection Temperature
Inlet Discrimination: Reduced response of later eluting compounds due to their lower volatility at a given temperature.
Higher Inlet Temperatures = Greater Response for Late Eluting Compounds
Ratio of last PAH over middle PAH Benzo(g,h,i)perylene / Pyrene
PAH Response vs. Inlet Temperature
0
0.2
0.4
0.6
0.8
1
1.2
1.4
150 170 190 210 230 250 270 290 310 330 350
Inlet Temperature
Rel
ativ
e Pe
ak In
tens
ity (B
enzi
[g,h
,i]Pe
ryle
ne /
Pyre
ne)
Active Analytes vs. Temperature
Relative Analyte Resopnse vs. Inlet Temperature
0
0.2
0.4
0.6
0.8
1
1.2
1.4
170 190 210 230 250 270 290 310 330 350Inlet Temperature
Rea
ltive
Res
opns
e to
DFT
PP
PCP RatioBenzidine RatioDDT Ratio
Kinetics states that reactions occur faster at higher temperatures. Expected less relative response at higher temperatures. Liner was new and CLEAN!
Note: Conditions were for Pulsed Split injection parameters; inlet liner with wool at bottom
Chromatography Optimization • 60 ºC gave much better initial peak shape • Initial peak shape still not perfect • Naphthalene at 4.5 min, last PAH at 15.2 min • Benzo[b]fluoranthene / Benzo[k]fluoranthene = 6.45% valley height
(8270D passing requirement = 50%) • Indeno[1,2,3-cd]pyrene / Dibenz[a,h]anthracene = 15.8%
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
Time-->
Abundance
TIC: PAH005.D
Guard Columns • Guard significantly improved initial peak shape • Higher initial temperature still resulted in split peaks • No change in retention times (programmed 35 m column) • Increased intensity of earlier peaks – better peak shape • Larger injections still caused a decrease in intensity with guard
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH006.D
4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.300
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH006.DTIC: PAH005.D
Confirmation of Pressure Pulse • Pressure pulse reduced inlet discrimination • Does not alter retention times
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
Time-->
Abundance
TIC: LVI003.DTIC: LVI004.D
1 1 . 5 0 1 2 . 0 0 1 2 . 5 0 1 3 . 0 0 1 3 . 5 0 1 4 . 0 0 1 4 . 5 0 1 5 . 0 00
1 0 0 0 0 0
2 0 0 0 0 0
3 0 0 0 0 0
4 0 0 0 0 0
5 0 0 0 0 0
6 0 0 0 0 0
7 0 0 0 0 0
8 0 0 0 0 0
9 0 0 0 0 0
1 0 0 0 0 0 0
1 1 0 0 0 0 0
1 2 0 0 0 0 0
1 3 0 0 0 0 0
1 4 0 0 0 0 0
1 5 0 0 0 0 0
1 6 0 0 0 0 0
1 7 0 0 0 0 0
1 8 0 0 0 0 0
1 9 0 0 0 0 0
2 0 0 0 0 0 0
2 1 0 0 0 0 0
2 2 0 0 0 0 0
2 3 0 0 0 0 0
2 4 0 0 0 0 0
2 5 0 0 0 0 0
2 6 0 0 0 0 0
2 7 0 0 0 0 0
2 8 0 0 0 0 0
2 9 0 0 0 0 0
3 0 0 0 0 0 0
3 1 0 0 0 0 0
3 2 0 0 0 0 0
3 3 0 0 0 0 0
T i m e - - >
A b u n d a n c e
T I C : L V I 0 0 3 . DT I C : L V I 0 0 4 . D
Pressure Pulse (30 p.s.i.) No pressure pulse
Peak # Ananlyte Rt % Difference 1 Naphthalene 4.339 25.5% 2 2-Methylnaphthalene 4.888 28.0% 3 Acenaphthylene 5.63 29.9% 4 Acenapthtene 5.77 29.7% 5 Fluorene 6.21 30.6% 6 Phenanthrene 7.02 34.0% 7 Anthracene 7.06 36.6% 8 Fluoranthene 8.07 50.5% 9 Pyrene 8.31 52.4% 10 Benz[a]anthracene 9.88 71.2% 11 Chrysene 9.94 70.8% 12 Benzo[b]fluoranthene 11.85 81.7% 13 Benzo[k]fluoranthene 11.91 81.3% 14 Benzo[a]pyrene 12.51 83.4% 15 Indeno[1,2,3-cd]pyrene 14.58 87.5% 16 Dibenz[a,h]anthracene 14.63 85.8% 17 Benzo[g,h,i]perylene 14.97 85.2%
