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ISCCE 2010
Considerations for Implementation and
Optimization of Capillary Column Backflush
Matthew S. KleeAgilent Technologies, Inc., Wilmington, DE 19808, USA
ISCCE 2010
Why Should You Implement Capillary Column Backflushing?• More samples/day/instrument• It is “green”; less gases + less power per sample• Lower costs per sample• Less frequent and faster GC & MSD maintenance• Longer column life• Better data quality
• less chemical background (better detection limits)• cleaner mass spectra (better ion ratios)• better peak integration (less intervention)
• More rugged multidimensional methods• more stable peak retention times• less background
ISCCE 2010
10 Runs With Backflush
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
Residual sample matrix builds up in the column…
1
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
2
Residual sample matrix builds up in the column…
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
3
Residual sample matrix builds up in the column…
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
4
Baseline increases, ghost peaks appear ...
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
5
Baseline increases, ghost peaks appear ...
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
6
Baseline increases, ghost peaks appear ...
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
7
Retention times shift out of quantitation windows …
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
8
Retention times shift out of quantitation windows …
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
9
Retention times shift out of quantitation windows …
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
10
Retention times shift out of quantitation windows …
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Res
pons
e
11
Retention times shift out of quantitation windows …
ISCCE 2010
10 Runs With Backflush
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
Late-eluting compounds are eliminated
1
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
Late-eluting compounds are eliminated
2
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
Late-eluting compounds are eliminated
3
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
baselines remain clean…
4
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
baselines remain clean…
5
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
baselines remain clean…
6
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
peak positions and ion ratios remain stable.
7
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
peak positions and ion ratios remain stable.
8
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
peak positions and ion ratios remain stable.
9
Res
pons
e
min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1
peak positions and ion ratios remain stable.
10
ISCCE 2010
Applications meriting consideration
Any analysis with• Extended temperature programs and/or bakeout
times after the last peak of interest elutes• Frequent maintenance due to dirty samples• Significant carry-over, ghost peaks, increasing
baseline• Late eluting compounds of no analytical interest• Long overall analysis times
ISCCE 2010
Purged devices that enable backflush
2-WayPurged SplittersDeans switch
Flow modulator
QuickSwap
Purged Ultimate Union
3-Way
PCM
EPC
ISCCE 2010
Setting up Backflush
1. Start with working method2. Extract pertinent method information from method files3. Select BF configuration4. Set up and verify hardware 5. Install columns and test configuration6. Screen standard on new configuration7. Lock with migrated RTL calibration if RTL method8. Pick last peak of interest using graphical tool, from
which BF timing and conditions are recommended9. Verify BF works10. Fine tune BF conditions if necessary
ISCCE 2010
Three Configurations and Two Modes of BFPost-Column
• Post-run backflushUncoated Pre-Column
• Concurrent backflushCoated Pre-Column (split column)
• Post-run• Concurrent
ISCCE 2010
Post-Column Configuration
Column
Purged Device
S/Sl Inlet
Inlet EPC
P1
Aux EPC
P2
Split Vent
Det.Restrictor
ISCCE 2010
Post-Column Analysis
ColumnSplit Vent
Inlet EPC Aux
EPC
Det.Restrictor
ISCCE 2010
Post-Column Backflush
ColumnSplit Vent
Inlet EPC
Det.Restrictor
Aux EPC
ISCCE 2010
Post-Column Configuration
Column
Purged Device
S/Sl Inlet
Inlet EPC
P1
Aux EPC
P2
Split Vent
Det.Restrictor
ISCCE 2010
What are the benefits of post-column backflush?
• Easiest mode to understand, and implement; Backflushing is very simple– Immediately after the last component of analytical interest elutes (obvious),
data acquisition ends and backflush is initiated at that temperature. – The determination and verification of backflush timing is the fastest of the three
approaches– Run has ended and data acquisition is stopped (MSD filaments, quad and
detector are OFF) during backflush
• RTL implementation is very straightforward and rugged to apply
• The analytical column is one single unit with constant mass flow through its full length (no degradation in original separation efficiency)
• Compatible with pressure pulsed injections
ISCCE 2010
What are the downsides of post-column backflush mode and use of QuickSwap?
