Column Bleed and method change help

[chromatonoob]

Hello All,

I have a few challenges here.

This is the TIC I have for a column DB-23, 60 m length, 0.250 mm id, 0.15 um film. We have a set method for this column - FAMES, C8-C24 etc.


This is the TIC after we cut that 60 m column in half to 30 m. C8-C12:0 not on chromatogram. AND major column bleed.


I've been told that I need to alter the method at this point. What do I need to change about the method in order to obtain the same results as I did before?
How should I fix the increasing baseline (I've already baked it out)?

Any suggestions?

[Don_Hilton]

1) Be sure the new column dimensions are included in your method. The linear velocity needs to stay the same.

2) With a shorter column, compunds elute more quickly - even with the correct linear velocity. You need to start acquiring data earlier in the run. The early eluters are present, but have eluted before you turned the detector on.

3) Look at separation of desired compunds and be sure that you have the separation you want - if not, you may need ot adjust the temperature profile.

[chromatonoob]

1) Be sure the new column dimensions are included in your method. The linear velocity needs to stay the same.

2) With a shorter column, compunds elute more quickly - even with the correct linear velocity. You need to start acquiring data earlier in the run. The early eluters are present, but have eluted before you turned the detector on.

3) Look at separation of desired compunds and be sure that you have the separation you want - if not, you may need ot adjust the temperature profile.

Thank you so much! How do I start acquiring data early on? I'm very new to the chemstation software.

[cleh]

Please post a spectrum of the baseline at 25 minutes.

[Consumer Products Guy]

Baseline rising can result from varied things. Since you have used the column before, just cut it in half, shouldn't seem to be column bleed. Septa can also cause this issue, one can try different type of septum, or bake that in advance. Are the inlet split or inlet conditions the same. I also question what was the driving force to shorten the column? You did save a little time.

Anyway, even with the rise in baseline, your data station should have no issues in integrating those peaks.

[chromatonoob]

Baseline rising can result from varied things. Since you have used the column before, just cut it in half, shouldn't seem to be column bleed. Septa can also cause this issue, one can try different type of septum, or bake that in advance. Are the inlet split or inlet conditions the same. I also question what was the driving force to shorten the column? You did save a little time.

Anyway, even with the rise in baseline, your data station should have no issues in integrating those peaks.

Actually, the first TIC is from the old column. We switched that column out with a new (and complete) 60 m column. And we had this same upward drift. Another coworker suggested we cut the column in half. Saves time, like you said. And also, now we have two. Shouldn't affect resolution by a significant amount. I changed the S/Sl inlet yesterday. As it is Good Friday, I don't go back to work until Monday to see the results.

And I apologize. I got thrown into this with basic "make these solutions, push these buttons, inject" and not a lot of GC maintenance. Can you explain to me what you mean by "inlet split or inlet conditions are the same?"

[jdezeeuw]

Hi
You have different challenges:

first Your initial run you ave a double injection. So there is an injection challenge you have to look at.

the analysis after a month shows a clear different pattern and perak s elute MUCH faster. It looks like your column has lost a LOT of phase + also the selectivity is changed. You have to be VERY careful as this may even results in wrong identifucation.

Problem is that many cyano phases do show a stability problem. There should be more stable phases out there.. The stripping off of the polymer causes the change in selectivity.
THis is catalyzed by water/Oxygen in carrier ags or via leaks. check that using leak detection and try not to use thes phase at their highest temperature. It looks like you are exceeding that, as I consider this "stripping" nin a month not acceptable.
May use some FLOW programming so you can stop at a lower final temperature.

are you sure you NEED 60m? this analytsis can be done with much shorter columns. Uisng a30/0.32 give you a lot more flexibility, also with flow programming.

If cpeak start to tail/column gets contaminated, you need to use pre-/ guard columns, or cut a 30 cm off the inlet;

jaap de Zeeuw, Restek Corporation

[James_Ball]

1) Be sure the new column dimensions are included in your method. The linear velocity needs to stay the same.