Optimization of Pressure Pulse • Optimum was found to be 30 psi • Higher pressures decreased initial responses • Lower pressures decreased all signals – less transfer on-column • Decreasing pressure time at 40 psi did NOT bring back naphthalene
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
Time-->
Abundance
TIC: PAH013.D
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH011.D
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH010.D
25 psi 30 psi 40 psi
10.0010.0210.0410.0610.0810.1010.1210.1410.1610.1810.2010.22
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
Time-->
Abundance
TIC: PAH010.DTIC: PAH011.DTIC: PAH013.D
25 psi
40 psi
30 psi
Optimization of Pressure Pulse • Zoom in on Benz[a]anthracene and Chyrsene
Non-Pulsed Consideration • Expected that standard splitless injection might allow for longer
splitless hold times to improve response • Longer splitless hold times gave better responses, but not
comparable to the pulsed splitless injection
1 1 . 6 0 1 1 . 7 0 1 1 . 8 0 1 1 . 9 0 1 2 . 0 0 1 2 . 1 0 1 2 . 2 0 1 2 . 3 0 1 2 . 4 0 1 2 . 5 0 1 2 . 6 0 1 2 . 7 00
2 0 0 0 0 0
4 0 0 0 0 0
6 0 0 0 0 0
8 0 0 0 0 0
1 0 0 0 0 0 0
1 2 0 0 0 0 0
1 4 0 0 0 0 0
1 6 0 0 0 0 0
1 8 0 0 0 0 0
2 0 0 0 0 0 0
2 2 0 0 0 0 0
2 4 0 0 0 0 0
2 6 0 0 0 0 0
2 8 0 0 0 0 0
T im e - - >
A b u n d a n c e
T I C : L V I 0 0 3 . DT I C : 0 5 1 1 1 2 - M 0 1 . D
T I C : L V I 0 0 4 . DT I C : L V I 0 0 6 . D
Later Eluting PAH’s
Pulsed Splitless for 0.6 min
Splitless for 1.0 min
Splitless for 0.6 min
Splitless for 0.5 min
Larger ID Guard – 0.32 mm ID • Larger ID guard has larger volume, with same retention time means larger mL/min • Later peaks NOT taller – did not increase loading of any peaks • Lost several early eluting peaks with 5 m x 0.32 mm ID guard
– 1 m x 0.32 mm ID guard did not help • Decreasing pulse pressure and/or time helped bring back peaks 4 & 5, but not peaks 1 &
2, and decreased all other peaks • Effect would be exaggerated for 0.53 mm ID guard
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
Time-->
Abundance
TIC: PAH011.DTIC: PAH015.DTIC: PAH019.D
(5m x 0.25mm ID guard) (5m x 0.32mm ID guard) (1m x 0.32mm ID guard)
Pulsed Splitless vs. Non-pulsed 0.32 mm ID Guard
Pulsed splitless on 30 x 0.25 x 0.25 with 0.32 mm ID guard (black) vs. normal splitless with 30 x 0.25 x 0.25 with 0.32 mm ID guard (green) • Improved early eluting peaks
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.0016.000
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
1.1e+07
1.2e+07
1.3e+07
Time-->
Abundance
TIC: E23002.DTIC: E23004.D
Smaller ID Column • Hoped that improved efficiency would give higher response • Retention times were earlier with last PAH before 14 minutes • All responses were lower, especially late eluting PAHs • Changing flow rates, pressure pulses, and injection volumes did not give
equivalent responses
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.000
