36
Large Volume Injection Presented By Tim Anderson GC Product Manager Phenomenex EPA Region 6 QA Conference October, 2015
Large Volume Injection
Presented By
Tim Anderson
GC Product Manager
Phenomenex
EPA Region 6 QA Conference
October, 2015
Goal
Develop a method to use the least amount of sample and achieve the highest possible s/n response
2
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
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
3
PreliminaryAssumptions
• Pressure Pulsed injection tend to improve analyte responses
• Improves transfer of analytes from the inlet into the column
• Started with Pressure Pulse and tested this assumption during throughout the experiment
Analytes:• Started with a relatively simple list of Polycyclic Aromatic
Hydrocarbons (PAHs)
• Contains relatively wide range of retention times
• Subsequent work includes other compounds
4
Column: Zebron ZB-SemiVolatiles 30 m x 0.25 mm 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
Initial Conditions
5
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 Experiments
What is the best liner?
Different styles
Compared early, mid, and late eluters
6
Liner Performance
DirectConnect Dual Taper, bottom hole
• Maximum Injection volume = 10 µL
• Maximum intensity = 1.67e7
• Better response for higher MW
• Response decreases after 10 µL!
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
Are
a
Injection Volume (uL)
Fluorene
Chrysene
Benzo[g,h,i]perylene
7
DirectConnect Top Hole
• Maximum Injection volume = 12.5 µL+
• intensity = 1.40e7
• Better response for lower MW 0.0E+00
5.0E+06
1.0E+07
1.5E+07
2.0E+07
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
Are
a
Injection Volume (uL)
Fluorene
Chrysene
Benzo[g,h,i]perylene
Liner PerformanceSingle Taper With Wool
• Maximum Injection volume = 10 µL
• Maximum intensity = 2.07e7
• Consistent response, except at larger volumes which benefit lower MW
• Response decreases after 10 µL!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
Are
a
INjectin Volume (uL)
Fluorene
Chrysene
Benzo[g,h,i]perylene
8
Cup Liner
• Maximum Injection volume = 7.5 µL
• Maximum intensity = 2.71e7
• Slightly better response for higher MW
• Response decreases after 7.5 µL 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
Are
a
Injection Volume (uL)
Fluorene
Chrysene
Benzo[g,h,i]perylene
Liner Performance
FocusLiner (single taper with wool in middle)
• Maximum Injection volume = 2.5 µL
• Maximum intensity = 6.23e6
• Much better response for lower MW
• Possible cooling of glass wool
9
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
Are
a
Injection Volume
Fluorene
chrysene
Benzo[g,h,i]perylene
Average Liner Performance
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
Direct ConnectBottom Hole dualtaperDirect Connect TopHole
Cup Liner
Single Taper WoolBotom
Focusliner
10
Single Taper with Wool at bottom
provides highest response at 7.5 µL
injection volume
Analytes1. Pentachlorophenol
2. DFTPP
3. Hexadecanoic acid
4. Octadecanoic acid
5. Benzidine
6. Hexanedioic acid, bis(2-ethylhexyl)ester
7. DDT
Liner Activity
11
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
7 . 0 0 7 . 5 0 8 . 0 0 8 . 5 0 9 . 0 0 9 . 5 0 1 0 . 0 0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 2 0 0 0 0
1 4 0 0 0 0
1 6 0 0 0 0
1 8 0 0 0 0
2 0 0 0 0 0
2 2 0 0 0 0
2 4 0 0 0 0
2 6 0 0 0 0
2 8 0 0 0 0
3 0 0 0 0 0
3 2 0 0 0 0
3 4 0 0 0 0
3 6 0 0 0 0
3 8 0 0 0 0
T im e -->
A b u n d a n c e
T I C : A G 8 4 9 9 _ 2 . D
7 .0 0 7 .5 0 8 .0 0 8 .5 0 9 .0 0 9 .5 0 1 0 .0 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
T im e -->
A b u n d a n c e
T IC : A G 4 6 5 7 _ 2 .D
Normal Liner