2) With a shorter column, compunds elute more quickly - even with the correct linear velocity. You need to start acquiring data earlier in the run. The early eluters are present, but have eluted before you turned the detector on.

3) Look at separation of desired compunds and be sure that you have the separation you want - if not, you may need ot adjust the temperature profile.

Thank you so much! How do I start acquiring data early on? I'm very new to the chemstation software.

To begin acquiring earlier you need to adjust your solvent delay. Depending which version of Chemstation you are using either in the Istrument menu under Sim/Scan Parameters or the icon with the Yellow Quadrupole to open the window were you will find Solvent Delay and a box to enter the desired time. Looks as you are starting now at 7.5 minutes and C14:0 went from about 13.25 minutes back to 8.5 minutes retention time so try setting solvent delay to 4 minutes. That should give you the other peaks you are missing. Then from there make sure your flow is correct. If using constant flow make sure you have entered the new column length into the column menu so that it will adjust the head pressure to give the same flow you had on the longer column.

I think what has happened is the column length was not set correctly and you are flowing far too fast compared to the previous run, just by looking at the sharper peaks along with reduced retention times. Chemstation sets column flow by calculating the column length and diameter with film thickness and adjust the inlet headpressure accordingly to give the correct flow rate.

The extra bleed could be coming from the tip of the column in the inlet. Are you using a guard column? If not it could be the fresh cut of the column is exposing phase that has not been heated to the temperature of the inlet before and causing it to bleed excessively. What is the inlet temperature versus the highest oven temperature of the run? If the inlet temperature is near the upper limit of the column or above, try adding a 1 to 5 meter guard column to help protect the phase, also be sure not to exceed the column maximum at the MS interface as that can cause excessive bleed also. I would set the MS interface (AUX on newer versions of software) to 10-20 degrees below the column maximum temperature to prevent bleed.

[chromatonoob]

1) Be sure the new column dimensions are included in your method. The linear velocity needs to stay the same.

2) With a shorter column, compunds elute more quickly - even with the correct linear velocity. You need to start acquiring data earlier in the run. The early eluters are present, but have eluted before you turned the detector on.

3) Look at separation of desired compunds and be sure that you have the separation you want - if not, you may need ot adjust the temperature profile.

Thank you so much! How do I start acquiring data early on? I'm very new to the chemstation software.

To begin acquiring earlier you need to adjust your solvent delay. Depending which version of Chemstation you are using either in the Istrument menu under Sim/Scan Parameters or the icon with the Yellow Quadrupole to open the window were you will find Solvent Delay and a box to enter the desired time. Looks as you are starting now at 7.5 minutes and C14:0 went from about 13.25 minutes back to 8.5 minutes retention time so try setting solvent delay to 4 minutes. That should give you the other peaks you are missing. Then from there make sure your flow is correct. If using constant flow make sure you have entered the new column length into the column menu so that it will adjust the head pressure to give the same flow you had on the longer column.

I think what has happened is the column length was not set correctly and you are flowing far too fast compared to the previous run, just by looking at the sharper peaks along with reduced retention times. Chemstation sets column flow by calculating the column length and diameter with film thickness and adjust the inlet headpressure accordingly to give the correct flow rate.

The extra bleed could be coming from the tip of the column in the inlet. Are you using a guard column? If not it could be the fresh cut of the column is exposing phase that has not been heated to the temperature of the inlet before and causing it to bleed excessively. What is the inlet temperature versus the highest oven temperature of the run? If the inlet temperature is near the upper limit of the column or above, try adding a 1 to 5 meter guard column to help protect the phase, also be sure not to exceed the column maximum at the MS interface as that can cause excessive bleed also. I would set the MS interface (AUX on newer versions of software) to 10-20 degrees below the column maximum temperature to prevent bleed.