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
Time-->
Abundance
TIC: PAH011.DTIC: PAH022.D
30x0.25x0.25 20x0.18x0.36
Final Conditions for PAHs
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH011.D
Column Dimension: Zebron ZB-SemiVolatiles 30 m + 5 m Guard x 0.25 mm ID x 0.25 µm Part Number: 7HG-G027-GGA Column Flow: 1.4 mL/min Helium (constant flow) Oven Program: 60 ºC for 0.75 to 260 ºC @ 30 ºC/min to 295 ºC @ 6 ºC/min to 325 ºC @ 25 ºC/min for 2 min Inlet Liner: Single Taper with wool at bottom (AG0-8499) Injection: 7.5 µL Pulsed Splitless, Pressure Pulse @ 30 psi for 0.66 min, splitless for 0.60 min Detector: MSD; 46-450amu, transfer line = 320 ºC Analytes: PAHs at 5 ppm in dichloromethane
4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.0015.0016.0017.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
3800000
4000000
4200000
4400000
4600000
4800000
5000000
5200000
Time-->
Abundance
TIC: LVIB007.D
Method Development Started for 8270 • Used PAH conditions as a starting point • Lowered initial temperature to 40 ºC for 2
minutes • 5 ppm Calibration Point with 1,4-Dioxane • Separation of Benzo[b]/[k]fluoranthene • Not the best separation of later PAHs • 17 min run
3.603.703.803.904.004.104.204.304.404.504.604.704.804.905.005.10
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
Time-->
Abundance
TIC: LVIB007.D
Pyr
idin
e N
-Nitr
osod
imet
hyla
min
e
1,4-
Dio
xane
5.5 Million Counts!
Injection Volume Effects
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
Time-->
Abundance
TIC: LVIG178.D\ data.msTIC: LVIG179.D\ data.ms (*)
Overlay of 7.5 µL (file179) and 5.0 µL (file178)
Injection Volume Effects Overlay of 7.5 µL (file179) and 5.0 µL (file178)
7 . 7 0 7 . 8 0 7 . 9 0 8 . 0 0 8 . 1 0 8 . 2 0 8 . 3 0 8 . 4 0 8 . 5 0 8 . 6 0 8 . 7 0 8 . 8 0 8 . 9 0 9 . 0 0 9 . 1 0 9 . 2 0 9 . 3 0 9 . 4 0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
4 0 0 0 0 0
4 5 0 0 0 0
5 0 0 0 0 0
5 5 0 0 0 0
6 0 0 0 0 0
6 5 0 0 0 0
7 0 0 0 0 0
7 5 0 0 0 0
8 0 0 0 0 0
T i m e - - >
A b u n d a n c e
T I C : L V I G 1 7 8 . D \ d a t a . m sT I C : L V I G 1 7 9 . D \ d a t a . m s ( * )
Middle and late chromatogram are similar
Injection Volume Effects Overlay of 7.5 µL (file179) and 5.0 µL (file178)
Early chromatogram is NOT identical
3 . 4 0 3 . 6 0 3 . 8 0 4 . 0 0 4 . 2 0 4 . 4 0 4 . 6 0 4 . 8 0 5 . 0 00
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
4 0 0 0 0 0
4 5 0 0 0 0
5 0 0 0 0 0
5 5 0 0 0 0
6 0 0 0 0 0
6 5 0 0 0 0
7 0 0 0 0 0
7 5 0 0 0 0
8 0 0 0 0 0
8 5 0 0 0 0
9 0 0 0 0 0
9 5 0 0 0 0
1 0 0 0 0 0 0
1 0 5 0 0 0 0
1 1 0 0 0 0 0
1 1 5 0 0 0 0
1 2 0 0 0 0 0
1 2 5 0 0 0 0
1 3 0 0 0 0 0
T i m e - - >
A b u n d a n c e
T I C : L V I G 1 7 8 . D \ d a t a . m sT I C : L V I G 1 7 9 . D \ d a t a . m s ( * )
Calibration Curve
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
Time-->
Abundance
TIC: LVIG112.D\data.ms
4.00 5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.0015.0016.0017.0018.00
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
Time-->
Abundance
TIC: LVIG112.D\data.ms
zoom
0.1 ppm injection – Lowest Concentration