• Flow is added to the column effluent – The choice of restrictor dictates the flow that to the MSD – Even a prudent choice in restrictor can reduce sensitivity 15-20%– Larger than necessary i.d. (bad choice in restrictor) can decrease
sensitivity and S/N much more dramatically
• The restrictor is uncoated, so there is a possibility of increased loss of labile compounds.
ISCCE 2010
Reference TIC – CF Screening Conditions
Time--> 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
0.818 1.400
2.213
3.136
4.012
4.823 5.570 6.2616.588
6.900
7.495
8.049
8.570
9.059 9.959
10.771
11.771
n-C10
n-C12
n-C14
n-C16
n-C18
n-C20
n-C30
n-C26
n-C23
n-C36 n-C40 n-C44
330 oC
ISCCE 2010
Reference Chromatogram
Time--> 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.000
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
0.818 1.400
2.213
3.136
4.012
4.823 5.570 6.2616.588
6.900
7.495
8.049
8.570
9.059 9.959
10.771
11.771
n-C10
n-C12
n-C14
n-C16
n-C18
n-C20
n-C30
n-C26
n-C23
n-C36 n-C40 n-C44
219 oC
ISCCE 2010
5 Runs Stopping @ 6.25 min, no BF, Showing Carryover Into Subsequent Runs
Time-->0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
C18C16 C20
C23 , C24 from previous run
C26 from 3 runs prior
ISCCE 2010
Solvent Blank Full Temperature-Programmed Analysis After Five 6.25 min Abbreviated Runs
Time--> 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
0
100000
200000
300000
400000
500000
600000
700000
800000 Grouping of same components from each run
35 5
5
5
5
5C44
C40
C36C23
C24
C26
CC2828
CC3030
CC3232
ISCCE 2010
Ten Sequential Runs with BF @ 6.25 min
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
6500000
7000000
7500000
Time-->
Abundance
ISCCE 2010
BF Signals from 10 sequential BF Runs
Signal: screen_024.D\FID1A.CHSignal: screen_025.D\FID1A.CH (*)Signal: screen_026.D\FID1A.CH (*)Signal: screen_027.D\FID1A.CH (*)Signal: screen_028.D\FID1A.CH (*)Signal: screen_029.D\FID1A.CH (*)Signal: screen_030.D\FID1A.CH (*)Signal: screen_031.D\FID1A.CH (*)Signal: screen_032.D\FID1A.CH (*)Signal: screen_033.D\FID1A.CH (*)
6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55 6.60
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
Time (miin)
Response BF initiated at 6.25 min
~2.5 voids
C22
C23
C24
C26
C40←C28
ISCCE 2010
Ten 6.25 min Runs without BF
Time-->
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
ISCCE 2010
Backflush after 10 runs vs. each run
5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
Time (min)
Response
BF initiated at 6.25 min
BF after 10 runs w/o BFBF after single run
ISCCE 2010
Uncoated Pre-Column Configuration
ColumnUncoatedPre-Column
FIDS/Sl Inlet
Inlet EPC
P1
Aux EPC
P2
Split Vent
Purged Union
ISCCE 2010
Uncoated Pre-Column Analysis
UncoatedPre-Column
Split Vent
Inlet EPC Aux
EPC
Column
FID
ISCCE 2010
Uncoated Pre-Column Backflush
ColumnUncoatedPre-Column
Split Vent
FID
Aux EPC
0
Inlet EPC
ISCCE 2010
Uncoated Pre-Column Configuration
ColumnUncoatedPre-Column
FIDS/Sl Inlet
Inlet EPC
P1
Aux EPC
P2
Split Vent
Purged Union
ISCCE 2010
What are the benefits of using uncoated (but deactivated) pre-columns for backflush?
• Decoupling of retention of pre-column from analytical column, therefore more suitable for replication on multiple systems/locations, more rugged.– Sample components are transferred to the analytical column at low
temperatures and are focused by the stationary phase of the analytical column, decoupling retention of the two sections (resulting in more rugged conditions)
• The heavy components are backflushed more quickly (at lower temperatures and/or with fewer column void volumes), making this the fastest backflush technique (often components are completely backflushed within seconds).
