Increasing resolution, GC-FID, fatty acids, Omegawax column

[Phil Helmet]

Dear forum members,

I am analyzing fatty acid methyl esters on a GC-FID, using an Agilent 7890 system equipped with an Omegawax 250 column. I am experiencing a drift in the resolution between some of the analytes in my control sample which is a GLC744, consisting of a mixture of fatty acid methyl esters covering the whole range up to C24:1 n-9.

Can anyone give me some feedback on the possible cause of this drift. I installed a new column two months ago, and the resolution between two peaks that I monitor in my control chart has increased from 1,47 to 2,63 in that period.

The samples are dissolved in n-hexane.

Samples are injected in a 1/100 split.

GC settings:
Injector port at 250 degress centigrade.
Constant flow 1.9336 mL/min.
Pressure 75 kPa

Detector at 280 degree centigrade.
H2 flow = 40 ml/min
Air flow = 450 ml/min
Makeup flow = 28 ml/min

Oven program:
Initial temp = 50 held at 1 min.
Ramp to 180 at 20/min
Ramp to 220 a 1/min
Total run time 47,5 min.


/Philip

[chromatographer1]

Philip

It might be helpful for a diagnosis to share with us the two peaks in question.

chromatography is not just physics but chemistry.

A Question: have any other resolutions changed during this time?

best wishes,

Rod

[Phil Helmet]

Rod,

We are monitoring two pairs of peaks in our control charts, in order to see if the column deteriorates over time. These two pairs are C20:3 n-3 and C21:0 AND C22:6 n-3 and C24:1 n-9. Both resolutions have increased during the course of two months, where I installed a brand new column. We have had the same problem before where C24:0 and C24:1 n-9 were getting too close, and so I replaced the column. Now the same pattern is observed and I would lkke to find out the cause of this. I dont want to change the column every two months

/Philip

[chromatographer1]

Your column is losing polarity.
Why?

low probability:
It could be damaged from oxygen that is present or is leaking into your carrier gas.

Higher probability:
It could be caused by less polar deposits from your samples.
It could be caused by some of your unsaturated FAMEs decomposing on the column.

I would remove 5 meters of the front of your column when this becomes noticable and see if the drift is reversed.

I don't know how many samples you test in two months but if it is damaged from use then lowering the final temperature of the analysis (costing you time) might save you column life (saving your money), but it might be cheaper to do it faster and buy columns more often.

I can remember being able to perform this analysis was a joy. (early 1980s)

Cost then was not an issue. Imagine trying to do this test on packed columns !

best wishes,

Rod

[Phil Helmet]

Rod,

I installed a guard column along with the new column. I might get better results by cutting 5 metres from that?
With respect to samples then we are running samples enough to have the system running constantly, exept for sunday evening. To lengthen the run time by 10 minutes could be expensive for us. I actually spent many days shortening the method by 5 minutes

I can imagine how it was with packed columns, but maybe you had fewer samples then

/Philip

[wiclef]

Dear forum members,

I am analyzing fatty acid methyl esters on a GC-FID, using an Agilent 7890 system equipped with an Omegawax 250 column. I am experiencing a drift in the resolution between some of the analytes in my control sample which is a GLC744, consisting of a mixture of fatty acid methyl esters covering the whole range up to C24:1 n-9.

Can anyone give me some feedback on the possible cause of this drift. I installed a new column two months ago, and the resolution between two peaks that I monitor in my control chart has increased from 1,47 to 2,63 in that period.

The samples are dissolved in n-hexane.

Samples are injected in a 1/100 split.




GC settings:
Injector port at 250 degress centigrade.
Constant flow 1.9336 mL/min.
Pressure 75 kPa

Detector at 280 degree centigrade.
H2 flow = 40 ml/min
Air flow = 450 ml/min
Makeup flow = 28 ml/min

Oven program:
Initial temp = 50 held at 1 min.
Ramp to 180 at 20/min
Ramp to 220 a 1/min
Total run time 47,5 min.


/Philip

Dear Phil,

I have analyzed FAMEs in different foodstuff and I have had good chromatograms.I used a different column which is not your brand.I was thinking that the column you used before was not the same as this one (Omegawax).If I understand well, those values you gave are the retention times (1,47 and 2,63)Does this column affect the retention time of your products?Can you explain more about your concern?

[chromatographer1]

Goodness !

It is time to replace that guard column. You can try removing 5 meters from it of course, if it is longer than 5 meters, this to show the effect of removal of 5 meters, but I would just replace it NOW.

Hope this fixes the issue.

best wishes,

Rod

[Phil Helmet]

Rod,

Today I replaced the guard column, and it had some effect (it is a 5 m guard column). Unfortunately it did not get the performance back to when the column was new. The retention of most peaks was lowered with a few seconds and the resolution of the pairs I record was reduced by 0.1 to 0.2 which is not satisfactory. The resolution has been as low as 1.8 initially.
The samples from todays preparations are now running, so I will have to wait for tommorow to see if cutting 5 metres from the front will change something.
I will also have a look at the oxygen filter to see if there is some kind of fault there.

I can add some bonus info: We have a second system running the same kind of samples, although not as many, on a 100m column for testing trans fatty acids. I have never touched that system, nor have I installed guard columns and the system is running flawlessly. This indicatates (with the help of your initial remarks) that there might be some kind of leak in the system allowing oxygen to enter.

Thanks for all the help

/Philip

[aldehyde]

I wouldn't want to touch the running-perfect machine if I was in your shoes (well actually I'm crazy so I probably would invite the risk lol), but it would be interesting to put the known good column in this system and see if suddenly your chromatographic problems go away.

Do both instruments share a common gas supply? And have you checked your gas filters (if you have them installed) lately? Trace moisture, oxygen, and hydrocarbons contaminating your carrier supply could certainly modify the surface chemistry of your inlet and column enough to cause negative effects to your chromatography.

[Appsii]

sounds like oxygen on the column to me.

try a new new ferrule on the column? perhaps there is a leak somewhere?

[chromatographer1]

The suggestions you have received are good. I hope you also check your septum. Leaks there can be subtle.

Good luck. I think you are on the right track.

Rod

[Phil Helmet]

Dear all,

Thank you for all the good reccomandations.

Heres the good news:

I trimmed 50 centimeters from the column at the injector side. I changed the septum and liner, and replaced the ferule at the detector side while I was cleaning the jet (I had spikes in the chromatograms).
My resolution are now 2.0 which is at the same level as when the column was new. This is the good news.
The bad news is that I still dont know why the column deteriorates which it will probably continue doing until I find the solution. I actually found a leak in the gas supply, at a connection where H2 enters the GC-system on the outside of the housing. I assume that this does not pose a risk for the column, since H2 enters the system after the column because it acts as a fuel for the FID, am I right about that?

Replacement of ferules and septa (which was very worn) might reduce the risk for leaks, but other than that I am still a little bit distressed.

/Philip

[BZK]

Hmmm. It sound as a bad metilation, with a some level of unmetilated fat. In that case by removing of some cm of column RT of high boiling compounds are restored.
I agree also with the previous posts regard oxigen damage.
If you run very fast analysis also uneluted cholesterol (or other sterols in case of vegetables) can increase the stationary phase of precolumn/column, exactly as unmetilated fat.

Have a good fun to find the right cause of resolution shift.

Robertino Barcarolo, Italy