10.0 ppm injection – Later Peaks (overloading)
11.5012.0012.5013.0013.5014.0014.5015.0015.5016.0016.5017.0017.5018.00
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
1e+07
1.1e+07
1.2e+07
Time-->
Abundance
TIC: LVIG118.D\data.ms
Zoom
5.00 6.00 7.00 8.00 9.0010.0011.0012.0013.0014.0015.0016.0017.0018.000
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
750000
800000
850000
900000
950000
1000000
1050000
1100000
1150000
1200000
1250000
1300000
1350000
1400000
Time-->
Abundance
TIC: LVIG09206.D
Large Volume Injection – 8270D
Chromatogram (1 ppm)
Conditions: Column: Zebron ZB-SemiVolatiles Dimensions: 30 m x 0.25 mm x 0.50 µm with 5 m Guardian Order #: 7HG-G027-17-GGA Liner: Single Taper with Wool at the bottom Order #: AG0-8499 Injection: 5.0 µL pulsed splitless at 30 psi for 0.66 min; 260 ºC Column Flow: 1.4 mL/min Helium (constant flow) Oven Program: 45 ºC for 3.0 min to 280 ºC @ 30 ºC/min to 325 ºC @ 9 ºC/min for 5 min
Calibration Curve: Standards at 0.1 – 10.0 ppm; IS and Surr @ 1.0 ppm
4.30 4.35 4.40 4.45 4.50 4.55 4.60 4.650
2000
4000
6000
8000
10000
12000
14000
16000
Time-->
Abundance
Ion 88.00 (87.70 to 88.70): LVIG09206.D
1,4-Dioxane
4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.400
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
Time-->
Abundance
Ion 74.00 (73.70 to 74.70): LVIG09206.DIon 79.00 (78.70 to 79.70): LVIG09206.D
N-Nitrosoodimethylamine Pyridine
6.40 6.45 6.50 6.55 6.60 6.65 6.70 6.75 6.80 6.850
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
85000
90000
95000
Time-->
Abundance
Ion 93.00 (92.70 to 93.70): LVIG09206.D
Mass 93: Aniline & bis(2-chloroethyl)ether
15.0015.0515.1015.1515.2015.2515.3015.3515.4015.4515.5015.5515.6015.6515.700
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
Time-->
Abundance
Ion 252.00 (251.70 to 252.70): LVIG09206.D 15.30 15.35
Mass 252: Benzo[b]fluoranthene & Benzo[k]fluoranthene
10.10 10.20 10.30 10.40 10.50 10.60 10.70 10.80 10.90 11.000
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
38000
Time-->
Abundance
Ion 266.00 (265.70 to 266.70): LVIG09206.D
Pentachlorophenol
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Ion 276.00 (275.70 to 276.70): LVIPAH08.DIon 278.00 (277.70 to 278.70): LVIPAH08.DIon 252.00 (251.70 to 252.70): LVIPAH08.DIon 228.00 (227.70 to 228.70): LVIPAH08.DIon 202.00 (201.70 to 202.70): LVIPAH08.DIon 178.00 (177.70 to 178.70): LVIPAH08.D
Large Volume Injection – PAHs Conditions: Column: Zebron ZB-SemiVolatiles Dimensions: 30 m x 0.25 mm x 0.50 µm with 5 m Guardian Order #: 7HG-G027-17-GGA Liner: Single Taper with Wool at the bottom Order #: AG0-8499 Injection: 5.0 µL pulsed splitless at 30 psi for 0.66 min; 300 ºC Column Flow: 1.4 mL/min Helium (constant flow) Oven Program: 60 ºC for 1.0 min to 280 ºC @ 30 ºC/min to 325 ºC @ 9 ºC/min for 5 min
Calibration Curve: Standards at 0.01 – 1.0 ppm; IS and Surr @ 1.0 ppm
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TIC: LVIPAH14.D
Calibration Standard of Later Eluting PAHs @ 10 ppb (extracted ions)
Calibration of Later Eluting PAHs @ 1 ppm
This is SCAN data, not SIM!
