Chromatography Forum

Hello,

I have been running a 3800 Varian with a 2200 saturn (MS) for some time until my turbo pump just crashed. SO, now I am using the back up GC FID 450 (Varian).
I have been having the hardest time when running standards. I am finding that my low standards peaks are taller and have more area than my high end standards.
I have replaced the flame tip assembly with a new one, changed out columns with the MS I had on, scored both ends of columns, repositioned column in injector, and detector and still I get the same results, not being able to run a cal curve.
Anyone run into this? I do have to say I am fairly new at GC FID compared to GCMS.
Thanks,
Anamari

The problem sounds like it is with your standards.

Things you never saw with a Mass Spec now you see with a FID.

Rod

What compounds do you analyse ?

What solvent, GC and detector conditions, injection volume, split ?

This may help to find the culprit.

@dblux- I am looking for taste and odor compounds in drinking water (MIB and Geosmin)..
I use SPME extraction.
20,30,300 gas mix using He as make up. Detector setpoint 300C
Split ratio 50 at 3.00 minutes and at 10.00 minutes 5 split ratio.

Thanks!

@chromatographer 1 I initially thought it could be my standards, but after multiple attempts to rule them out, they are clearly ruled out. I ran this same instrument several months back having no problems and detecting down to 5ppt. Ever since I changed the flame tip etc.. I am having these issues.

Thanks all for you speedy responses.

@dblux- I am looking for taste and odor compounds in drinking water (MIB and Geosmin)..
I use SPME extraction.
20,30,300 gas mix using He as make up. Detector setpoint 300C
Split ratio 50 at 3.00 minutes and at 10.00 minutes 5 split ratio.

I'm not familiar with SPME. Just a guess - inlet temp not stable ?
I would start with investigating what happens in the inlet.

I have racked my brains for anything to do with the instrument that could cause a larger peak for a smaller quantity - with no success at all. Anything that reduced the size of the high standard peaks would do the same to the low standard peaks, and anything that increased the size of the low standard would have the same effect on the high standard.

This, of course, assumes that everything except the concentration of the target analytes in the standard is kept constant.

What trouble shooting did you do that made you decide that the standards were not the problem ?

If you run replicate standards at the same concentration do the peak areas stay the same ?

How repeatable is the problem - how many times have you seen large peaks for low standards and small ones for high standards ?

If you are using an autosampler - what kind is it ?

Peter

@Peter apps

I have re-made my standards 3 times now, from different stock lots, and every run I have made with the different standards have given me the same results. Lower concentration standards gives me bigger peak than the highest one.
I think if it was standard making error I would have caught it, hence I believe the problem doesn't lie within the standards themselves.
Yes, I tried replicates of the same standard and got decent repeatability. Its only when I run a set of different concentrations (5ppt-100ppt) that I see this insanity!
Yes, it has happened everytime I have tried to run a cal curve, this is probably my 8th or 9th appempt.
Autosampler is a CTC combipal. Great autosampler.
Temps in injector seen consistant, at least this is what I see.

Just to be completely clear - a 5 "ppt" (presumably ng/l) gives a bigger peak than a 100 ppt standard ? or is it just that the peaks are bigger than they are expected to be for some of the standards ?

The smallest peak that you can expect to see with an FID is about 0.1 ng, let me guess at a sample volume of 10 ml and assume 100 % recovery into the SPME fiber. Your lowest standard contains only 50 pg (= 0.05 ng) of analyte, so whatever peak you are seeing with the FID it is not your analyte. Your highest standard will give you a clearly detectable peak if you have 100 % extraction to the SPME fiber.

What is your dilution scheme when making up standards, what are the various materials dissolved in an diluted with at each stage ?

Peter

Just to be completely clear - a 5 "ppt" (presumably ng/l) gives a bigger peak than a 100 ppt standard ? or is it just that the peaks are bigger than they are expected to be for some of the standards ?

The smallest peak that you can expect to see with an FID is about 0.1 ng, let me guess at a sample volume of 10 ml and assume 100 % recovery into the SPME fiber. Your lowest standard contains only 50 pg (= 0.05 ng) of analyte, so whatever peak you are seeing with the FID it is not your analyte. Your highest standard will give you a clearly detectable peak if you have 100 % extraction to the SPME fiber.

What is your dilution scheme when making up standards, what are the various materials dissolved in an diluted with at each stage ?