Competing Liner
1
2
34
5
6
7
1
2
5 7
Inlet Temperature = 275 ºC
Ratio of over middle PAH
Benzo(g,h,i)perylene / Pyrene
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
Injection 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
Re
lati
ve
Pe
ak
In
ten
sit
y (
Be
nzi[
g,h
,i]P
ery
len
e /
Pyre
ne
)
Inlet Temperature
PAH Response vs. Inlet Temperature
12
Active Analytes vs. Temperature
13
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
0
0.2
0.4
0.6
0.8
1
1.2
1.4
170 190 210 230 250 270 290 310 330 350
Rea
ltiv
e R
es
op
ns
e t
o D
FT
PP
Inlet Temperature
Relative Analyte Resopnse vs. Inlet Temperature
PCP Ratio
Benzidine Ratio
DDT Ratio
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 requirement = 50%)
Indeno[1,2,3-cd]pyrene / Dibenz[a,h]anthracene = 15.8%
Chromatography Optimization
143.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
Time-->
Abundance
TIC: PAH005.D
Guard Columns
15
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH006.D
4 .4 0 4 .5 0 4 .6 0 4 .7 0 4 .8 0 4 .9 0 5 .0 0 5 .1 0 5 .2 0 5 .3 00
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
4 5 0 0 0 0 0
5 0 0 0 0 0 0
5 5 0 0 0 0 0
T im e -->
A b u n d a n c e
T IC : P A H 0 0 6 .DT IC : P A H 0 0 5 .D
No Guard
Guard
Confirmation of Pressure Pulse
3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 1 1 .0 0 1 2 .0 0 1 3 .0 0 1 4 .0 0 1 5 .0 0
0
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
T ime -->
A b u n d a n c e
T IC: L V I0 0 3 .D
T IC: L V I0 0 4 .D
Pressure Pulse (30 p.s.i.)
No pressure pulse
Benzo[b]fluoranthene
82% Diff.
Benzo[k]fluoranthene
81% Diff.
Benzo[a]pyrene
83% Diff.
Indeno[1,2,3-cd]pyrene
88% Diff.
Dibenz[a,h]anthracene
86% Diff.
Benzo[g,h,i]perylene
85% Diff.
Optimization of Pressure PulseHigher pressures decreased initial responses
Lower pressures decreased all signals – less transfer on-column
Optimum was found to be 30 psi
Decreasing pressure time at 40 psi did NOT bring back naphthalene
17
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
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 10.00 11.00 12.00 13.00 14.00 15.00
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 10.00 11.00 12.00 13.00 14.00 15.00
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
Time-->
Abundance
TIC: PAH010.D
25 psi 30 psi 40 psi
10.00 10.02 10.04 10.06 10.08 10.10 10.12 10.14 10.16 10.18 10.20 10.22
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 PulseZoom in on Benz[a]anthracene and Chyrsene
18
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
Non-Pulsed Consideration
19
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 i m 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
(5m x 0.25mm ID guard)
(5m x 0.32mm ID guard)(1m x 0.32mm ID guard)
Larger ID Guard: 0.32 mm ID
20
Later peaks NOT taller;
did not increase
loading of any peaks
Lost early
eluters
Pulsed vs. Non-pulsed 0.32 mm ID Guard
21
Non-pulse
Improved
Early
Eluters
Pressure Pulse
No pressure pulse
Non-pulse
Reduced
Later Eluters
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
Experimented with flow rates, pressure pulses, and injection volumes
Smaller ID Column
22
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
4500000
5000000
Time-->
Abundance
TIC: PAH011.DTIC: PAH022.D
30x0.25x0.2520x0.18x0.36
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
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
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
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
Final Conditions for PAHs
23
4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 01 0 .0 01 1 .0 01 2 .0 01 3 .0 01 4 .0 01 5 .0 01 6 .0 01 7 .0 0
