Dear Hui Wei

I am also doing PLFA analysis on South African soils. I never had this problem since our soils are very poor. The aminopropyl columns that I am using have a limit of 10 mg of material. As long as you syay below that yiu will probably not experience overloading problems.

Regards

Arno Hugo

I do not really understand the problem: Column overloading is seen in a broadening and distortion of peaks.This of course effects integration and hence quantification. It also effects separation because the peaks tend to broaden and smear into the next compounds. I definitely would run the samples under higher dilution again! We do a lot of PLFA analyses but overloading is seldom a problem. Are we talking here about something else??

Dear Arnold and Wolf-Rainer,

Thanks for your answers. Yes we are talking the same thing which only occurs when PLFA concentration is too high. Overloading is really seldom a problem and did not occur in my PLFA analysis before. Unfortunately it happened in my friend's experiment. I actually agree with wolf-Rainer and suggested that my friend should dilute and re-run the samples again for a more precise result, but I was not very sure about the effect of overloading peaks and want to verify before she restarts......

Thank you both again.

Regards,

Hui

Dear Hui Wei

I do also a lot of PLFA-analysis. I never had the problem of overloading the column! I totally agree with Wolf-Rainer, but I think it's strange that the column was overloaded. I would definitly check if nothing went wrong during the extraction. I would also dilute and re-run the samples to be sure,

Retention time is frequently dependant on the type of fatty acid the column characteristics, the mass loaded and the temperature program, when considering results your reference identity standards should be of the same concentration as the sample if possible. If this is not possible then increased concentration will generally result in peak broadening and therefore a longer retention time this can be quite significant if the fatty acid is very concentrated and result in miss-labelling of the fatty acid. Peak shape will distort if the column is overloaded and washout between peaks can be poor eg a large peak may actually hide a smaller following peak eg Vaccenic after Oleic. PLFA determination is frequently a study of fatty acid patterns, miss identification or loss of some fatty acids can cause significant problems when doing subsequent statistical analysis, if there is a consistent pattern of fatty acids present and then one disappears closely examine the peaks surrounding them for shoulders it may be that retention or resolving power may have been compromised. Do this before drawing conclusions based on perceived changes to the profile. 

Dear Pieter,

Yes it is strange the overloading pronblem happened in the PLFA analysis. I never had such a problem but my friend did. She makes sure nothing is wrong in the analysis; the same person conducting the analysis, the same method used for analysis, the same reagents......I guess it may be attributed to the high microbial biomass (indicated by microbial biomass carbon with fumigation-extraction method).

And dear Kenneth,

Thank you very much for your detailed explanation. Changing the following statistical analysis is the thing we worried about. To avoid drawing mistaked conclusions, we will re-extract and re-run the samples.

Regards,

Hui

The main problem with peak overload is that peaks become broader and retention time can shift significantly, the broader the peak the less likley that you will acheive full resolution between peaks with the potential that some key peaks that allow appropriate discrimination about characteristics are hidden under the enlarged peak - rerun on a lower dilution or a smaller injection volume may also assist.

Care also should be taken in the isolation of the plfa's from your neutral or glyco lipids that the column here also has not been overloaded causing some fractional loss of PLFA's.