Choice of Column - EPA Method 8270D
• EPA 8270D is a challenging method • Contains Bases, Neutrals, and Acids • Need very inert column for Bases, Neutrals, AND
Acids
EPA Method 8270D (cont’d)
DFTPP Tune – Decafluorotriphenylphospine, Pentachlorophenol, DDT, Benzidine • Instrument must pass the DFTPP Tune before running
samples – DFTPP (tests MS response)
• MS must meet ion ratio criteria – Pentachlorophenol (test acidic response)
• Peak skew < 2.0 – Benzidine (test basic response)
• Peak skew < 2.0 – DDT (tests other activity)
• Breakdown < 20%
Why is Pyridine Important? • “Pyridine may perform poorly … Therefore, if pyridine is to be
determined in addition to other target analytes, it may be necessary to perform separate analyses.” – EPA Method 8270D
• Labs don’t want to run additional analyses = it costs them more money! • Pyridine is also an indicator of column activity for other compounds.
ZB-SemiVolatiles Test Mixes Traditional Test Mix • Efficiency • Polarity • Bleed • Activity
Method Specific Test Mix • Better measure of activity • This is the DFTPP Tuning Standard • Also includes Pyridine, more
sensitive base than benzidine
Comparison of Column Activity • Measure peak height of active compounds
and compare against DFTPP as an internal standard
• This Response Factor (RF) is used to directly compare column activity
The ZB-SemiVolatiles Advantage
• Better initial peak shapes for active compounds – Gives better RSD values when calibrating instrument – Makes the 8270D Tuning Mix requirements easy to pass – Stands up to contamination better to give longer lifetime – Provides better quantitation for active compounds across all
concentrations • Low concentrations – stronger response • All concentrations – better peak shape for easier and more
consistent integration
Summary • For Pulsed Splitless conditions, a Single Taper Liner with Wool at
Bottom (AG0-8499) provides the best results for expansion capacity and directing sample on to the column.
• Pressure pulse of 30 psi for 0.66 min works best for controlling expansion volume
• Guard column of same ID as analytical column helps the peak shape of initial compounds and is expected to extend the column lifetime
• A (constant) flow of 1.4 mL/min gave the best separation for the PAHs and improved the run times
• Initial temperature needs to be 60 ºC or lower to fully focus the naphthalene peak
• The ZB-SemiVolatiles 30 m x 0.25 mm x 0.25 µm with a Guardian (7HG-G027-11-GGA) provided the best separation for Benzo[b]fluoranthene / Benzo[k]fluoranthene and Indeno[1,2,3-cd]perylene and Dibenz[a,h]anthracene in the shortest time (<16 min)
![Monitoring Naphthalene Catabolism Bioluminescence with ... · Mineralization. Naphthalene metabolism was monitored byusing a mineralization procedure to measure conversion of["4C]naphthalene](https://img.dokumen.tips/doc/110x75/5e86f458fffce403b43df98f/monitoring-naphthalene-catabolism-bioluminescence-with-mineralization-naphthalene.jpg)