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
3 0 0 0 0 0 0
3 2 0 0 0 0 0
3 4 0 0 0 0 0
3 6 0 0 0 0 0
3 8 0 0 0 0 0
4 0 0 0 0 0 0
4 2 0 0 0 0 0
4 4 0 0 0 0 0
4 6 0 0 0 0 0
4 8 0 0 0 0 0
5 0 0 0 0 0 0
5 2 0 0 0 0 0
T im e -->
A b u n d a n c e
T I C : L V IB 0 0 7 . D
Method Development for Semivolatiles• Used PAH conditions as a starting point
• 17 min run
• 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
24
3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
T ime-->
Abundanc e
T IC: LVIB007.D
Pyridin
e
N-N
itro
sodim
eth
yla
min
e
1,4
-Dio
xan
e
5.5 Million Counts!
Injection Volume Effects
25
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
T ime-->
Abundanc e
T IC: LVIG178.D \ data.ms
T IC: LVIG179.D \ data.ms (*)
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 s
T I C : L V I G 1 7 9 . D \ d a t a . m s ( * )
Middle and late chromatogram are similar
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 0
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
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 s
T I C : L V I G 1 7 9 . D \ d a t a . m s ( * )
Early Chromatogram is NOT Similar
0.1ppm Calibration Curve
26
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 . 0 05 . 0 06 . 0 07 . 0 08 . 0 09 . 0 01 0 . 0 01 1 . 0 01 2 . 0 01 3 . 0 01 4 . 0 01 5 . 0 01 6 . 0 01 7 . 0 01 8 . 0 0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 2 0 0 0 0
1 4 0 0 0 0
1 6 0 0 0 0
1 8 0 0 0 0
2 0 0 0 0 0
2 2 0 0 0 0
2 4 0 0 0 0
2 6 0 0 0 0
2 8 0 0 0 0
3 0 0 0 0 0
3 2 0 0 0 0
3 4 0 0 0 0
3 6 0 0 0 0
3 8 0 0 0 0
4 0 0 0 0 0
4 2 0 0 0 0
T im e -->
A b u n d a n c e
T I C : L V I G 1 1 2 . D \ d a t a . m s
zoom
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
T ime-->
Abundanc e
T IC: LVIG118.D \ data.ms
Column: Zebron ZB-SemiVolatiles 30 m x 0.25 mm x 0.50 µm with 5 m Guardian
Liner: Single Taper with Wool at the bottom
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: 0.1 – 10.0ppm; IS & Surrogate at 1.0ppm
LVI: Semivolatiles
275 .0 06 .0 07 .0 08 .0 09 .0 01 0 .0 01 1 .0 01 2 .0 01 3 .0 01 4 .0 01 5 .0 01 6 .0 01 7 .0 01 8 .0 0
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
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
1 3 5 0 0 0 0
1 4 0 0 0 0 0
T im e -->
A b u n d a n c e
T IC : L V IG 0 9 2 0 6 .D
Key Performance Characteristics
5 .0 06 .0 07 .0 08 .0 09 .0 01 0 .0 01 1 .0 01 2 .0 01 3 .0 01 4 .0 01 5 .0 01 6 .0 01 7 .0 01 8 .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
1 3 5 0 0 0 0
1 4 0 0 0 0 0
T im e -->
A b u n d a n c e
T IC : L V IG 0 9 2 0 6 .D
284.30 4.35 4.40 4.45 4.50 4.55 4.60 4.65
0
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 5.40
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
Time-->
Abundance
Ion 74.00 (73.70 to 74.70): LVIG09206.D
Ion 79.00 (78.70 to 79.70): LVIG09206.D
N-Nitrosoodimethylamine
Pyridine
6 .4 0 6 .4 5 6 .5 0 6 .5 5 6 .6 0 6 .6 5 6 .7 0 6 .7 5 6 .8 0 6 .8 50
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
3 0 0 0 0
3 5 0 0 0
4 0 0 0 0
4 5 0 0 0
5 0 0 0 0
5 5 0 0 0
6 0 0 0 0
6 5 0 0 0
7 0 0 0 0
7 5 0 0 0
8 0 0 0 0
8 5 0 0 0
9 0 0 0 0
9 5 0 0 0
T im e -->
A b u n d a n c e
Io n 9 3 .0 0 (9 2 .7 0 to 9 3 .7 0 ): L V IG 0 9 2 0 6 .D
Mass 93:
Aniline &
bis(2-chloroethyl)ether
15.0015.0515.1015.1515.2015.2515.3015.3515.4015.4515.5015.5515.6015.6515.70