• Fastest backflush of all modes and concurrent with the analysis so when the last compound of interest has eluted, the run can stop and get ready for the next analysis (maximizing lab throughput)
• Analytical column mass flow is constant through total analytical column length (maximum efficiency)
ISCCE 2010
What are the benefits of using uncoated (but deactivated) pre-columns for backflush?
• Flow to detectors during backflush is the least of all BF configurations – exactly the same as original method
• Most flexibility in choice of pre-column dimensions (especially i.d.) and flow rates
• Retained matrix has least influence on initial peak shapes and retention times
• Cheapest to replace when necessary.
• Highest accuracy in transfer of retention time locking methods
ISCCE 2010
What are the limitations of using uncoated pre-columns for backflush?
• More difficult to determine backflush timing than post-column approach (but easier than coated column approach).
• Wider peaks than with coated column approach
• RT dependent on amount and sample matrix
• Highest possibility of sample degradation (least inert)
• Retention time most dependent on residual sample matrix
ISCCE 2010
Retention in Retention Gaps360 oC
(5 min)50 oC
10 oC/min
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000Response
5.00 10.00 15.00 20.00 25.00 30.00
C44
C10
C12
C14
C16
C18
C20
C30C26
C23
C36 C40
C22C24 C28 C32
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
200000
300000
400000
500000
600000
Time
C44
C16
C18C20
C30C26
C23
C36 C40C22
C24C28 C32
Retention Gap Effluent
ISCCE 2010
12 min BF vs. reference
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.000
2e+0
4e+0
6e+06
8e+06
1e+07
1.2e+07
1.4e+07
1.6e+07
Time
ISCCE 2010
12 min BF vs. reference
Time 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
Partial loss/backflush
ISCCE 2010
Coated Pre-Column Configuration
MSD
Coated Pre-Column
Restrictor
Column
AuxEPC
P2P1
S/S Inlet
Inlet EPC
Split Vent
Purged Union
ISCCE 2010
Coated Pre-Column Analysis
Coated Pre-Column
Split Vent
MSDPurged Union
Restrictor
Column
AuxEPCInlet
EPC
ISCCE 2010
Column
Coated Pre-Column Backflush
Coated Pre-Column
Split Vent
MSD
Restrictor
AuxEPCInlet
EPC
Purged Union
ISCCE 2010
Coated Pre-Column Configuration
MSD
Coated Pre-Column
Restrictor
Column
AuxEPC
P2P1
S/S Inlet
Inlet EPC
Split Vent
Purged Union
ISCCE 2010
Purged Ultimate Union
58 2010 April rev HP1PCT Introduction PREST – Internal / Restricted
ISCCE 2010
100 OFN fg in 1% diesel
59 2010 April rev HP1PCT Introduction PREST – Internal / Restricted
5 replicate injections
5.95 6.05 6.15 6.25 6.350
50
100
150
200
250
300
350
400
450
500
550
injections 8 & 11
Injection 3
Injection 2
5.95 6.05 6.15 6.25 6.350
200
400
600
800
1000
1200
1400
1600
1800
Using Backflush NOT using Backflush
ISCCE 2010
What are the benefits of using coated pre-columns for backflush?
• Most inert approach when analyzing “active” compounds
• Ability to tune onset of backflush between closely eluting components
• Backflushing can happen concurrent with the analysis (when short pre-columns are used)
• Compatible with diffusion pump MSDs
• Fairly simple to implement
– Cut sections of existing columns to use as pre-columns
– Use commercially available coated columns that total the length of original column (e.g., two 15 m columns instead of the original 30 m column)
ISCCE 2010
What is the downside of using coated pre-columns for backflush?
• Most difficult approach to determine backflush temperature/time
• More expensive to replace than uncoated columns
• Backflushing occurs later into the run than with uncoated columns, so less time for concurrent backflush
• Longer backflushing times than with uncoated pre-columns
– When using long pre-columns, lower reversed flow during backflush
– Components are retained more because of stationary phase so needmore column void volumes to backflush than with uncoated pre-columns
• Contaminants have more influence on retention times of subsequent runs than with uncoated pre-columns (same issue as post-column configuration).
ISCCE 2010
Conclusions
Capillary column backflush provides many potential benefits for routine GC analysesThree possible configurations offer some flexibility depending on sample and method constraintsSeveral available purged devices enable rugged and reliable implementation and automated control of capillary column backflushing
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