Chromatography Forum

Hi, I'm back for more help. Currently, I'm running my set up from the other thread:

viewtopic.php?f=2&t=18208

Currently, everything(including the once broken backpressure valve) is working equipment-wise. I inject my solution, which contains 4 target substances including the ITSD, and it runs. After about 4.5 minutes, the ITSD(MW ~270) elutes and presents a peak of several hundred to a few thousand. However, nothing of the other compounds(MW ~310) elute according to the FID signal. Can anyone help me troubleshoot this, please?

EDIT: I should also add that I changed out the inlet liner to be only wool, as to not filter out the analytes(which I suspected originally as the problem because I had Chromosorb W in there).

bump?

What exactly are your internal standard and target substances?

What exactly are your internal standard and target substances?

ITSD: 0.5mg/ml tribenzylamine/ethanol
Targets: cannabinol, tetrahydracannabinol, cannabidiol

What is the temperature program you are using?
Pending you temperature range, you simply may not be setting your GC at a temperature high enough or time long enough for the other materials to elute.A DB-5 column can handle temperatures up to 350C, though obviously I would not go that high unless needed. If this is the case you will want to bake out you column and set a method up that does allow for these heaver MW compounds to elute. Also, you can keep you inlet (depending on what it is) and detector (in this case) at the highest temperature you use on your method.

What is the Flow?

Have you run the other target compounds only to see if still nothing appears?

What is the temperature program you are using?
Pending you temperature range, you simply may not be setting your GC at a temperature high enough or time long enough for the other materials to elute.A DB-5 column can handle temperatures up to 350C, though obviously I would not go that high unless needed. If this is the case you will want to bake out you column and set a method up that does allow for these heaver MW compounds to elute. Also, you can keep you inlet (depending on what it is) and detector (in this case) at the highest temperature you use on your method.

What is the Flow?

Have you run the other target compounds only to see if still nothing appears?

I'm using:

inlet: 280
detector: 300
oven: 200, 10 degrees/min for 4 min to 240

The flow is splitless, 20:1, ~20ml/min:1ml/min.

I ran it without the standard and it gave me one small peak. I'm assuming this means they are all eluting together. I am going to let the oven cool off and then I'm going to try resetting the column in the oven(per this: https://docs.google.com/viewer?a=v&q=ca ... E7bchbVt3w). But that's where I am so far.

I re-set the column. I had the column only going about 8mm past the ferrule and not in to the detector. I adjusted that(probably about 70mm in now?). I tested it with both ITSD and no-ITSD samples. With both, I am getting one peak.

I should also take this time to clarify that I meant I'm only getting on peak. I'm assuming its the solvent peak instead of the ITSD peak since I still get the peak with just solvent and nothing else.

I am wondering if you have a concentrated standard, since you have one peak it may be solvent peak and there is a leak around the inlet or the split is not working well.

I am wondering if you have a concentrated standard, since you have one peak it may be solvent peak and there is a leak around the inlet or the split is not working well.

I am going to be disassembling the split inlet tomorrow to check for problems.

Few comments.
1. Check that your system works properly using your routine System Suitability (performance checking) solution. If you do not have any, use something that worked before, re-cap and keep in the fridge for the next time.
2. Selection of ISTD is not optimal. Try something more inert and closer to chemistry of your analytes. I also would not recommend EtOH as solvent.
3. Most people do derivatization.
4. Check that LOD is adequate to your task/concentration.

There are digital flow meters, that when you turn all the flows off, FID off and only allow column flow, you can check the flow out on the FID and see if it matches the flow given from the method.

good luck

Alright, I've finally disassembled the inlet, and it appears that the inlet seal is missing a washer on top. Would this be a cause of my problems, or should I fix that and keep looking?

There are digital flow meters, that when you turn all the flows off, FID off and only allow column flow, you can check the flow out on the FID and see if it matches the flow given from the method.

good luck

Everything seems to be flowing fine. The one peak I do get is approx 4.8 minutes in and that's about what I'm shooting for for elution time. I will take the soap film flowmeter to it later to verify, though.

Few comments.
1. Check that your system works properly using your routine System Suitability (performance checking) solution. If you do not have any, use something that worked before, re-cap and keep in the fridge for the next time.
2. Selection of ISTD is not optimal. Try something more inert and closer to chemistry of your analytes. I also would not recommend EtOH as solvent.
3. Most people do derivatization.
4. Check that LOD is adequate to your task/concentration.

1. Don't have one and have never used this machine before. It was purchased used. I will look into getting a performance checking solution from my supplier.
2. The choice of ISTD and ethanol was made for me by the validated procedure I'm following. I'm going to switch to methanol after this ethanol runs out though because the standard solution I have is in methanol.
3. I will implement that.
4. Suggestions on what it should be?

Start from the beginning. Check all gases, quality, and feeding pressure. Can you ignite? Establish a baseline just for flame. You may need to wait half a day to fully stabilize the system as it is of unknown state as you mentioned.

Performance solution is good but if it is too long to get, try something that definitely will work. Check glossy catalogues or Google. Check what is in your drawers, inject this if it sounds right. Something like toluene, decane. Keep correct split/concentration ratio.

I would walk away from alcohols completely, try something non reactive. If you are planning to do derivatization you will do it anyway.

Regarding LOD, if you know expected concentration, try to match it, no need to go too low if your expected concentration is high. I would say at least 3 to 10 times better than expected lowest concentration of your drugs.

The inlet washer goes under the seal. You will not get a good seal if the washer is on top.
Make sure the column is properly installed in the injection port ( 5-7 mm above the ferrule tip).
Try lowering your inital temp to 20 degrees below the boiling point of your solvent. Hold this temp. for at least 2X your split time ( if using).
When I insert a column into an FID I push it up ( gently) until I feel resistance then pull it back 1-2 mm.

I just re-set the columns...again. However, this time I re-cut the ends(the FID end was 1/2 obstructed I found). I also upped the column flow(it was originally going at like 0.5 ml/min but I upped it to ~1.2ml/min). Also, I just performed a capillary column conditioning(column didn't get conditioned when installed originally).

I tried starting the oven out at 70 and 60 degrees(ethanol boils at 78). The first run I did showed two, overlapping peaks, however, I used an in-syringe aliquot of ethanol to rinse. In the second one, I used no ethanol to rinse and there was no second peak.

Due to financial constraints, I've been trying to hold off with purchasing a performance checking solution(I don't have anything, literally, else to run through there either) and hexane or toluene as a solvent. However, if things keep up this way, I may have to find some money for those just because I seem to be running out of other options.

The inlet washer goes under the seal. You will not get a good seal if the washer is on top.
Make sure the column is properly installed in the injection port ( 5-7 mm above the ferrule tip).
Try lowering your inital temp to 20 degrees below the boiling point of your solvent. Hold this temp. for at least 2X your split time ( if using).
When I insert a column into an FID I push it up ( gently) until I feel resistance then pull it back 1-2 mm.

I'm using split, but I'm leaving it on for the whole run. I changed my initial temp. And so checked for the washer and the column-FID connection.

EDIT: I finally got one run to show me something else, barely:



That was done starting at 80 degrees, working up to 230 at 30/min.