Hi James_Ball:

Ok, I changed the solvent delay from 7.50 to 4 minutes. Our inlet temperature is 280 C, highest oven temperature of the run is 230 C.

Aux T was initially 240, I changed it to 230 C.

In the first image, we were using a guard column. Upon column change, we kept the old guard column and connected that with the new column. We obtained the same results we have now, so we took the guard column off assuming it was old and time for a new one - but same problem. I changed the inlet liner, o-ring, septum, and gold seal, hoping this would solve it as well. I came back today to the same results - upward drift.

[James_Ball]

Looking that column up is lists the temperature limit as 250c isocratic and 260c programmed. For this without a guard would be best to keep the injector below 250c, with guard it should be ok at 280c. The Aux would be ok at 240c but give you even less bleed at 230c as you have it now.

If the guard connector had a small leak when it was on before, air can damage the column to give the higher bleed. Sometimes baking it out or just running it over time can reduce the bleed, sometimes not. If you have the other half of the column and it hasn't been exposed to high temperatures with possible air in it then you could switch to it and see if the bleed continues.

One thing I did notice with the first two chromatograms is that the peak heights are half what they were before going to the short version of the column. If this is the same standard they should have nearly the same height. If the flow is wrong because the length was not correctly entered for the shorter column that would not only explain the shorter retention times but lower peak heights since the overall pressure in the analyzer would be higher due to too much carrier flow. Do you have the same vacuum reading now as before? If the flow was correct the lower peak heights could suggest there was a leak at the connector for the guard column. When I attach a guard column I set the flow and place the whole connector into a beaker of methanol to see if there are any bubbles, if I don't have a helium leak detector handy, the leak detector will find even smaller leaks.

A good idea with the vacuum is to make a note of what it is when you have a good setup, that will help trouble shoot leaks or problems with flow in the future. It should be 5*10^-5torr or less. Even better if you can run in the 10^-6torr ranges.

[chromatonoob]

Looking that column up is lists the temperature limit as 250c isocratic and 260c programmed. For this without a guard would be best to keep the injector below 250c, with guard it should be ok at 280c. The Aux would be ok at 240c but give you even less bleed at 230c as you have it now.

If the guard connector had a small leak when it was on before, air can damage the column to give the higher bleed. Sometimes baking it out or just running it over time can reduce the bleed, sometimes not. If you have the other half of the column and it hasn't been exposed to high temperatures with possible air in it then you could switch to it and see if the bleed continues.

One thing I did notice with the first two chromatograms is that the peak heights are half what they were before going to the short version of the column. If this is the same standard they should have nearly the same height. If the flow is wrong because the length was not correctly entered for the shorter column that would not only explain the shorter retention times but lower peak heights since the overall pressure in the analyzer would be higher due to too much carrier flow. Do you have the same vacuum reading now as before? If the flow was correct the lower peak heights could suggest there was a leak at the connector for the guard column. When I attach a guard column I set the flow and place the whole connector into a beaker of methanol to see if there are any bubbles, if I don't have a helium leak detector handy, the leak detector will find even smaller leaks.

A good idea with the vacuum is to make a note of what it is when you have a good setup, that will help trouble shoot leaks or problems with flow in the future. It should be 5*10^-5torr or less. Even better if you can run in the 10^-6torr ranges.

I just finished a run today - same results after those changes. I did however change the inlet T to 250 C - I will try another run again... After looking at the TIC from a month ago vs. recent results, you mentioned the peaks doubling. That would also change the scale. The TIC from a month ago shows a slight upward drift at about where we are seeing it now. If changing the inlet temperature doesn't work, what else can I do?

Here is the history on baking out the column. 2 hours the first day - which apparently wasn't long enough. On the second day (a Friday), baked out for 4 hours. That following Monday, we decided to recondition over night for about 10 hours. Then we cut the column in half. Unfortunately, the other half of the column has been exposed to high temperatures.

I don't suspect any leaks. Or am I wrong?

