1. No! Not acceptable.

2. The technician may have cleaned everything appropriately. I think you need a 'new or replacement' column.

Before replacement of the column, cut first 15-20 cm from the injector site. After that connect it back and make a test.

Check few blank runs with and without injection of clean solvent.

Clean the column in the gas flow at highest acceptable temperature of its usage (check the max temperature on the column box) and monitor the baseline - normally a few hours allow to remove all impurities.

Good luck!

G.

Also inject a 'lot' of Hexane so you clean any injector carryover. Where did this column come from? Was it dedicated for FAME analysis only?

First, inject 1 uL of air (no sample) and run the method to see if it is nothing from the sample that may cause the problem.

Secondly, cut the column per the recommendation above.

Thirdly, I think it may be the column issue, Can you find another column and test it without the sample to see if you have the same issue. If no issue with the second column, your first column is bad (looks like column bleeding to me). You can also check by running isothermal at high temp (final temp of your gradient) to see if you have a stable baseline. If you do, it is the column bleeding.

I think you need a serious cleaning of the column.But first as previously said, cut the column but make sure you do not have particules of copper entering in the column during this step. Do what is preconized on the previous comments by washing with solvents and so one. Then the next step would also be to 'bake the column by increasing the temperature of the oven so you get rid of everything not wanted in the column.

The main problem is definitely the column itself, the stationnary phase worns off after years of use. You might have to buy a new one... I have seen that on 5-15 years old columns.

Your injector may also be contaminated with a polar solute which Hexane alone will not solubilized. Thus, injected 100% Hexane, then 100% IPA, water, 100% IPA, and finally Hexane again.

Thx for all ur suggests. By the way, my experiments just force on the FA composition for Salami, not for microbes fingerprinting dear Bruce Neagle. By the way, what does ur too much hexane mean? Do you mean their dissolve other compounds which caused an interference? My other colleagues thought there was too much water and suggest me to dry the hexane before using. Again, thx for ur suggestions.

I suggest you to first check with the column manufacturer to see if this column is compatible with the chemicals you injected. If you think it is the water, you should add a guard column. I assume you have a capillary column, you should also try to 'bake the column' . Set the temperature slightly below the maximum column temperature and bake it for half hour. You can do it isothermally. If you do not see any improvement, then you should replace the column. Also , check the leak from the sample loop/rotation valve.

You can try to clean the column, leaving it with an isothermal method at 250°C overnight with a carrier flow. Also check if the column is fine, maybe you need to change it.

to run the sample in GC folow this

Ø GC

1. Take the methylated samples from the freezer to a fume hood

2. Prepare brown vials and inserts

3. Vortex the samples, and put 50ul (30 ul minimum) to every vial

4. Store in a fridge

5. Teflon sticky tape and store in a freezer at -18 °C

Cut the column the main problem is definitely the column (instead of going to experimentation on old column, let try to experiment on new column) test it without the sample to see if you have the same issue. If no issue with the new column, then also check by running isothermal at high temp (final temp of your gradient) to see if you have a stable baseline.

Could be multiple of problems. Was the gas recently changed? Since this is gas chromatography the 1st place to look for problems is the gas supply. Carrier gas, make-up gas, and hydrogen for detector.

How old is the injection liner? The liner should be changed frequently! How does your septum look? Change it daily! You may have septum bits that have migrated from injection port down onto head of column. Think about using a little bit of glass wool in your injection liner to avoid this in the future.

Get some butane and do a u-opt on the column - adjust flow/pressure of carrier gas until you have optimized the flow rates. (add butane to septum vial, direct inject butane head space, and get RT of the butane peak. follow procedure outlined here http://www.team-cag.com/support/other_support/CarrierGases_GC.pdf

Many GC bad chromatography issues are caused by injecting too much. Make sure that the injection liner you are using is appropriate for the amount of sample you are injecting. It is quite easy to overload your injector port,- that overload contaminates the entire injection system and purge flow lines. Limit injections to 2uL - if that has happened you need to clean the entire injection port system, and sometimes replace purge vent lines.

FIDs require cleaning or changing of the jet frequently. During a run gently tap the detector with a screwdriver - do you see peaks on your chromatogram? If yes - you have a dirty detector.

Finally if this fails look at uninstalling column - cleaning injection port, clipping front of column (1/3 meter) re-installing - Install column to injection port 1st, make sure you have flow by placing detector end of column into vial of hexane, should see nice flow of bubbles indication good flow through column. Install into detector. Re-run u-opt with butane.

If you do all of the above it should be back to normal. This sounds like alot but you should be able to get this all done in less than 2 hours.

A key topic is related to periodic maintenance for GC. According your pics, all indicate that column is the problem. Please consider the following quiestions.

-How many injections have this column?

-When it was the last FID maintenance?

-Do you clean-up or dry your samples?

-Do you conditioning your column before analysis, when GC stands off during some time?

Do you use glasswool in liner?

good luck

Dear Majid

It is so clear that your GC column encountered with "Stationary Phase Bleeding". And as your GC spectrum shows this problem is critical on peak quantification and resulting wrong peak concentration.

1- This problem can be happen because of different reasons. The most possible one is "Oxidation of stationary phase at high temperatures. This can happen by leakage in injection port or in the gas lines entering the injector. Or by oxygen contamination in the carrier gas. For this problem you can use oxygen tarp on the carrier gas lines and check carefully any leakage in the gas line and flow system and also the injector inlet.

2- Running GC without carrier gas flow or not enough flow can hurt the stationary phase film in the column at high temperatures. So check your gas flow by submerging the outlet line in vial of methanol and check to see the gas bubbles. if the problems comes from flowing, check your carrier gas system cylinder or generator and the system lines for stuck or broken.

3- Run GC column with temperatures more than manufacturer maximum recommended temperature which will born out the stationary phase gradually with times.

4- sometimes contaminated sample with high molecular weight residues poorly separated in the sample fractionation can cause rising baseline. In this situation these compounds can elute from the column in form of separated and irregular peaks. The solution of this problem is cleaning the column by frequent running the solvent with high allowed temperatures. Check your sample preparation and fractionation carefully and check the samples for impurities.

5- contaminations of the sample by acids and bases also can degrade the stationary phase resulting elevated baseline.

Finally after checking all the possibilities of the reason , I think your column need to replace with new one, but care about all the cations before running the GC. Be careful about maximum temperature, purity of the sample, carrier gas and all the inlet, lines leakage.

There are some methods to estimate the bleeding and subtracting from the peak concentration but for using them the bleeding intensity should be less than expected value for that type of stationary phase and also depend on your column configuration.

Hi Everyone!!

I am attaching the gas chromatography analysis report here.

The shape of peaks in my calibration report doesn't look fine.is'nt it?

What in your opinion is the reason

1) of these irregular peaks?

2) Why the shape of baseline in detector 2 is not smooth near Nonane(time=36.6)

3) Reason of variation in response factor deviation in every calibration (of 4 heavy hydrocarbons in detector 2)

Will be grateful ,if anyone can help!!

not all the peaks are well separated and I think one of the peak at the beginning was too intense because you can see that the detector is saturated (or scale isnt good).

Otherwise I am not really sure what we are looking at so...