Peter

Yes, 5ppt is ng/L and yes my samples are 10ml.
Standards made in MEOH, I start off with a stock of 100 ng/uL of MIB and geiosmin (in MEOH), add 1ul of this to 1mL of MEOH for my internal standards and calibration. From here I make my 5ppt, 10ppt and so forth till I get to 100ppt to complete my cal curve.
I have used this instrument in the past briefly and got great resoluion at the 5ppt level, I always thought that it was my compounds since when I overlay all my peaks in different concentrations, there use to be spot on in the same Retention time etc...
Now, I have having this crazy issue that the 5ppt gives me a taller, bigger area count than my other standards.
Over night I ran another set of curves using the "original" flame tip (I had replaced it previously due to the ferrule disintegrating) and things look better. I have no idea why the flame tip would matter. But I am running some more standards changing gas ratios to 20(He), 40(H2), 350(air) see if this helps.
Thanks for the idea sharing. It helps me greatly to "talk" to someone about it!

Presumably your final dilution step is into water ? How much 0.1 ng/ul solution in methanol do you dilute with how much water for each standard ?

To verify the retention time of the geosmin and MIB do a liquid injection of 1 ul of your 100 ng/ul solution. This will give you nice big peaks to show where the two substances elute. There might be a minor shift compared to SPME injections.

Can you post chromatograms please - there is a how to sticky at the top of the sub-forum page.

Peter

Presumably your final dilution step is into water ? How much 0.1 ng/ul solution in methanol do you dilute with how much water for each standard ?

To verify the retention time of the geosmin and MIB do a liquid injection of 1 ul of your 100 ng/ul solution. This will give you nice big peaks to show where the two substances elute. There might be a minor shift compared to SPME injections.

Can you post chromatograms please - there is a how to sticky at the top of the sub-forum page.

Peter

for the 5ppt I inject 0.5uL, for the 10ppt 1uL etc... to 100ppt which I inject 10uL into 10 ml of DI water.
I will try injecting my stock manually like you say. Doesn't the sample have to be derivatized (extracted) in order to see it though? I was always under that impression I guess.
I will try posting a chromatogram in a while, Instrument is on a PC with no internet connection so I have to find thumbdriver etcc.
Thanks!

Unless there is a deivatization step that you have neglected to mention all that the SPME extraction does is to reverse the final dilution step - it takes (some of) the MIB geosmin and methanol back out of the water.

Peter

Unless there is a deivatization step that you have neglected to mention all that the SPME extraction does is to reverse the final dilution step - it takes (some of) the MIB geosmin and methanol back out of the water.

Peter

Thank you for that clarification. I thought derivitizing meant actually the extraction process/making the compounds "visible" basically.
No, no other steps omitted then.

Unless there is a deivatization step that you have neglected to mention all that the SPME extraction does is to reverse the final dilution step - it takes (some of) the MIB geosmin and methanol back out of the water.

Peter

Thank you for that clarification. I thought derivitizing meant actually the extraction process/making the compounds "visible" basically.
No, no other steps omitted then.

@Peter apps.

So, I manually injected my stock standards to get good idea of retention times, then I decided to manually inject my standards to make a cal curve but my chromatogram dipped far into the negatives, and am geting bad data, again.
So, my conclusion is the GC is running fine, detector detects and column seems ok. So my problem is with in the injection/SPME part then?
Thanks

Unless there is a deivatization step that you have neglected to mention all that the SPME extraction does is to reverse the final dilution step - it takes (some of) the MIB geosmin and methanol back out of the water.

Peter

Thank you for that clarification. I thought derivitizing meant actually the extraction process/making the compounds "visible" basically.
No, no other steps omitted then.

@Peter apps.

So, I manually injected my stock standards to get good idea of retention times are the retention times of the MIB and geosmin from the standard in methanol the same as the retention times of the peaks that you have assigned to MIB and geosmin when you do SPME sampling from standards in water ? What are the actual retention times in each case ?, then I decided to manually inject my standards are these the standards in water, or in methanol ?? if they are in water you are likely to have had some injector problems, if you are doing liquid injections then you must inject in methanolto make a cal curve but my chromatogram dipped far into the negatives does this mean that you see negative peaks i.e. a dip below the baseline, at the retention times of MIB and geosmin ?, and am geting bad data, again.
So, my conclusion is the GC is running fine, detector detects as I argue above, your lower standards contain too little MIB and geosmin for an FID to see them and column seems ok. So my problem is with in the injection/SPME I doubt that you are getting 100% recovery into the SPME fiber, and this further reduces the quantity of MIB and geosmin that the detector has to detect part then?
Thanks

If you run a blank i.e. SPME 10 ml of water with 5 ul of methanol in it containing no MIB or geosmin, do you see peaks at the retention times of MIB and geosmin ?

Peter