Thank you so much for all your help. I'm beginning to get discouraged and your responses make things a lot easier for me to deal with it.

EDIT: Wow, I just realized that when I conditioned the column over night, I had it connected to the MS interface. Could this be the problem? I may have potentially contaminated it then.

[Don_Hilton]

Suggestion:

Post your instrument conditions, as listed in ChemStation. Let's see if anything stands out. If you have conditions from before and after you made method changes, that may help.


As far as conditioning the column with it in the MS interface. Unless you are dumping a lot of stuff into the mass spec, you should be OK. It is not uncommon to run a column up to a high temperature (close to the upper program limit) and hold it there for a while - just to clear compunds that do not elute well. If you do dump a lot of stuff into the mass spec, you may find yourself cleaining the source sooner. Check the tune - and if it looks OK, drive on.

[chromatonoob]

Suggestion:

Post your instrument conditions, as listed in ChemStation. Let's see if anything stands out. If you have conditions from before and after you made method changes, that may help.


As far as conditioning the column with it in the MS interface. Unless you are dumping a lot of stuff into the mass spec, you should be OK. It is not uncommon to run a column up to a high temperature (close to the upper program limit) and hold it there for a while - just to clear compunds that do not elute well. If you do dump a lot of stuff into the mass spec, you may find yourself cleaining the source sooner. Check the tune - and if it looks OK, drive on.

Hi Don_Hilton:

Tune and air and water check look fine. As far as instrument conditions go, what should I list?

Thank you

[Don_Hilton]

Method parameters - the various settings for the GC method. Inlet and Oven conditions. I assume that you did not change carrier gas while shortening the column? And I assume column parameters are part of the ChemStation method? (It's been a while since I used ChemStation - perhaps on instruments too old to have electronic flow controllers.)

[chromatonoob]

Method parameters - the various settings for the GC method. Inlet and Oven conditions. I assume that you did not change carrier gas while shortening the column? And I assume column parameters are part of the ChemStation method? (It's been a while since I used ChemStation - perhaps on instruments too old to have electronic flow controllers.)

Here is the method with the 60 m column:

ALS
Injection volume: 1uL
Washes and Pumps: Sample Washes 3; Sample Pumps 6; Pre injection solvent (hexane) washes 2; post injection solvent (hexane) washes 2

S/Sl Inlet
Mode: split, split ratio 2:1
temperature: 280c
Pressure 17 psi
septum purge flow: 3 mL/min

Oven Temperature program: 50c, hold 1 min; 25c/min to 175c; 4c/min to 230 c, hold 10 min

Carrier gas, carrier gas pressure: He, 17 psi, constant pressure mode

MSD
Source T: 230c
Quad T: 150c
solvent Delay: 6 min
EMV Mode: Gain Factor
Acquisition Mode: Scan
Scan Speed: Normal
Low mass: 50.00
High mass: 550.00

Thermal Aux 2 T: 240c

Run Time: 29.75 min

Now a few changes for the 30 m column:

ALS
Injection volume: 1 uL
Washes and Pumps: sample washes 6 (not sure how or why that changed); sample pumps 6; pre injection solvent (hexane) washes 6 (again not sure how or why that changed); post injection solvent (hexane) washes 2

S/Sl Inlet
Mode: Split, split ratio 2:1, 2 mL/min
Temperature: 250c
Pressure: 7.6165 psi
Septum purge flow: 3 mL/min

Oven Temperature Program: 50c, hold 1 min; 25c/min to 175c; 4c/min to 230c, hold 5 min (hold for 5 min is the only thing I changed about the T programming/ramping - I need to do more research on a T program method)

Carrier gas, carrier gas pressure: He, But instead of constant pressure mode, constant flow mode was chosen at 1 mL/min; average velocity listed 36.445 cm/sec.

MSD
source T at 230c
quad T at 150c
solvent delay now at 4 min

Thermal Aux 2 Temp: 230 c
Run Time 24.75 min

Changes are in BOLD