0
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
1 0 .1 0 1 0 .2 0 1 0 .3 0 1 0 .4 0 1 0 .5 0 1 0 .6 0 1 0 .7 0 1 0 .8 0 1 0 .9 0 1 1 .0 00
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 2 0 0 0
1 4 0 0 0
1 6 0 0 0
1 8 0 0 0
2 0 0 0 0
2 2 0 0 0
2 4 0 0 0
2 6 0 0 0
2 8 0 0 0
3 0 0 0 0
3 2 0 0 0
3 4 0 0 0
3 6 0 0 0
3 8 0 0 0
T ime -->
A b u n d a n c e
Io n 2 6 6 .0 0 (2 6 5 .7 0 to 2 6 6 .7 0 ): L V IG 0 9 2 0 6 .D
Pentachlorophenol
Column: Zebron ZB-SemiVolatiles 30 m x 0.25 mm x 0.50 µm with 5 m Guardian
Liner: Single Taper with Wool at the bottom
Injection: 5.0 µL pulsed splitless at 30 psi for 0.66 min; 300ºC
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: 0.01 – 1.0 ppm; IS and Surr @ 1.0 ppm
LVI: PAHs
29
8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
0
500
1000
1500
2000
2500
3000
3500
T ime-->
Abundanc e
Ion 276.00 (275.70 to 276.70): LVIPAH 08.D
Ion 278.00 (277.70 to 278.70): LVIPAH 08.D
Ion 252.00 (251.70 to 252.70): LVIPAH 08.D
Ion 228.00 (227.70 to 228.70): LVIPAH 08.D
Ion 202.00 (201.70 to 202.70): LVIPAH 08.D
Ion 178.00 (177.70 to 178.70): LVIPAH 08.D
5 .0 0 6 .0 0 7 .0 0 8 .0 0 9 .0 0 1 0 .0 0 1 1 .0 0 1 2 .0 0 1 3 .0 0 1 4 .0 0 1 5 .0 0 1 6 .0 0
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 ime -->
A b u n d a n c e
T IC: L V IP A H 1 4 .D
Calibration Standard of Later Eluting PAHs @ 10 ppb
(extracted ions)
This is SCAN data, not SIM!
Choice of Column
Semivolatiles are challenging compounds
Contains Bases, Neutrals, and Acids
Need very inert column for Bases, Neutrals, AND Acids
30
EPA Method Requirements
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%
31
“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.
Why is Pyridine Important?
32
Traditional Test Mix Efficiency
Polarity
Bleed
Activity
ZB-SemiVolatiles Test Mixes
Method Specific Test Mix Better measure of activity
This is the DFTPP Tuning Standard
Also includes Pyridine, more sensitive base than benzidine
33
The ZB-SemiVolatiles Advantage
Better peak shapes for active compounds
Better calibration RSD values
Makes the 8270D Tuning requirements easy to pass
Resists contamination = longer lifetime
Better quantitation across all concentrations Low concentrations: Stronger response
All concentrations: Better peak shape for easier and more consistent integration
35
Summary
Single Taper Liner with Wool at Bottom provides
the best results for expansion capacity and directing sample on to
the column.
Pressure pulse of 30 psi for 0.66 min for controlling
expansion volume
Guard column of same ID as analytical column
helps the peak shape of initial compounds extend
column lifetime
Constant flow of 1.4 mL/min gave the best
separation and improved run times
60°C Initial temperature to fully focus early eluters
such as naphthalene
The ZB-SemiVolatiles 30 m x 0.25 mm x 0.25 µm with a 5m integrated Guard
best separation for crticalPAH pairs in the shortest
time (<16 min)
37
Thank You
Trademarks
Zebron is a trademark of Phenomenex. Rxi and Restek are trademarks of Restek Corporation.
Agilent and DB are trademarks of Agilent Technologies, Inc.
Disclaimer
Comparative separations are not representative of all applications. Phenomenex is in no way
affiliated with Restek Corporation or Agilent Technologies, Inc.
© 2015 Phenomenex, Inc. All rights reserved.
