Three years of experience with backflush and
retention time locking on
Agilent 7000B GC-MS/MS
Hans Ragnar Norli
Bioforsk, Plant Health and Plant Protection
Pesticide Team
The History of GC-MS Technology at
Bioforsk
2005
-Agilent 5973 MS, full scan, Targeted
-Agilent 5890 GC with 30 m HP5 MSI kolonne
-Pre column 2.5 m.
-Gerstel PTV injector air cooled, 20 uL inj. volume
-Liner: sintred
-RT-lock.
-Dataanalysis: ChemStation with
-DRS system and AMDIS database; 196 compounds
- Injection cycle: 55 min (injection to injection)
2010: Introduction of backflush on GC-MS
2012
-Agilent 7000 B GC/MS Triple Quad
-Agilent 7890 GC with 2X 15m HP5 MSUI
-Pre column 2.5 m.
-MMI inlet, air cooled, 5 uL inj. volume
-Liner: 2mm DIMPLED
-RT-lock
-Back-flush in post run (column midpoint)
-Data analysis: Masshunter; 122 compounds
-Injection cycle: 30 min
The Idea behind Backflush Technology
New Column
FLOW
Column after several injections
Column after backflush
FLOW
Quechers of Leek
Abundance
TIC: 00501007.D\data.ms
1.25e+07
Leek, no back flush
Time-->
10.00
15.00
20.00
25.00
30.00
35.00
40.00
TIC: 00401008.D\data.ms
280000
ACN, injected after
10.00
15.00
20.00
25.00
30.00
35.00
40.00
TIC: 00501001.D\data.ms
8500000
Leek, with back flush
10.00
15.00
20.00
25.00
30.00
35.00
TIC: 00401002.D\data.ms
280000
ACN, injected after
10.00
15.00
20.00
25.00
30.00
35.00
Control chart examples of Ortho-phenylphenol and Boscalid at spiking level
0.05 mg/kg
Back Flush
introduced
Lettuce
Apple
Orange
Decreasing the Measurement
Uncertainty
Measurement Uncertainty (2*CV%)
Pesticide (0.05
mg/kg)
Three months before
BF installation, N=49
Three months after BF
installation, N=49
Change (Before-After)
Orto-phenylphenol
72
38
-34
Diphenylamine
56
28
-28
Pirimiphos-methyl
64
31
-33
Chlorpyriphos
57
22
-35
Cyprodinil
48
16
-32
Prothiophos
45
20
-25
Boscalid
58
23
-35
Cypermethrin
79
65
-14
Bean(2)
Apple (8)
Lettuce (27)
Wheat (8)
Rice (4)
Bean(3)
Apple (13)
Lettuce (13)
Wheat (5)
Rice (3)
Orange (13)
Matrices
Agilent 7000 B GC-MS/MS:
-Number of analytes: 119
-Number of time segments: 19
-Largest segment consist of 30 transitions (15 analytes) 3 cycles/s
-RT-Lock with ditalimfos at 14.4 min
-PCM:10 psi (50 psi in post run)
-Column head pressure 16 psi
-Backflush time 2.7 min (column hold up time x 5)
-Injection cycle: 30 min
BACK- FLUSH:
-After last eluting compound postrun is enabled:
The pressure in the injector is lowered ->0.5 psi
PCM pressure and temp in oven and injector are increased-> 50 psi, 280 °C, 280 °C
Establishing a Constant Pressure Method
with Backflush in combination with RT-Lock
• For improving peak shape of early eluting peaks; include a pre column
• Establish method with constant pressure P:
• Make test runs of a high standard with all pesticides and establish your
chromatography with as few time segments as possible!
• Enable backflush in post run and set a pressure at your pressure controller
(PCM)
• Make a run and ensure that during the run the flow in Column 2 (Column 1+
0.04 ml/min) is always higher than Column 1
• Eventually adjust column head pressure or PCM pressure
2.5
Flow (mL/min
2
1.5
Col 1
1
Col 2
0.5
0
0
5
10
15
Run time (min)
20
25
Implementing RT-Lock
• Put the transitions of your Rt-lock compound into the neighboring time
segments
• RT-Lock program: select the pressures at P+2 and P-2
• Make runs at P+2 and P-2 and ensure that flow in Column 2 > Column 1
• Eventually adjust the P+2 or P-2 and re-inject
• Run the RT-Lock setup program with your RT-Lock compound
• Increase the number of time segments to improve the sensitivity
P
Due to increased injector pressure, the flow in
column 1 could be higher than in column 2
The PCM module may also not maintain its
pressure
P+2
P+1
P
P-1
P-2
Rt
Due to reduced injector pressure, the flow in
column 1 could be too low; constant backflush
P+1 =P+10%
Maintenance Routines
Re-Lock
Changing Changing
with
liner
preDitalimfos
column
Changing
column 1
Changing
column 2
Before
starting
samples
After 3-6
months
Once a year After 6-8
months
50-60
samples
50-60
samples
Cleaning
ion source
There is no need for venting the MS when changing column 1
Tuning
Every
month
Changing Columns
SilTite
UltiMetal Ferrules
The UltiMetal ferrules are not leaking, simpler to install and cause
no problems with snapping off the column when tightening!
http://www.chem.agilent.com/Library/usermanuals/Public/G3188-90001.pdf
Conclusion
Backflush:
• Protects the ion source
• Decrease the measurement uncertainty for
several compounds
• No need for venting the MS when changing the
first column #1
• Column #2 can last twice as long as column #1
• Easy change of columns with UltiMetal Ferrules
• Robust system: First installed and tuned it works
without technical problems; «hassle-free»
Conclusion
• Don't solve problems with traditional difficult
pesticides like captan, folpet, pyrethroids…
• We still have problems with certain matrices:
– Tea -> increased retention times
– Extraction at pH 1 for captan, folpet and
chlorothalonil -> reduced sensitivity
• You have to invest time and effort to establish
and understand how the system work
Thanks to:
Kari Stuveseth
Anita Glosli
Application notes from Agilent: 5990-4643EN, 5990-3578EN and 5990-9317EN