considerations for implementation and optimization of ... · considerations for implementation and...

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


min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2

Res

pons

e

3


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


min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2

Res

pons

e

6


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


min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2

Res

pons

e

9


min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2

Res

pons

e

10


min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2

Res

pons

e

11


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


2

Res

pons

e

min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1


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


5

Res

pons

e

min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1


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


8

Res

pons

e

min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1


9

Res

pons

e

min5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1


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


Split Vent

FID

Aux EPC

0

Inlet EPC

ISCCE 2010

Uncoated Pre-Column Configuration


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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